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Hu S, Xu J, Cui W, Jin H, Wang X, Maimaitiyiming Y. Post-Translational Modifications in Multiple Myeloma: Mechanisms of Drug Resistance and Therapeutic Opportunities. Biomolecules 2025; 15:702. [PMID: 40427595 PMCID: PMC12109037 DOI: 10.3390/biom15050702] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2025] [Revised: 05/01/2025] [Accepted: 05/08/2025] [Indexed: 05/29/2025] Open
Abstract
Multiple myeloma (MM) remains an incurable hematologic malignancy due to the inevitable development of drug resistance, particularly in relapsed or refractory cases. Post-translational modifications (PTMs), including phosphorylation, ubiquitination, acetylation, and glycosylation, play pivotal roles in regulating protein function, stability, and interactions, thereby influencing MM pathogenesis and therapeutic resistance. This review comprehensively explores the mechanisms by which dysregulated PTMs contribute to drug resistance in MM, focusing on their impact on key signaling pathways, metabolic reprogramming, and the tumor microenvironment. We highlight how PTMs modulate drug uptake, alter drug targets, and regulate cell survival signals, ultimately promoting resistance to PIs, IMiDs, and other therapeutic agents. Furthermore, we discuss emerging therapeutic strategies targeting PTM-related pathways, which offer promising avenues for overcoming resistance to treatment. By integrating preclinical and clinical insights, this review underscores the potential of PTM-targeted therapies to enhance treatment efficacy and improve patient outcomes in MM.
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Affiliation(s)
- Shuoyang Hu
- Department of Immunology, School of Basic Medical Sciences, Xinjiang Medical University, Urumqi 830011, China; (S.H.); (J.X.); (W.C.); (H.J.); (X.W.)
| | - Jirun Xu
- Department of Immunology, School of Basic Medical Sciences, Xinjiang Medical University, Urumqi 830011, China; (S.H.); (J.X.); (W.C.); (H.J.); (X.W.)
| | - Weiyan Cui
- Department of Immunology, School of Basic Medical Sciences, Xinjiang Medical University, Urumqi 830011, China; (S.H.); (J.X.); (W.C.); (H.J.); (X.W.)
| | - Haoran Jin
- Department of Immunology, School of Basic Medical Sciences, Xinjiang Medical University, Urumqi 830011, China; (S.H.); (J.X.); (W.C.); (H.J.); (X.W.)
| | - Xiaoyu Wang
- Department of Immunology, School of Basic Medical Sciences, Xinjiang Medical University, Urumqi 830011, China; (S.H.); (J.X.); (W.C.); (H.J.); (X.W.)
| | - Yasen Maimaitiyiming
- Department of Immunology, School of Basic Medical Sciences, Xinjiang Medical University, Urumqi 830011, China; (S.H.); (J.X.); (W.C.); (H.J.); (X.W.)
- Xinjiang Key Laboratory of Molecular Biology for Endemic Diseases, Xinjiang Medical University, Urumqi 830011, China
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Peris-Díaz MD, Deslignière E, Jager S, Mokiem N, Barendregt A, Bondt A, Heck AJR. Asymmetric N-Glycosylation in the Tailpiece of Recombinant IgA1. J Am Chem Soc 2024; 146:34720-34732. [PMID: 39641195 DOI: 10.1021/jacs.4c13156] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/07/2024]
Abstract
Here, we employed a variety of mass spectrometry (MS)-based approaches, both (glyco)peptide-centric and protein-centric, to resolve the complex glycoproteoform landscape of recombinant IgA1 produced in HEK293 cells. These key immunoglobulins harbor several N- and O-glycosylation sites, making them considerably more heterogeneous than their IgG counterparts. We provide quantitative data on the occupancy and glycan composition for each IgA1 glycosylation site. Combining all data, we revealed that IgA1 molecules consist of at least three distinct populations with varying N-glycosylation site occupancies at the C-terminal tailpiece, namely, one with both glycosylation sites occupied, another with both glycosylation sites unoccupied, and a third asymmetric population with one glycosylation site occupied and the other unoccupied, challenging the prevailing acceptance that IgA1 N-glycosylation is symmetrical. This finding is significant, given that the tailpiece is involved in interactions with the J-chain and the Polymeric Immunoglobulin Receptor, and in general as antibody glycosylation is a quality attribute that needs to be carefully monitored, as the presence and nature of these modifications can affect the antibody's efficacy, lifetime, stability, and binding and/or neutralizing capacities. Optimizing strategies to produce recombinant IgA1 requires efficient and specific quality control analytical strategies, as presented here, which is essential for therapeutic IgA1-based antibody development. We expect that the integrated MS-based strategy presented here may be beneficial to comprehensively characterize the glycoproteoform profiles of IgA1-based therapeutics, thereby improving their production and optimization processes and facilitating the pathway to bring more IgA1-based therapeutics into clinical applications.
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Affiliation(s)
- Manuel David Peris-Díaz
- Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht 3584 CH, The Netherlands
- Department of Chemical Biology, Faculty of Biotechnology, University of Wrocław, F. Joliot-Curie 14a, Wrocław 50-383, Poland
| | - Evolène Deslignière
- Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht 3584 CH, The Netherlands
| | - Shelley Jager
- Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht 3584 CH, The Netherlands
| | - Nadia Mokiem
- Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht 3584 CH, The Netherlands
| | - Arjan Barendregt
- Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht 3584 CH, The Netherlands
| | - Albert Bondt
- Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht 3584 CH, The Netherlands
| | - Albert J R Heck
- Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht 3584 CH, The Netherlands
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Novak J, King RG, Yother J, Renfrow MB, Green TJ. O-glycosylation of IgA1 and the pathogenesis of an autoimmune disease IgA nephropathy. Glycobiology 2024; 34:cwae060. [PMID: 39095059 PMCID: PMC11442006 DOI: 10.1093/glycob/cwae060] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2024] [Revised: 07/21/2024] [Accepted: 08/01/2024] [Indexed: 08/04/2024] Open
Abstract
IgA nephropathy is a kidney disease characterized by deposition of immune complexes containing abnormally O-glycosylated IgA1 in the glomeruli. Specifically, some O-glycans are missing galactose that is normally β1,3-linked to N-acetylgalactosamine of the core 1 glycans. These galactose-deficient IgA1 glycoforms are produced by IgA1-secreting cells due to a dysregulated expression and activity of several glycosyltransferases. Galactose-deficient IgA1 in the circulation of patients with IgA nephropathy is bound by IgG autoantibodies and the resultant immune complexes can contain additional proteins, such as complement C3. These complexes, if not removed from the circulation, can enter the glomerular mesangium, activate the resident mesangial cells, and induce glomerular injury. In this review, we briefly summarize clinical and pathological features of IgA nephropathy, review normal and aberrant IgA1 O-glycosylation pathways, and discuss the origins and potential significance of natural anti-glycan antibodies, namely those recognizing N-acetylgalactosamine. We also discuss the features of autoantibodies specific for galactose-deficient IgA1 and the characteristics of pathogenic immune complexes containing IgA1 and IgG. In IgA nephropathy, kidneys are injured by IgA1-containing immune complexes as innocent bystanders. Most patients with IgA nephropathy progress to kidney failure and require dialysis or transplantation. Moreover, most patients after transplantation experience a recurrent disease. Thus, a better understanding of the pathogenetic mechanisms is needed to develop new disease-specific treatments.
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Affiliation(s)
- Jan Novak
- Department of Microbiology, University of Alabama at Birmingham, 845 19th Street South, Birmingham, AL 35294, United States
| | - R Glenn King
- Department of Microbiology, University of Alabama at Birmingham, 845 19th Street South, Birmingham, AL 35294, United States
| | - Janet Yother
- Department of Microbiology, University of Alabama at Birmingham, 845 19th Street South, Birmingham, AL 35294, United States
| | - Matthew B Renfrow
- Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, 720 20th Street South, Birmingham, AL 35294, United States
| | - Todd J Green
- Department of Microbiology, University of Alabama at Birmingham, 845 19th Street South, Birmingham, AL 35294, United States
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Novak J. Pathogenesis of IgA nephropathy: Omics data inform glycomedicine. Nephrology (Carlton) 2024; 29 Suppl 2:18-22. [PMID: 39327757 PMCID: PMC11441619 DOI: 10.1111/nep.14350] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2023] [Revised: 03/11/2024] [Accepted: 06/25/2024] [Indexed: 09/28/2024]
Affiliation(s)
- Jan Novak
- Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, USA
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Scheurer S, Junker AC, He C, Schülke S, Toda M. The Role of IgA in the Manifestation and Prevention of Allergic Immune Responses. Curr Allergy Asthma Rep 2023; 23:589-600. [PMID: 37610671 PMCID: PMC10506939 DOI: 10.1007/s11882-023-01105-x] [Citation(s) in RCA: 14] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 08/07/2023] [Indexed: 08/24/2023]
Abstract
PURPOSE OF REVIEW Immunoglobulin A (IgA) mediates immune exclusion of antigens in the gut. Notably, IgA plays also a role in the prevention of IgE-mediated allergies and induction of immune tolerance. The present review addresses the role of IgA in the manifestation of IgE-mediated allergies, including allergen-specific immunotherapy (AIT), the regulation of IgA production, and the mechanism of IgA in immune cell activation. RECENT FINDINGS The majority of studies report an association of IgA with the induction of immune tolerance in IgE-mediated allergies. However, reports on the involvement of humoral and mucosal IgA, IgA subtypes, monomeric and polymeric IgA, and the mechanism of IgA-mediated immune cell activation are confounding. Effects by IgA are likely mediated by alteration of microbiota, IgE-blocking capacity, or activation of inhibitory signaling pathways. However, the precise mechanism of IgA-regulation, the contribution of serum and/or mucosal IgA, and IgA1/2 subtypes, on the manifestation of IgE-mediated allergies, and the underlying immune modulatory mechanism are still elusive.
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Affiliation(s)
- Stephan Scheurer
- Federal Institute for Vaccines and Biomedicines, Molecular Allergology, Paul-Ehrlich-Institut, Paul-Ehrlich Str., 51-58, 63225, Langen, Germany.
| | - Ann-Christine Junker
- Federal Institute for Vaccines and Biomedicines, Molecular Allergology, Paul-Ehrlich-Institut, Paul-Ehrlich Str., 51-58, 63225, Langen, Germany
| | - Chaoqi He
- Laboratory of Food and Biomolecular Science, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan
| | - Stefan Schülke
- Federal Institute for Vaccines and Biomedicines, Molecular Allergology, Paul-Ehrlich-Institut, Paul-Ehrlich Str., 51-58, 63225, Langen, Germany
- Division of Allergology, Paul-Ehrlich-Institut, Federal Institute for Vaccines and Biomedicines, Langen, Germany
| | - Masako Toda
- Laboratory of Food and Biomolecular Science, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan
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Gentile M, Sanchez-Russo L, Riella LV, Verlato A, Manrique J, Granata S, Fiaccadori E, Pesce F, Zaza G, Cravedi P. Immune abnormalities in IgA nephropathy. Clin Kidney J 2023; 16:1059-1070. [PMID: 37398689 PMCID: PMC10310525 DOI: 10.1093/ckj/sfad025] [Citation(s) in RCA: 21] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2022] [Indexed: 09/10/2023] Open
Abstract
Immunoglobulin A (IgA) nephropathy (IgAN) is the most common primary glomerulonephritis worldwide and it is characterized by mesangial IgA deposition. Asymptomatic hematuria with various degrees of proteinuria is the most common clinical presentation and up to 20%-40% of patients develop end-stage kidney disease within 20 years after disease onset. The pathogenesis of IgAN involves four sequential processes known as the "four-hit hypothesis" which starts with the production of a galactose-deficient IgA1 (gd-IgA1), followed by the formation of anti-gd-IgA1 IgG or IgA1 autoantibodies and immune complexes that ultimately deposit in the glomerular mesangium, leading to inflammation and injury. Although several key questions about the production of gd-IgA1 and the formation of anti-gd-IgA1 antibodies remain unanswered, a growing body of evidence is shedding light on the innate and adaptive immune mechanisms involved in this complex pathogenic process. Herein, we will focus on these mechanisms that, along with genetic and environmental factors, are thought to play a key role in disease pathogenesis.
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Affiliation(s)
- Micaela Gentile
- Translational Transplant Research Center and Department of Medicine, Icahn School of Medicine at Mount Sinai, NY, USA
- UO Nefrologia, Dipartimento di Medicina e Chirurgia, Università di Parma, Parma, Italy
| | - Luis Sanchez-Russo
- Translational Transplant Research Center and Department of Medicine, Icahn School of Medicine at Mount Sinai, NY, USA
| | - Leonardo V Riella
- Division of Nephrology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA
| | - Alberto Verlato
- Translational Transplant Research Center and Department of Medicine, Icahn School of Medicine at Mount Sinai, NY, USA
| | - Joaquin Manrique
- Nephrology Service, Complejo Hospitalario de Navarra, Pamplona, Spain
| | - Simona Granata
- Nephrology, Dialysis and Transplantation Unit, University of Foggia, Foggia, Italy
| | - Enrico Fiaccadori
- UO Nefrologia, Dipartimento di Medicina e Chirurgia, Università di Parma, Parma, Italy
| | - Francesco Pesce
- Nephrology, Dialysis and Transplantation Unit, Department of Emergency and Organ Transplantation, University of Bari “A. Moro”, Bari, Italy
| | - Gianluigi Zaza
- Nephrology, Dialysis and Transplantation Unit, University of Foggia, Foggia, Italy
| | - Paolo Cravedi
- Translational Transplant Research Center and Department of Medicine, Icahn School of Medicine at Mount Sinai, NY, USA
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Jemelkova J, Stuchlova Horynova M, Kosztyu P, Zachova K, Zadrazil J, Galuszkova D, Takahashi K, Novak J, Raska M. GalNAc-T14 may Contribute to Production of Galactose-Deficient Immunoglobulin A1, the Main Autoantigen in IgA Nephropathy. Kidney Int Rep 2023; 8:1068-1075. [PMID: 37180502 PMCID: PMC10166743 DOI: 10.1016/j.ekir.2023.02.1072] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2022] [Revised: 01/13/2023] [Accepted: 02/06/2023] [Indexed: 02/16/2023] Open
Abstract
Introduction Immunoglobulin A1 (IgA1) with galactose-deficient O-glycans (Gd-IgA1) play a key role in the pathogenesis of IgA nephropathy (IgAN). Mucosal-tissue infections increase IL-6 production and, in patients with IgAN, are often associated with macroscopic hematuria. IgA1-secreting cell lines derived from the circulation of patients with IgAN, compared to those of healthy controls (HCs), produce more IgA1 that has O-glycans with terminal or sialylated N-acetylgalactosamine (GalNAc). GalNAc residues are added to IgA1 hinge region by some of the 20 GalNAc transferases, the O-glycosylation-initiating enzymes. Expression of GALNT2, encoding GalNAc-T2, the main enzyme initiating IgA1 O-glycosylation, is similar in cells derived from patients with IgAN and HCs. In this report, we extend our observations of GALNT14 overexpression in IgA1-producing cell lines from patients with IgAN. Methods GALNT14 expression was analyzed in peripheral blood mononuclear cells (PBMCs) from patients with IgAN and from HCs. Moreover, the effect of GALNT14 overexpression or knock-down on Gd-IgA1 production in Dakiki cells was assessed. Results GALNT14 was overexpressed in PBMCs from patients with IgAN. IL-6 increased GALNT14 expression in PBMCs from patients with IgAN and HCs. We used IgA1-producing cell line Dakiki, a previously reported model of Gd-IgA1-producing cells, and showed that overexpression of GalNAc-T14 enhanced galactose deficiency of IgA1, whereas siRNA-mediated GalNAc-T14 knock-down reduced it. GalNAc-T14 was localized in trans-Golgi network, as expected. Conclusions Overexpression of GALNT14 due to inflammatory signals during mucosal infections may contribute to overproduction of Gd-IgA1 in patients with IgAN.
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Affiliation(s)
- Jana Jemelkova
- Department of Immunology, Faculty of Medicine and Dentistry, Palacky University Olomouc, Olomouc, Czech Republic
| | - Milada Stuchlova Horynova
- Department of Immunology, Faculty of Medicine and Dentistry, Palacky University Olomouc, Olomouc, Czech Republic
| | - Petr Kosztyu
- Department of Immunology, Faculty of Medicine and Dentistry, Palacky University Olomouc, Olomouc, Czech Republic
- Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University Olomouc, Olomouc, Czech Republic
| | - Katerina Zachova
- Department of Immunology, Faculty of Medicine and Dentistry, Palacky University Olomouc, Olomouc, Czech Republic
| | - Josef Zadrazil
- Department of Internal Medicine III Nephrology, Rheumatology and Endocrinology, Faculty of Medicine and Dentistry, Palacky University Olomouc and University Hospital Olomouc, Olomouc, Czech Republic
| | - Dana Galuszkova
- Department of Transfusion Medicine, University Hospital Olomouc, Olomouc, Czech Republic
| | - Kazuo Takahashi
- Department of Biomedical Molecular Sciences, School of Medicine, Fujita Health University, Nagoya, Aichi, Japan
| | - Jan Novak
- Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, USA
| | - Milan Raska
- Department of Immunology, Faculty of Medicine and Dentistry, Palacky University Olomouc, Olomouc, Czech Republic
- Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University Olomouc, Olomouc, Czech Republic
- Department of Immunology, University Hospital Olomouc, Olomouc, Czech Republic
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Habas E, Ali E, Farfar K, Errayes M, Alfitori J, Habas E, Ghazouani H, Akbar R, Khan F, Al Dab A, Elzouki AN. IgA nephropathy pathogenesis and therapy: Review & updates. Medicine (Baltimore) 2022; 101:e31219. [PMID: 36482575 PMCID: PMC9726424 DOI: 10.1097/md.0000000000031219] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
BACKGROUND IgA nephropathy (IgAN) is the most frequent type of primary glomerulonephritis since the first type was described more than four decades ago. It is the prevalent cause of primary glomerular disease that causes end-stage renal disease. In most patients with IgAN, hematuria is the most common reported symptom, particularly in those with a preceding upper respiratory tract infection. Although the pathogenesis of IgAN is usually multifactorial, autoimmune complex formation and inflammatory processes are the most widely recognized pathogenic mechanisms. Multiple approaches have been trialed as a therapy for IgAN, including tonsillectomy, steroids, other immune-suppressive therapy in different regimens, and kidney transplantation. AIM AND METHOD PubMed, Google, Google Scholar, Scopus, and EMBASE were searched by the authors using different texts, keywords, and phrases. A non-systemic clinical review is intended to review the available data and clinical updates about the possible mechanism(s) of IgAN pathogenesis and treatments. CONCLUSION IgAN has a heterogeneous pattern worldwide, making it difficult to understand its pathogenesis and treatment. Proteinuria is the best guide to follow up on the IgAN progression and treatment response. Steroids are the cornerstone of IgAN therapy; however, other immune-suppressive and immune-modulative agents are used with a variable response rate. Kidney transplantation is highly advisable for IgAN patients, although the recurrence rate is high. Finally, IgAN management requires collaborative work between patients and their treating physicians for safe long-term outcomes.
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Affiliation(s)
- Elmukhtar Habas
- Hamad General Medicine, Doha, Qatar
- *Correspondence: Elmukhtar Habas, Internal Medicine, Hamad Medical Corporation, AL-Rayyan Road, PO Box 3050, Doha, Qatar (e-mail: )
| | - Elrazi Ali
- Hamad General Hospital, Medicine Department, Doha, Qatar
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Development of an enrichment-free one-pot sample preparation and ultra-high performance liquid chromatography-tandem mass spectrometry method to identify Immunoglobulin A1 hinge region O-glycoforms for Immunoglobulin A nephropathy. J Chromatogr A 2022; 1685:463589. [DOI: 10.1016/j.chroma.2022.463589] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2022] [Revised: 09/24/2022] [Accepted: 10/18/2022] [Indexed: 11/07/2022]
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Ohyama Y, Yamaguchi H, Ogata S, Chiurlia S, Cox SN, Kouri NM, Stangou MJ, Nakajima K, Hayashi H, Inaguma D, Hasegawa M, Yuzawa Y, Tsuboi N, Renfrow MB, Novak J, Papagianni AA, Schena FP, Takahashi K. Racial heterogeneity of IgA1 hinge-region O-glycoforms in patients with IgA nephropathy. iScience 2022; 25:105223. [PMID: 36277451 PMCID: PMC9583103 DOI: 10.1016/j.isci.2022.105223] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2022] [Revised: 08/22/2022] [Accepted: 09/23/2022] [Indexed: 11/23/2022] Open
Abstract
Galactose (Gal)-deficient IgA1 (Gd-IgA1) is involved in IgA nephropathy (IgAN) pathogenesis. To reflect racial differences in clinical characteristics, we assessed disease- and race-specific heterogeneity in the O-glycosylation of the IgA1 hinge region (HR). We determined serum Gd-IgA1 levels in Caucasians (healthy controls [HCs], n = 31; IgAN patients, n = 63) and Asians (HCs, n = 20; IgAN patients, n = 60) and analyzed profiles of serum IgA1 HR O-glycoforms. Elevated serum Gd-IgA1 levels and reduced number of Gal residues per HR were observed in Caucasians. Reduced number of N-acetylgalactosamine (GalNAc) residues per HR and elevated relative abundance of IgA1 with three HR O-glycans were common features in IgAN patients; these features were associated with elevated blood pressure and reduced renal function. We speculate that the mechanisms underlying the reduced GalNAc content in IgA1 HR may be relevant to IgAN pathogenesis.
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Affiliation(s)
- Yukako Ohyama
- Department of Biomedical Molecular Sciences, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan
- Department of Nephrology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan
| | - Hisateru Yamaguchi
- Department of Nursing, Yokkaichi Nursing and Medical Care University, Yokkaichi, Mie 512-8045, Japan
| | - Soshiro Ogata
- Preventive Medicine and Epidemiology, National Cerebral and Cardiovascular Center, Suita, Osaka 564-8565, Japan
| | - Samantha Chiurlia
- University of Bari and Schena Foundation, Valenzano, Bari 70010, Italy
| | - Sharon N. Cox
- University of Bari and Schena Foundation, Valenzano, Bari 70010, Italy
| | - Nikoletta-Maria Kouri
- Department of Nephrology, Aristotle University of Thessaloniki, Thessaloniki, 54642, Greece
| | - Maria J. Stangou
- Department of Nephrology, Aristotle University of Thessaloniki, Thessaloniki, 54642, Greece
| | - Kazuki Nakajima
- Institute for Glyco-core Research, Gifu University, Gifu, Gifu 501-1193, Japan
| | - Hiroki Hayashi
- Department of Nephrology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan
| | - Daijo Inaguma
- Department of Nephrology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan
| | - Midori Hasegawa
- Department of Nephrology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan
| | - Yukio Yuzawa
- Department of Nephrology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan
| | - Naotake Tsuboi
- Department of Nephrology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan
| | - Matthew B. Renfrow
- Departments of Biochemistry and Molecular Genetics and Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA
| | - Jan Novak
- Departments of Biochemistry and Molecular Genetics and Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA
| | | | | | - Kazuo Takahashi
- Department of Biomedical Molecular Sciences, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan
- Department of Nephrology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan
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Ding L, Chen X, Cheng H, Zhang T, Li Z. Advances in IgA glycosylation and its correlation with diseases. Front Chem 2022; 10:974854. [PMID: 36238099 PMCID: PMC9552352 DOI: 10.3389/fchem.2022.974854] [Citation(s) in RCA: 32] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2022] [Accepted: 08/23/2022] [Indexed: 11/29/2022] Open
Abstract
Immunoglobulin A (IgA) is the most abundant immunoglobulin synthesized in the human body. It has the highest concentration in the mucosa and is second only to IgG in serum. IgA plays an important role in mucosal immunity, and is the predominant antibody used to protect the mucosal surface from pathogens invasion and to maintain the homeostasis of intestinal flora. Moreover, The binding IgA to the FcαRI (Fc alpha Receptor I) in soluble or aggregated form can mediate anti- or pro- inflammatory responses, respectively. IgA is also known as one of the most heavily glycosylated antibodies among human immunoglobulins. The glycosylation of IgA has been shown to have a significant effect on its immune function. Variation in the glycoform of IgA is often the main characteration of autoimmune diseases such as IgA nephropathy (IgAN), IgA vasculitis (IgAV), systemic lupus erythematosus (SLE), and rheumatoid arthritis (RA). However, compared with the confirmed glycosylation function of IgG, the pathogenic mechanism of IgA glycosylation involved in related diseases is still unclear. This paper mainly summarizes the recent reports on IgA's glycan structure, its function, its relationship with the occurrence and development of diseases, and the potential application of glycoengineered IgA in clinical antibody therapeutics, in order to provide a potential reference for future research in this field.
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Affiliation(s)
| | | | | | | | - Zheng Li
- Laboratory for Functional Glycomics, College of Life Sciences, Northwest University, Xi’an, China
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12
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Development and Evaluation of a Robust Sandwich Immunoassay System Detecting Serum WFA-Reactive IgA1 for Diagnosis of IgA Nephropathy. Int J Mol Sci 2022; 23:ijms23095165. [PMID: 35563555 PMCID: PMC9104065 DOI: 10.3390/ijms23095165] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2022] [Revised: 04/28/2022] [Accepted: 05/03/2022] [Indexed: 02/04/2023] Open
Abstract
Aberrant glycosylation of IgA1 is involved in the development of IgA nephropathy (IgAN). There are many reports of IgAN markers focusing on the glycoform of IgA1. None have been clinically applied as a routine test. In this study, we established an automated sandwich immunoassay system for detecting aberrant glycosylated IgA1, using Wisteria floribunda agglutinin (WFA) and anti-IgA1 monoclonal antibody. The diagnostic performance as an IgAN marker was evaluated. The usefulness of WFA for immunoassays was investigated by lectin microarray. A reliable standard for quantitative immunoassay measurements was designed by modifying a purified IgA1 substrate. A validation study using multiple serum specimens was performed using the established WFA-antibody sandwich automated immunoassay. Lectin microarray results showed that WFA specifically recognized N-glycans of agglutinated IgA1 in IgAN patients. The constructed IgA1 standard exhibited a wide dynamic range and high reactivity. In the validation study, serum WFA-reactive IgA1 (WFA+-IgA1) differed significantly between healthy control subjects and IgAN patients. The findings indicate that WFA is a suitable lectin that specifically targets abnormal agglutinated IgA1 in serum. We also describe an automated immunoassay system for detecting WFA+-IgA1, focusing on N-glycans.
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13
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Bondt A, Dingess KA, Hoek M, van Rijswijck DMH, Heck AJR. A Direct MS-Based Approach to Profile Human Milk Secretory Immunoglobulin A (IgA1) Reveals Donor-Specific Clonal Repertoires With High Longitudinal Stability. Front Immunol 2021; 12:789748. [PMID: 34938298 PMCID: PMC8685336 DOI: 10.3389/fimmu.2021.789748] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2021] [Accepted: 11/17/2021] [Indexed: 12/29/2022] Open
Abstract
Recently, a mass spectrometry-based approach was introduced to directly assess the IgG1 immunoglobulin clonal repertoires in plasma. Here we expanded upon this approach by describing a mass spectrometry-based technique to assess specifically the clonal repertoire of another important class of immunoglobulin molecules, IgA1, and show it is efficiently and robustly applicable to either milk or plasma samples. Focusing on two individual healthy donors, whose milk was sampled longitudinally during the first 16 weeks of lactation, we demonstrate that the total repertoire of milk sIgA1 is dominated by only 50-500 clones, even though the human body theoretically can generate several orders of magnitude more clones. We show that in each donor the sIgA1 repertoire only changes marginally and quite gradually over the monitored 16-week period of lactation. Furthermore, the observed overlap in clonal repertoires between the two individual donors is close to non-existent. Mothers provide protection to their newborn infants directly by the transfer of antibodies via breastfeeding. The approach introduced here, can be used to visualize the clonal repertoire transferred from mother to infant and to detect changes in-time in that repertoire adapting to changes in maternal physiology.
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Affiliation(s)
- Albert Bondt
- Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, Netherlands.,Netherlands Proteomics Center, Utrecht, Netherlands
| | - Kelly A Dingess
- Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, Netherlands.,Netherlands Proteomics Center, Utrecht, Netherlands
| | - Max Hoek
- Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, Netherlands.,Netherlands Proteomics Center, Utrecht, Netherlands
| | - Danique M H van Rijswijck
- Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, Netherlands.,Netherlands Proteomics Center, Utrecht, Netherlands
| | - Albert J R Heck
- Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, Netherlands.,Netherlands Proteomics Center, Utrecht, Netherlands
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14
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Zhang Z, Zhang Y, Zhang H. IgA Nephropathy: A Chinese Perspective. GLOMERULAR DISEASES 2021; 2:30-41. [PMID: 36751266 PMCID: PMC9677733 DOI: 10.1159/000520039] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/04/2021] [Accepted: 10/01/2021] [Indexed: 11/19/2022]
Abstract
Background IgA nephropathy (IgAN) is the most common primary glomerular disease worldwide and remains a leading cause of chronic kidney disease and end-stage renal disease. The disease prevalence, clinical and pathological phenotypes, the underlying pathogenic molecular mechanisms, and the response to treatments are highly heterogeneous in different ethnic populations, which raise the concern that IgAN may differ across different parts of the world. Summary From a Chinese perspective, we stated the disease burden of IgAN, summarized genome-wide association studies and research into pathological molecules, and compared them with findings based on other populations. The emerging biomarkers, indigenous clinical trials, and major challenges for Chinese researchers and nephrologists in studying IgAN are also discussed. Key Messages In this review, we described a higher risk of major susceptible loci in mucosal immunity, IgA production, and complement activation pathways in Chinese patients with IgAN. With our understanding of the pathogenesis of IgAN, novel biomarkers are emerging. Although there are challenges for conducting high-quality clinical trials in China, it is still feasible to conduct innovative and well-designed studies of IgAN. In the future, international collaborations on research infrastructure would be helpful to advance clinical and basic research in China.
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Affiliation(s)
- Zhao Zhang
- Renal Division, Department of Medicine, Peking University First Hospital, Beijing, China,Institute of Nephrology, Peking University, Beijing, China,Key Laboratory of Renal Disease, Ministry of Health of China, Beijing, China,Key Laboratory of Chronic Kidney Disease Prevention and Treatment, Peking University, Ministry of Education, Beijing, China
| | - Yuemiao Zhang
- Renal Division, Department of Medicine, Peking University First Hospital, Beijing, China,Institute of Nephrology, Peking University, Beijing, China,Key Laboratory of Renal Disease, Ministry of Health of China, Beijing, China,Key Laboratory of Chronic Kidney Disease Prevention and Treatment, Peking University, Ministry of Education, Beijing, China
| | - Hong Zhang
- Renal Division, Department of Medicine, Peking University First Hospital, Beijing, China,Institute of Nephrology, Peking University, Beijing, China,Key Laboratory of Renal Disease, Ministry of Health of China, Beijing, China,Key Laboratory of Chronic Kidney Disease Prevention and Treatment, Peking University, Ministry of Education, Beijing, China,*Hong Zhang,
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15
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Ohyama Y, Renfrow MB, Novak J, Takahashi K. Aberrantly Glycosylated IgA1 in IgA Nephropathy: What We Know and What We Don't Know. J Clin Med 2021; 10:jcm10163467. [PMID: 34441764 PMCID: PMC8396900 DOI: 10.3390/jcm10163467] [Citation(s) in RCA: 30] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2021] [Revised: 07/26/2021] [Accepted: 08/02/2021] [Indexed: 12/17/2022] Open
Abstract
IgA nephropathy (IgAN), the most common primary glomerular disease worldwide, is characterized by glomerular deposition of IgA1-containing immune complexes. The IgA1 hinge region (HR) has up to six clustered O-glycans consisting of Ser/Thr-linked N-acetylgalactosamine usually with β1,3-linked galactose and variable sialylation. Circulating levels of IgA1 with abnormally O-glycosylated HR, termed galactose-deficient IgA1 (Gd-IgA1), are increased in patients with IgAN. Current evidence suggests that IgAN is induced by multiple sequential pathogenic steps, and production of aberrantly glycosylated IgA1 is considered the initial step. Thus, the mechanisms of biosynthesis of aberrantly glycosylated IgA1 and the involvement of aberrant glycoforms of IgA1 in disease development have been studied. Furthermore, Gd-IgA1 represents an attractive biomarker for IgAN, and its clinical significance is still being evaluated. To elucidate the pathogenesis of IgAN, it is important to deconvolute the biosynthetic origins of Gd-IgA1 and characterize the pathogenic IgA1 HR O-glycoform(s), including the glycan structures and their sites of attachment. These efforts will likely lead to development of new biomarkers. Here, we review the IgA1 HR O-glycosylation in general and the role of aberrantly glycosylated IgA1 in the pathogenesis of IgAN in particular.
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Affiliation(s)
- Yukako Ohyama
- Department of Biomedical Molecular Sciences, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan;
| | - Matthew B. Renfrow
- Departments of Biochemistry and Molecular Genetics and Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA; (M.B.R.); (J.N.)
| | - Jan Novak
- Departments of Biochemistry and Molecular Genetics and Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA; (M.B.R.); (J.N.)
| | - Kazuo Takahashi
- Department of Biomedical Molecular Sciences, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan;
- Correspondence: ; Tel.: +81-(562)-93-2430; Fax: +81-(562)-93-1830
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16
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Chiu YL, Lin WC, Shu KH, Fang YW, Chang FC, Chou YH, Wu CF, Chiang WC, Lin SL, Chen YM, Wu MS. Alternative Complement Pathway Is Activated and Associated with Galactose-Deficient IgA 1 Antibody in IgA Nephropathy Patients. Front Immunol 2021; 12:638309. [PMID: 34177889 PMCID: PMC8223746 DOI: 10.3389/fimmu.2021.638309] [Citation(s) in RCA: 26] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2020] [Accepted: 05/20/2021] [Indexed: 12/30/2022] Open
Abstract
Background Galactose-deficient IgA1 (Gd-IgA1) and alternative complement pathway activation are considered to be involved in the pathogenesis of IgA nephropathy (IgAN). Nevertheless, the relationships between alternative pathway activation and disease activity or Gd-IgA1 level remains unclear. Methods Ninety-eight biopsy-diagnosed IgAN, twenty-five primary focal segmental sclerosis (FSGS) patients and forty-two healthy individuals were recruited in this study. Among them, fifty IgAN patients received immunosuppression. Follow-up blood samples at 1 and 3~6 months after immunosuppression were collected. Plasma levels of complement C5a, factor Ba and Gd-IgA1 were measured and analyzed. Immunostaining for complement was performed in twenty-five IgAN and FSGS patients. Results At baseline, IgAN patients had higher levels of plasma C5a, factor Ba and Gd-IgA1 than control subjects. Gd-IgA1 levels positively correlated with plasma C5a and factor Ba. In addition, levels of factor Ba and Gd-IgA1 were positively associated with proteinuria and negatively associated with renal function. Immunostaining revealed positive staining for factor Bb and C3c in glomeruli in IgAN patients, but not in FSGS patients. At baseline, patients receiving immunosuppression had more severe proteinuria and higher factor Ba. After 6 months, eGFR declined and proteinuria persisted in patients without immunosuppression. In contrast, patients who received immunosuppression exhibited decreased plasma levels of C5a, factor Ba, and Gd-IgA1 as early as 1 month after treatment. Proteinuria decreased and renal function also remained stable 6 months after immunosuppression. Conclusions Our results indicate a close relationship between alternative complement pathway activation, Gd-IgA1 concentration and clinical severity of IgAN. Level of complement factor B may be a potential marker for disease activity and therapeutic target in IgAN patients.
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Affiliation(s)
- Yen-Ling Chiu
- Graduate Program in Biomedical Informatics and Graduate Institute of Medicine, Yuan Ze University, Taoyuan, Taiwan.,Department of Medical Research, Far Eastern Memorial Hospital, New Taipei City, Taiwan.,Graduate Institute of Clinical Medicine, National Taiwan University, Taipei, Taiwan
| | - Wei-Chou Lin
- Department of Pathology, National Taiwan University Hospital, Taipei, Taiwan
| | - Kai-Hsiang Shu
- Graduate Program in Biomedical Informatics and Graduate Institute of Medicine, Yuan Ze University, Taoyuan, Taiwan
| | - Yi-Wen Fang
- Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan
| | - Fan-Chi Chang
- Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan
| | - Yu-Hsiang Chou
- Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan
| | - Ching-Fang Wu
- Department of Internal Medicine, E-Da Hospital, Kaohsiung, Taiwan
| | - Wen-Chih Chiang
- Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan
| | - Shuei-Liong Lin
- Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan.,Graduate Institute of Physiology, College of Medicine, National Taiwan University, Taipei, Taiwan
| | - Yung-Ming Chen
- Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan
| | - Ming-Shiou Wu
- Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan
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17
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Stewart TJ, Takahashi K, Xu N, Prakash A, Brown R, Raska M, Renfrow MB, Novak J. Quantitative assessment of successive carbohydrate additions to the clustered O-glycosylation sites of IgA1 by glycosyltransferases. Glycobiology 2021; 31:540-556. [PMID: 33295603 PMCID: PMC8176776 DOI: 10.1093/glycob/cwaa111] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2020] [Revised: 12/01/2020] [Accepted: 12/02/2020] [Indexed: 11/12/2022] Open
Abstract
Mucin-type O-glycosylation occurs on many proteins that transit the Golgi apparatus. These glycans impact structure and function of many proteins and have important roles in cellular biosynthetic processes, signaling and differentiation. Although recent technological advances have enhanced our ability to profile glycosylation of glycoproteins, limitations in the understanding of the biosynthesis of these glycan structures remain. Some of these limitations stem from the difficulty to track the biosynthetic process of mucin-type O-glycosylation, especially when glycans occur in dense clusters in repeat regions of proteins, such as the mucins or immunoglobulin A1 (IgA1). Here, we describe a series of nano-liquid chromatography (LC)-mass spectrometry (MS) analyses that demonstrate the range of glycosyltransferase enzymatic activities involved in the biosynthesis of clustered O-glycans on IgA1. By utilizing nano-LC-MS relative quantitation of in vitro reaction products, our results provide unique insights into the biosynthesis of clustered IgA1 O-glycans. We have developed a workflow to determine glycoform-specific apparent rates of a human UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltrasnfersase (GalNAc-T EC 2.4.1.41) and demonstrated how pre-existing glycans affect subsequent activity of glycosyltransferases, such as core 1 galactosyltransferase and α2,3- and α2,6-specific sialyltransferases, in successive additions in the biosynthesis of clustered O-glycans. In the context of IgA1, these results have potential to provide insight into the molecular mechanisms implicated in the pathogenesis of IgA nephropathy, an autoimmune renal disease involving aberrant IgA1 O-glycosylation. In a broader sense, these methods and workflows are applicable to the studies of the concerted and competing functions of other glycosyltransferases that initiate and extend mucin-type core 1 clustered O-glycosylation.
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Affiliation(s)
- Tyler J Stewart
- Department of Microbiology, University of Alabama at Birmingham, 845 19th Street South, BBRB 761A, Birmingham, AL 35294, USA
- Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, 720 20th Street South, KAUL 524, Birmingham, AL 35294, USA
| | - Kazuo Takahashi
- Department of Biomedical Molecular Sciences, Fujita Health University School of Medicine, 1-98 Dengakugakubo, Kutsukake, Aichi, Toyoake 470-1192, Japan
- Department of Microbiology, University of Alabama at Birmingham, 845 19th Street South, BBRB 761A, Birmingham, AL 35294, USA
| | - Nuo Xu
- Department of Management, Information Systems & Quantitative Methods, 710 13th Street South, University of Alabama at Birmingham, Birmingham, AL 35233, USA
| | - Amol Prakash
- Optys Tech Corporation, Shrewsbury, MA 01545, USA
| | - Rhubell Brown
- Department of Microbiology, University of Alabama at Birmingham, 845 19th Street South, BBRB 761A, Birmingham, AL 35294, USA
| | - Milan Raska
- Department of Immunology, Palacky University and University Hospital, Hnevotinska 3, Olomouc 775 15, Czech Republic
| | - Matthew B Renfrow
- Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, 720 20th Street South, KAUL 524, Birmingham, AL 35294, USA
| | - Jan Novak
- Department of Microbiology, University of Alabama at Birmingham, 845 19th Street South, BBRB 761A, Birmingham, AL 35294, USA
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18
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Hansen AL, Reily C, Novak J, Renfrow MB. Immunoglobulin A Glycosylation and Its Role in Disease. EXPERIENTIA SUPPLEMENTUM (2012) 2021; 112:433-477. [PMID: 34687019 DOI: 10.1007/978-3-030-76912-3_14] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/13/2023]
Abstract
Human IgA is comprised of two subclasses, IgA1 and IgA2. Monomeric IgA (mIgA), polymeric IgA (pIgA), and secretory IgA (SIgA) are the main molecular forms of IgA. The production of IgA rivals all other immunoglobulin isotypes. The large quantities of IgA reflect the fundamental roles it plays in immune defense, protecting vulnerable mucosal surfaces against invading pathogens. SIgA dominates mucosal surfaces, whereas IgA in circulation is predominately monomeric. All forms of IgA are glycosylated, and the glycans significantly influence its various roles, including antigen binding and the antibody effector functions, mediated by the Fab and Fc portions, respectively. In contrast to its protective role, the aberrant glycosylation of IgA1 has been implicated in the pathogenesis of autoimmune diseases, such as IgA nephropathy (IgAN) and IgA vasculitis with nephritis (IgAVN). Furthermore, detailed characterization of IgA glycosylation, including its diverse range of heterogeneity, is of emerging interest. We provide an overview of the glycosylation observed for each subclass and molecular form of IgA as well as the range of heterogeneity for each site of glycosylation. In many ways, the role of IgA glycosylation is in its early stages of being elucidated. This chapter provides an overview of the current knowledge and research directions.
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Affiliation(s)
- Alyssa L Hansen
- Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL, USA
| | - Colin Reily
- Departments of Medicine and Microbiology, University of Alabama at Birmingham, Birmingham, AL, USA
| | - Jan Novak
- Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL, USA.
| | - Matthew B Renfrow
- Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL, USA.
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19
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Ohyama Y, Nakajima K, Renfrow MB, Novak J, Takahashi K. Mass spectrometry for the identification and analysis of highly complex glycosylation of therapeutic or pathogenic proteins. Expert Rev Proteomics 2020; 17:275-296. [PMID: 32406805 DOI: 10.1080/14789450.2020.1769479] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
INTRODUCTION Protein glycosylation influences characteristics such as folding, stability, protein interactions, and solubility. Therefore, glycan moieties of therapeutic proteins and proteins that are likely associated with disease pathogenesis should be analyzed in-depth, including glycan heterogeneity and modification sites. Recent advances in analytical methods and instrumentation have enabled comprehensive characterization of highly complex glycosylated proteins. AREA COVERED The following aspects should be considered when analyzing glycosylated proteins: sample preparation, chromatographic separation, mass spectrometry (MS) and fragmentation methods, and bioinformatics, such as software solutions for data analyses. Notably, analysis of glycoproteins with heavily sialylated glycans or multiple glycosylation sites requires special considerations. Here, we discuss recent methodological advances in MS that provide detailed characterization of heterogeneous glycoproteins. EXPERT OPINION As characterization of complex glycosylated proteins is still analytically challenging, the function or pathophysiological significance of these proteins is not fully understood. To reproducibly produce desired forms of therapeutic glycoproteins or to fully elucidate disease-specific patterns of protein glycosylation, a highly reproducible and robust analytical platform(s) should be established. In addition to advances in MS instrumentation, optimization of analytical and bioinformatics methods and utilization of glycoprotein/glycopeptide standards is desirable. Ultimately, we envision that an automated high-throughput MS analysis will provide additional power to clinical studies and precision medicine.
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Affiliation(s)
- Yukako Ohyama
- Department of Nephrology, Fujita Health University School of Medicine , Toyoake, Japan.,Department of Biomedical Molecular Sciences, Fujita Health University School of Medicine , Toyoake, Japan
| | - Kazuki Nakajima
- Center for Research Promotion and Support, Fujita Health University , Toyoake, Japan
| | - Matthew B Renfrow
- Departments of Biochemistry and Molecular Genetics and Microbiology, University of Alabama at Birmingham , Birmingham, AL, USA
| | - Jan Novak
- Departments of Biochemistry and Molecular Genetics and Microbiology, University of Alabama at Birmingham , Birmingham, AL, USA
| | - Kazuo Takahashi
- Department of Nephrology, Fujita Health University School of Medicine , Toyoake, Japan.,Department of Biomedical Molecular Sciences, Fujita Health University School of Medicine , Toyoake, Japan.,Departments of Biochemistry and Molecular Genetics and Microbiology, University of Alabama at Birmingham , Birmingham, AL, USA
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20
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Analysis of O-glycoforms of the IgA1 hinge region by sequential deglycosylation. Sci Rep 2020; 10:671. [PMID: 31959827 PMCID: PMC6971281 DOI: 10.1038/s41598-020-57510-z] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2019] [Accepted: 12/30/2019] [Indexed: 12/21/2022] Open
Abstract
A common renal disease, immunoglobulin A (IgA) nephropathy (IgAN), is associated with glomerular deposition of IgA1-containing immune complexes. IgA1 hinge region (HR) has up to six clustered O-glycans consisting of Ser/Thr-linked N-acetylgalactosamine with β1,3-linked galactose and variable sialylation. IgA1 glycoforms with some galactose-deficient (Gd) HR O-glycans play a key role in IgAN pathogenesis. The clustered and variable O-glycans make the IgA1 glycomic analysis challenging and better approaches are needed. Here, we report a comprehensive analytical workflow for IgA1 HR O-glycoform analysis. We combined an automated quantitative analysis of the HR O-glycopeptide profiles with sequential deglycosylation to remove all but Gd O-glycans from the HR. The workflow was tested using serum IgA1 from healthy subjects. Twelve variants of glycopeptides corresponding to the HR with three to six O-glycans were detected; nine glycopeptides carried up to three Gd O-glycans. Sites with Gd O-glycans were unambiguously identified by electron-transfer/higher-energy collision dissociation tandem mass spectrometry. Extracted ion chromatograms of isomeric glycoforms enabled quantitative assignment of Gd sites. The most frequent Gd site was T236, followed by S230, T233, T228, and S232. The new workflow for quantitative profiling of IgA1 HR O-glycoforms with site-specific resolution will enable identification of pathogenic IgA1 HR O-glycoforms in IgAN.
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21
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Stewart TJ, Takahashi K, Whitaker RH, Raska M, Placzek WJ, Novak J, Renfrow MB. IgA1 hinge-region clustered glycan fidelity is established early during semi-ordered glycosylation by GalNAc-T2. Glycobiology 2019; 29:543-556. [PMID: 30759204 PMCID: PMC6583770 DOI: 10.1093/glycob/cwz007] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2018] [Revised: 01/13/2019] [Accepted: 02/04/2019] [Indexed: 01/03/2023] Open
Abstract
GalNAc-type O-glycans are often added to proteins post-translationally in a clustered manner in repeat regions of proteins, such as mucins and IgA1. Observed IgA1 glycosylation patterns show that glycans occur at similar sites with similar structures. It is not clear how the sites and number of glycans added to IgA1, or other proteins, can follow a conservative process. GalNAc-transferases initiate GalNAc-type glycosylation. In IgA nephropathy, an autoimmune disease, the sites and O-glycan structures of IgA1 hinge-region are altered, giving rise to a glycan autoantigen. To better understand how GalNAc-transferases determine sites and densities of clustered O-glycans, we used IgA1 hinge-region (HR) segment as a probe. Using LC-MS, we demonstrated a semi-ordered process of glycosylation by GalNAc-T2 towards the IgA1 HR. The catalytic domain was responsible for selection of four initial sites based on amino-acid sequence recognition. Both catalytic and lectin domains were involved in multiple second site-selections, each dependent on initial site-selection. Our data demonstrated that multiple start-sites and follow-up pathways were key to increasing the number of glycans added. The lectin domain predominately enhanced IgA1 HR glycan density by increasing synthesis pathway exploration by GalNAc-T2. Our data indicated a link between site-specific glycan addition and clustered glycan density that defines a mechanism of how conserved clustered O-glycosylation patterns and glycoform populations of IgA1 can be controlled by GalNAc-T2. Together, these findings characterized a correlation between glycosylation pathway diversity and glycosylation density, revealing mechanisms by which a single GalNAc-T isozyme can limit and define glycan heterogeneity in a disease-relevant context.
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Affiliation(s)
- Tyler J Stewart
- Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL, USA
- Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL, USA
| | - Kazuo Takahashi
- Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL, USA
- Department of Nephrology, Fujita Health University, Toyoake, Japan
| | - Robert H Whitaker
- Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL, USA
| | - Milan Raska
- Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL, USA
- Department of Immunology, Palacky University and University Hospital, Olomouc, Czech Republic
| | - William J Placzek
- Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL, USA
| | - Jan Novak
- Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL, USA
| | - Matthew B Renfrow
- Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL, USA
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22
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Lomax-Browne HJ, Robertson C, Antonopoulos A, Leathem AJC, Haslam SM, Dell A, Dwek MV. Serum IgA1 shows increased levels of α2,6-linked sialic acid in breast cancer. Interface Focus 2019; 9:20180079. [PMID: 30842877 DOI: 10.1098/rsfs.2018.0079] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 01/03/2019] [Indexed: 12/12/2022] Open
Abstract
The lectin Helix pomatia agglutinin (HPA) recognizes altered glycosylation in solid cancers and the identification of HPA binding partners in tumour tissue and serum is an important aim. Among the many HPA binding proteins, IgA1 has been reported to be the most abundant in liver metastases. In this study, the glycosylation of IgA1 was evaluated using serum samples from patients with breast cancer (BCa) and the utility of IgA1 glycosylation as a biomarker was assessed. Detailed mass spectrometric structural analysis showed an increase in disialo-biantennary N-linked glycans on IgA1 from BCa patients (p < 0.0001: non-core fucosylated; p = 0.0345: core fucosylated) and increased asialo-Thomsen-Friedenreich antigen (TF) and disialo-TF antigens in the O-linked glycan preparations from IgA1 of cancer patients compared with healthy control individuals. An increase in Sambucus nigra binding was observed, suggestive of increased α2,6-linked sialic acid on IgA1 in BCa. Logistic regression analysis showed HPA binding to IgA1 and tumour size to be significant independent predictors of distant metastases (χ 2 13.359; n = 114; p = 0.020) with positive and negative predictive values of 65.7% and 64.6%, respectively. Immunohistochemical analysis of tumour tissue samples showed IgA1 to be detectable in BCa tissue. This report provides a detailed analysis of serum IgA1 glycosylation in BCa and illustrates the potential utility of IgA1 glycosylation as a biomarker for BCa prognostication.
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Affiliation(s)
- Hannah J Lomax-Browne
- School of Life Sciences, University of Westminster, 115 New Cavendish Street, London W1W 6UW, UK
| | - Claire Robertson
- School of Life Sciences, University of Westminster, 115 New Cavendish Street, London W1W 6UW, UK
| | - Aristotelis Antonopoulos
- Department of Life Sciences, Imperial College London, South Kensington Campus, London SW7 2AZ, UK
| | - Anthony J C Leathem
- School of Life Sciences, University of Westminster, 115 New Cavendish Street, London W1W 6UW, UK
| | - Stuart M Haslam
- Department of Life Sciences, Imperial College London, South Kensington Campus, London SW7 2AZ, UK
| | - Anne Dell
- Department of Life Sciences, Imperial College London, South Kensington Campus, London SW7 2AZ, UK
| | - Miriam V Dwek
- School of Life Sciences, University of Westminster, 115 New Cavendish Street, London W1W 6UW, UK
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Nagy G, Veličković D, Chu RK, Carrell AA, Weston DJ, Ibrahim YM, Anderton CR, Smith RD. Towards resolving the spatial metabolome with unambiguous molecular annotations in complex biological systems by coupling mass spectrometry imaging with structures for lossless ion manipulations. Chem Commun (Camb) 2019; 55:306-309. [PMID: 30534702 PMCID: PMC6537888 DOI: 10.1039/c8cc07482h] [Citation(s) in RCA: 26] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
We demonstrate the coupling of liquid extraction surface analysis (LESA) to structures for lossless ion manipulations in conjunction with serpentine ultralong path with extending routing (SLIM SUPER) ion mobility-mass spectrometry (IM-MS) for the unambiguous annotation of important isomeric glycoforms in carbon-fixing communities.
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Affiliation(s)
- Gabe Nagy
- Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
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24
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Yamasaki K, Suzuki H, Yasutake J, Yamazaki Y, Suzuki Y. Galactose-Deficient IgA1-Specific Antibody Recognizes GalNAc-Modified Unique Epitope on Hinge Region of IgA1. Monoclon Antib Immunodiagn Immunother 2018; 37:252-256. [PMID: 30570353 PMCID: PMC6338562 DOI: 10.1089/mab.2018.0041] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023] Open
Abstract
Galactose-deficient IgA1 (Gd-IgA1) that exposes GalNAc or sialylated GalNAc has been shown to be associated with disease activity of IgA nephropathy (IgAN). In a previous report, we established an enzyme-linked immunosorbent assay that measures human Gd-IgA1 using a specific monoclonal antibody KM55 (KM55 mAb), and showed that patients with IgAN contain a higher level of serum Gd-IgA1 than other types of renal diseases. Recently, we also found that the KM55 mAb specifically recognized the glomerular-deposited Gd-IgA1 in renal biopsy. In this study, we aimed to analyze the epitope of KM55 mAb using synthesized peptides corresponding to the hinge region of IgA1 with GalNAc moiety on putative glycosylated Ser/Thr residues, which are Thr225, Thr228, Ser230, Ser232, and Thr236. Binding analysis to single GalNAc-modified hinge region peptide of IgA1 showed that Thr225 with GalNAc is required for recognition of KM55. PST(GalNAC)PP motif was required for KM55 mAb to recognize hinge region peptide of IgA1 which is shown by binding assay with deletion peptide. This result was confirmed by binding of KM55 mAb against peptide with GalNAc at Thr233, which resulted in containing another PST(GalNAC)PP motif. Taken together, we concluded that the epitope of Gd-IgA1-specific KM55 mAb is PST(GalNAc)PP motif.
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Affiliation(s)
- Kohei Yamasaki
- 1 Department of Nephrology, Juntendo University Faculty of Medicine, Tokyo, Japan.,2 Nephrology R&D Unit, Kyowa Hakko Kirin Co., Ltd., Tokyo, Japan
| | - Hitoshi Suzuki
- 1 Department of Nephrology, Juntendo University Faculty of Medicine, Tokyo, Japan
| | - Junichi Yasutake
- 2 Nephrology R&D Unit, Kyowa Hakko Kirin Co., Ltd., Tokyo, Japan
| | - Yuji Yamazaki
- 2 Nephrology R&D Unit, Kyowa Hakko Kirin Co., Ltd., Tokyo, Japan
| | - Yusuke Suzuki
- 1 Department of Nephrology, Juntendo University Faculty of Medicine, Tokyo, Japan
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25
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Abstract
IgA nephropathy, the most common primary glomerulonephritis in the world and a frequent cause of end-stage renal disease, is characterized by typical mesangial deposits of IgA1, as described by Berger and Hinglaise in 1968. Since then, it has been discovered that aberrant IgA1 O-glycosylation is involved in disease pathogenesis. Progress in glycomic, genomic, clinical, analytical, and biochemical studies has shown autoimmune features of IgA nephropathy. The autoimmune character of the disease is explained by a multihit pathogenesis model, wherein overproduction of aberrantly glycosylated IgA1, galactose-deficient in some O-glycans, by IgA1-secreting cells leads to increased levels of circulatory galactose-deficient IgA1. These glycoforms induce production of autoantibodies that subsequently bind hinge-region of galactose-deficient IgA1 molecules, resulting in the formation of nephritogenic immune complexes. Some of these complexes deposit in the kidney, activate mesangial cells, and incite glomerular injury. Thus, galactose-deficient IgA1 is central to the disease process. In this article, we review studies concerning IgA1 O-glycosylation that have contributed to the current understanding of the role of IgA1 in the pathogenesis of IgA nephropathy.
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Affiliation(s)
- Jan Novak
- Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL..
| | - Jonathan Barratt
- Department of Infection, Immunity and Inflammation, University of Leicester, Leicester, UK
| | - Bruce A Julian
- Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL.; Department of Medicine, University of Alabama at Birmingham, Birmingham, AL
| | - Matthew B Renfrow
- Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL
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26
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Reily C, Rizk DV, Julian BA, Novak J. Assay for galactose-deficient IgA1 enables mechanistic studies with primary cells from IgA nephropathy patients. Biotechniques 2018; 65:71-77. [PMID: 30091383 PMCID: PMC6152805 DOI: 10.2144/btn-2018-0042] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2018] [Accepted: 06/19/2018] [Indexed: 11/23/2022] Open
Abstract
AIMS IgA nephropathy, the most common primary glomerulonephritis worldwide, is characterized by glomerular deposition of galactose-deficient IgA1 and elevated serum levels of this IgA1 glycoform. Current ELISA methods lack sensitivity to assess galactose deficiency using small amounts of IgA1, which limits studies in primary cells due to modest IgA1 production in isolated peripheral-blood lymphocytes. METHODS Lectin from Helix pomatia was conjugated to biotin or acridinium ester and used in ELISA to detect galactose deficiency of IgA1 using small amounts of IgA1. RESULTS Lectin conjugated to acridinium had an approximately a log-fold increased sensitivity compared with biotin-labeled lectin. CONCLUSIONS The new method of using lectin from Helix pomatia conjugated to acridinium increased assay sensitivity, allowing future mechanistic studies with cultured primary cells.
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Affiliation(s)
- Colin Reily
- University of Alabama at Birmingham, Department of Medicine, Birmingham, AL, USA
| | - Dana V Rizk
- University of Alabama at Birmingham, Department of Medicine, Birmingham, AL, USA
| | - Bruce A Julian
- University of Alabama at Birmingham, Department of Medicine, Birmingham, AL, USA
- University of Alabama at Birmingham, Department of Microbiology, Birmingham, AL, USA
| | - Jan Novak
- University of Alabama at Birmingham, Department of Microbiology, Birmingham, AL, USA
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27
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Darula Z, Medzihradszky KF. Analysis of Mammalian O-Glycopeptides-We Have Made a Good Start, but There is a Long Way to Go. Mol Cell Proteomics 2018; 17:2-17. [PMID: 29162637 PMCID: PMC5750848 DOI: 10.1074/mcp.mr117.000126] [Citation(s) in RCA: 69] [Impact Index Per Article: 9.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2017] [Indexed: 12/18/2022] Open
Abstract
Glycosylation is perhaps the most common post-translational modification. Recently there has been growing interest in cataloging the glycan structures, glycoproteins, and specific sites modified and deciphering the biological functions of glycosylation. Although the results are piling up for N-glycosylation, O-glycosylation is seriously trailing behind. In our review we reiterate the difficulties researchers have to overcome in order to characterize O-glycosylation. We describe how an ingenious cell engineering method delivered exciting results, and what could we gain from "wild-type" samples. Although we refer to the biological role(s) of O-glycosylation, we do not provide a complete inventory on this topic.
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Affiliation(s)
- Zsuzsanna Darula
- From the ‡Laboratory of Proteomics Research, Biological Research Centre, Hungarian Academy of Sciences, H-6726, 62 Temesvari krt, Szeged, Hungary
| | - Katalin F Medzihradszky
- From the ‡Laboratory of Proteomics Research, Biological Research Centre, Hungarian Academy of Sciences, H-6726, 62 Temesvari krt, Szeged, Hungary;
- §Department of Pharmaceutical Chemistry, School of Pharmacy, University of California San Francisco, Genentech Hall, N472A, MC 2240, 600 16th Street, San Francisco, California 94158-2517
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28
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Zhang Y, Xie X, Zhao X, Tian F, Lv J, Ying W, Qian X. Systems analysis of singly and multiply O-glycosylated peptides in the human serum glycoproteome via EThcD and HCD mass spectrometry. J Proteomics 2017; 170:14-27. [PMID: 28970103 DOI: 10.1016/j.jprot.2017.09.014] [Citation(s) in RCA: 63] [Impact Index Per Article: 7.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2017] [Revised: 09/26/2017] [Accepted: 09/28/2017] [Indexed: 01/20/2023]
Abstract
Human serum has been intensively studied to identify biomarkers via global proteomic analysis. The altered O-glycoproteome is associated with human pathological state including cancer, inflammatory and degenerative diseases and is an attractive source of disease biomarkers. Because of the microheterogeneity and macroheterogeneity of O-glycosylation, site-specific O-glycosylation analysis in human serum is still challenging. Here, we developed a systematic strategy that combined multiple enzyme digestion, multidimensional separation for sample preparation and high-resolution tandem MS with Byonic software for intact O-glycopeptide characterization. We demonstrated that multiple enzyme digestion or multidimensional separation can make sample preparation more efficient and that EThcD is not only suitable for the identification of singly O-glycosylated peptides (50.3%) but also doubly (21.2%) and triply (28.5%) O-glycosylated peptides. Totally, with the strict scoring criteria, 499 non-redundant intact O-glycopeptides, 173 O-glycosylation sites and 6 types of O-glycans originating from 49 O-glycoprotein groups were identified in human serum, including 121 novel O-glycosylation sites. Currently, this is the largest data set of site-specific native O-glycoproteome from human serum samples. We expect that the strategies developed by this study will facilitate in-depth analyses of native O-glycoproteomes in human serum and provide opportunities to understand the functional roles of protein O-glycosylation in human health and diseases. BIOLOGICAL SIGNIFICANCE The altered O-glycoproteome is associated with human pathological state and is an attractive source of disease biomarkers. However, site-specific O-glycosylation analysis is challenging because of the microheterogeneity (different glycoforms attached to one glycosylation site) and macroheterogeneity (site occupancy) of O-glycosylation. In this work, we developed a systematic strategy for intact O-glycopeptide characterization. This study took advantage of the inherent properties of the new fragmentation method called EThcD, which provides more complete fragmentation information about O-glycosylated peptides and a more confident site localization of O-glycans than collision-induced dissociation (HCD). We demonstrated that multiple enzyme digestion or multidimensional separation can make sample preparation more efficient and that EThcD was not only suitable for the identification of singly O-glycosylated peptides (50.3%) but also doubly (21.2%) and triply (28.5%) O-glycosylated peptides. Finally, we got a largest data set of site-specific native O-glycoproteome from human serum samples. Furthermore, quantitative analysis of intact O-glycopeptides from the serum samples of IgA nephropathy (IgAN) patients and healthy donors was performed, and the results showed the potential of the strategy to discover O-glycosylation biomarkers. We expect that the strategies developed by this study will facilitate in-depth analyses of native O-glycoproteomes in human serum and lead to exciting opportunities to understand the functional roles of protein O-glycosylation in human health and diseases.
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Affiliation(s)
- Yong Zhang
- State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Radiation Medicine, Beijing 102206, China
| | - Xinfang Xie
- Renal Division, Peking University First Hospital, Peking University Institute of Nephrology, Key Laboratory of Renal Disease, Ministry of Health of China, Beijing, China; Key Laboratory of Chronic Kidney Disease Prevention and Treatment, Peking University, Ministry of Education, Beijing, China
| | - Xinyuan Zhao
- State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Radiation Medicine, Beijing 102206, China
| | - Fang Tian
- State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Radiation Medicine, Beijing 102206, China
| | - Jicheng Lv
- Renal Division, Peking University First Hospital, Peking University Institute of Nephrology, Key Laboratory of Renal Disease, Ministry of Health of China, Beijing, China; Key Laboratory of Chronic Kidney Disease Prevention and Treatment, Peking University, Ministry of Education, Beijing, China
| | - Wantao Ying
- State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Radiation Medicine, Beijing 102206, China.
| | - Xiaohong Qian
- State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Radiation Medicine, Beijing 102206, China.
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IgA N- and O-glycosylation profiling reveals no association with the pregnancy-related improvement in rheumatoid arthritis. Arthritis Res Ther 2017; 19:160. [PMID: 28679431 PMCID: PMC5498977 DOI: 10.1186/s13075-017-1367-0] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2017] [Accepted: 06/09/2017] [Indexed: 02/01/2023] Open
Abstract
Background The Fc glycosylation of immunoglobulin G (IgG) is well known to associate with rheumatoid arthritis (RA) disease activity. The same may be true for other classes of Igs. In the present study, we sought to determine whether the glycosylation of IgA was different between healthy subjects and patients with RA, as well as whether it was associated with RA disease activity, in particular with the pregnancy-associated improvement thereof or the flare after delivery. Methods A recently developed high-throughput method for glycoprofiling of IgA1 was applied to affinity-captured IgA from sera of patients with RA (n = 252) and healthy control subjects (n = 32) collected before, during and after pregnancy. Results IgA1 O-glycans bore more sialic acids in patients with RA than in control subjects. In addition, levels of bisecting N-acetylglucosamine of the N-glycans at asparagine 144 were higher in the patients with RA. The levels of several N-glycosylation traits were shown to change with pregnancy, similar to what has been shown before for IgG. However, the changes in IgA glycosylation were not associated with improvement or a flare of disease activity. Conclusions The glycosylation of IgA differs between patients with RA and healthy control subjects. However, our data suggest only a minor, if any, association of IgA glycosylation with RA disease activity. Electronic supplementary material The online version of this article (doi:10.1186/s13075-017-1367-0) contains supplementary material, which is available to authorized users.
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30
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Renfrow MB, Novak J. What insights can proteomics give us into IgA nephropathy (Berger's disease)? Expert Rev Proteomics 2017; 14:645-647. [PMID: 28535694 DOI: 10.1080/14789450.2017.1331738] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/19/2022]
Affiliation(s)
- Matthew B Renfrow
- a Department of Biochemistry and Molecular Genetics , University of Alabama at Birmingham , Birmingham , AL , USA
| | - Jan Novak
- b Department of Microbiology , University of Alabama at Birmingham , Birmingham , AL , USA
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31
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Lehoux S, Ju T. Separation of Two Distinct O-Glycoforms of Human IgA1 by Serial Lectin Chromatography Followed by Mass Spectrometry O-Glycan Analysis. Methods Enzymol 2016; 585:61-75. [PMID: 28109443 DOI: 10.1016/bs.mie.2016.10.003] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/03/2022]
Abstract
Human immunoglobulin A1 (IgA1), which carries four to six mucin-type O-glycans (O-glycans) on its hinge region (HR), is the most abundant O-glycoprotein in plasma or serum. While normal O-glycans from hematopoietic-originated cells are core 1-based complex structures, many reports showed that the IgA1 from patients with IgA nephropathy (IgAN) carries undergalactosylated or truncated O-glycans such as the Tn antigen and its sialylated version the SialylTn (STn) antigen on the HR. Yet, there is still a debate whether Tn/STn on the HR of IgA1 is specific to the IgA1 from patients with IgAN since these antigens have also been seen in serum IgA1 of healthy individuals. An additional question is whether the O-glycans at all sites on the two HRs of one IgA1 molecule are homogeneous (either all normal or all Tn/STn) or heterogeneous (both normal and Tn/STn O-glycans). To address these questions, we conducted a systematic study on the O-glycans of plasma IgA1 from both IgAN patients and healthy controls using serial HPA and PNA lectin chromatography followed by western blotting and further analysis of O-glycans from HPA-bound and PNA-bound IgA1 fractions by mass spectrometry. Unexpectedly, we found that a variable minor fraction of IgA1 from both IgAN patients and healthy controls had Tn/STn antigens, and that the O-glycoprotein IgA1 molecules from most samples had only two distinct O-glycoforms: one major glycoform with homogeneous normal core 1-based O-glycans and one minor glycoform with homogeneous Tn/STn antigens. These results raised a serious question about the role of Tn/STn antigens on IgA1 in pathogenesis of IgAN, and there is a demand for a practical methodology that any laboratory can utilize to analyze the O-glycans of IgA1. Herein, we describe the methodology we developed in more detail. The method could also be applied to the analysis of any other O-glycosylated proteins.
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Affiliation(s)
- S Lehoux
- Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, United States.
| | - T Ju
- Emory University School of Medicine, Atlanta, GA, United States.
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32
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Fabiano RCG, Pinheiro SVB, Simões E Silva AC. Immunoglobulin A nephropathy: a pathophysiology view. Inflamm Res 2016; 65:757-770. [PMID: 27351940 DOI: 10.1007/s00011-016-0962-x] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2015] [Revised: 03/07/2016] [Accepted: 06/13/2016] [Indexed: 01/10/2023] Open
Abstract
BACKGROUND AND AIM IgA nephropathy is one of the leading causes of primary glomerulonephritis worldwide and an important etiology of renal disease in young adults. IgA nephropathy is considered an immune complex-mediated disease. METHODS This review article summarizes recent evidence on the pathophysiology of IgA nephropathy. RESULTS Current studies indicate an ordered sequence of multi-hits as fundamental to disease occurrence. Altered glycan structures in the hinge region of the heavy chains of IgA1 molecules act as auto-antigens, potentially triggering the production of glycan-specific autoantibodies. Recognition of novel epitopes by IgA and IgG antibodies leads to the formation of immune complexes galactose deficient-IgA1/anti-glycan IgG or IgA. Immune complexes of IgA combined with FcαRI/CD89 have also been implicated in disease exacerbation. These nephritogenic immune complexes are formed in the circulation and deposited in renal mesangium. Deposited immune complexes ultimately induce glomerular injury, through the release of pro-inflammatory cytokines, secretion of chemokines and the resultant migration of macrophages into the kidney. The TfR1/CD71 receptor has a pivotal role in mesangial cells. New signaling intracellular mechanisms have also been described. CONCLUSION The knowledge of the whole pathophysiology of this disease could provide the rational bases for developing novel approaches for diagnosis, for monitoring disease activity, and for disease-specific treatment.
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Affiliation(s)
| | - Sérgio Veloso Brant Pinheiro
- Unit of Pediatric Nephrology, Department of Pediatrics, Faculty of Medicine, Federal University of Minas Gerais, Belo Horizonte, Brazil
| | - Ana Cristina Simões E Silva
- Unit of Pediatric Nephrology, Department of Pediatrics, Faculty of Medicine, Federal University of Minas Gerais, Belo Horizonte, Brazil.
- Pediatric Branch, Interdisciplinary Laboratory of Medical Investigation, Faculty of Medicine, Federal University of Minas Gerais (UFMG), Avenida Alfredo Balena, 190, Room# 281, Belo Horizonte, MG, 30130-100, Brazil.
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Xie YX, He LY, Chen X, Peng XF, Ye MY, Zhao YJ, Yan WZ, Liu C, Shao J, Peng YM. Potential diagnostic biomarkers for IgA nephropathy: a comparative study pre- and post-tonsillectomy. Int Urol Nephrol 2016; 48:1855-1861. [PMID: 27465795 DOI: 10.1007/s11255-016-1372-2] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2016] [Accepted: 07/11/2016] [Indexed: 01/08/2023]
Abstract
OBJECTIVE The proteins BAFF, ST6GALNAC2, C1GALT1, and COSMC in peripheral blood mononuclear cells (PBMCs) and plasma levels of IgA1 and galactose-deficient IgA1 (Gd-IgA1) are potential biomarkers for IgAN nephropathy. In this study, we comparatively studied the changes of those biomarkers before and after tonsillectomy. METHODS Peripheral blood samples were obtained from 16 IgAN patients with pre- and post-tonsillectomy. IgAN was diagnosed based on results from analysis of percutaneous renal biopsy tissue. Peripheral blood samples from three patients without renal diseases (non-IgAN), before and after tonsillectomy, and 16 healthy controls were also examined. BAFF, ST6GALNAC2, C1GALT1, and COSMC mRNA levels in PBMCs were detected using real-time PCR. Plasma IgA1 content was measured by ELISA. Gd-IgA1 levels were determined using the VV lectin-ELISA method. RESULTS BAFF, ST6GALNAC2, C1GALT1, and COSMC mRNA levels and the plasma concentrations of IgA1 and Gd-IgA1 in IgAN patients before tonsillectomy were significantly higher than those in healthy controls (P < 0.05). Tonsillectomy significantly increased the expression of BAFF and ST6GALNAC2, and plasma IgA1 level, while it downregulated that of C1GALT1 and COSMC (P < 0.05). However, in non-IgAN patients, tonsillectomy did not affect the mRNA levels of BAFF, ST6GALNAC2, C1GALT1, and COSMC, plasma IgA1 content and Gd-IgA1 level. Positive correlations were established between BAFF and IgA1 (r = 0.604, P < 0.01) and between ST6GALNAC2 and Gd-IgA1 (r = 0.623, P < 0.01). CONCLUSIONS Tonsillectomy changes the mRNA levels of BAFF, ST6GALNAC2, C1GALT1, and COSMC in PBMCs, as well as the plasma IgA1 level in IgAN patients. BAFF and ST6GALNAC2 might regulate IgA1 secretion and O-glycosylation.
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Affiliation(s)
- Ying-Xin Xie
- Nephrology Department, 2nd Xiangya Hospital, Central South University, Key Lab of Kidney Disease and Blood Purification in Hunan, 139 Renmin Road, Changsha, 410011, Hunan, People's Republic of China.,Nephrology Department, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China
| | - Li-Yu He
- Nephrology Department, 2nd Xiangya Hospital, Central South University, Key Lab of Kidney Disease and Blood Purification in Hunan, 139 Renmin Road, Changsha, 410011, Hunan, People's Republic of China
| | - Xian Chen
- Nephrology Department, 2nd Xiangya Hospital, Central South University, Key Lab of Kidney Disease and Blood Purification in Hunan, 139 Renmin Road, Changsha, 410011, Hunan, People's Republic of China
| | - Xiao-Fei Peng
- Nephrology Department, 2nd Xiangya Hospital, Central South University, Key Lab of Kidney Disease and Blood Purification in Hunan, 139 Renmin Road, Changsha, 410011, Hunan, People's Republic of China
| | - Mu-Yao Ye
- Nephrology Department, 2nd Xiangya Hospital, Central South University, Key Lab of Kidney Disease and Blood Purification in Hunan, 139 Renmin Road, Changsha, 410011, Hunan, People's Republic of China
| | - Yu-Jing Zhao
- Nephrology Department, 2nd Xiangya Hospital, Central South University, Key Lab of Kidney Disease and Blood Purification in Hunan, 139 Renmin Road, Changsha, 410011, Hunan, People's Republic of China
| | - Wen-Zhe Yan
- Nephrology Department, 2nd Xiangya Hospital, Central South University, Key Lab of Kidney Disease and Blood Purification in Hunan, 139 Renmin Road, Changsha, 410011, Hunan, People's Republic of China
| | - Chan Liu
- Nephrology Department, 2nd Xiangya Hospital, Central South University, Key Lab of Kidney Disease and Blood Purification in Hunan, 139 Renmin Road, Changsha, 410011, Hunan, People's Republic of China
| | - Jing Shao
- Nephrology Department, 2nd Xiangya Hospital, Central South University, Key Lab of Kidney Disease and Blood Purification in Hunan, 139 Renmin Road, Changsha, 410011, Hunan, People's Republic of China
| | - You-Ming Peng
- Nephrology Department, 2nd Xiangya Hospital, Central South University, Key Lab of Kidney Disease and Blood Purification in Hunan, 139 Renmin Road, Changsha, 410011, Hunan, People's Republic of China.
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Longitudinal monitoring of immunoglobulin A glycosylation during pregnancy by simultaneous MALDI-FTICR-MS analysis of N- and O-glycopeptides. Sci Rep 2016; 6:27955. [PMID: 27302155 PMCID: PMC4908400 DOI: 10.1038/srep27955] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2016] [Accepted: 05/24/2016] [Indexed: 12/13/2022] Open
Abstract
Immunoglobulin A (IgA) is a glycoprotein of which altered glycosylation has been associated with several pathologies. Conventional methods for IgA N- and O-glycosylation analysis are tedious, thus limiting such analyses to small sample sizes. Here we present a high-throughput strategy for the simultaneous analysis of serum-derived IgA1 N- and O-glycopeptides using matrix-assisted laser/desorption ionisation Fourier transform ion cyclotron resonance (MALDI-FTICR) mass spectrometry (MS). Six non-fucosylated diantennary complex type glycoforms were detected on the Asn144-containing glycopeptide. Thirteen distinct glycoforms were identified for the Asn340-containing tailpiece glycopeptide, mainly of the diantennary complex type, and low amounts of triantennary glycoforms. Simultaneously with these N-glycopeptides, 53 compositional glycoforms of the hinge region O-glycopeptide were profiled in a single high resolution MALDI-FTICR spectrum. Since many pregnancy associated changes have been recognized for immunoglobulin G, we sought to demonstrate the clinical applicability of this method in a cohort of 29 pregnant women, from whom samples were collected at three time points during pregnancy and three time points after delivery. Pregnancy associated changes of N-glycan bisection were different for IgA1 as compared to IgG-Fc described earlier. We foresee further applications of the developed method for larger patient cohorts to study IgA N- and O-glycosylation changes in pathologies.
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Knoppova B, Reily C, Maillard N, Rizk DV, Moldoveanu Z, Mestecky J, Raska M, Renfrow MB, Julian BA, Novak J. The Origin and Activities of IgA1-Containing Immune Complexes in IgA Nephropathy. Front Immunol 2016; 7:117. [PMID: 27148252 PMCID: PMC4828451 DOI: 10.3389/fimmu.2016.00117] [Citation(s) in RCA: 134] [Impact Index Per Article: 14.9] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2016] [Accepted: 03/15/2016] [Indexed: 12/12/2022] Open
Abstract
IgA nephropathy (IgAN) is the most common primary glomerulonephritis, frequently leading to end-stage renal disease, as there is no disease-specific therapy. IgAN is diagnosed from pathological assessment of a renal biopsy specimen based on predominant or codominant IgA-containing immunodeposits, usually with complement C3 co-deposits and with variable presence of IgG and/or IgM. The IgA in these renal deposits is galactose-deficient IgA1, with less than a full complement of galactose residues on the O-glycans in the hinge region of the heavy chains. Research from the past decade led to the definition of IgAN as an autoimmune disease with a multi-hit pathogenetic process with contributing genetic and environmental components. In this process, circulating galactose-deficient IgA1 (autoantigen) is bound by antiglycan IgG or IgA (autoantibodies) to form immune complexes. Some of these circulating complexes deposit in glomeruli, and thereby activate mesangial cells and induce renal injury through cellular proliferation and overproduction of extracellular matrix components and cytokines/chemokines. Glycosylation pathways associated with production of the autoantigen and the unique characteristics of the corresponding autoantibodies in patients with IgAN have been uncovered. Complement likely plays a significant role in the formation and the nephritogenic activities of these complexes. Complement activation is mediated through the alternative and lectin pathways and probably occurs systemically on IgA1-containing circulating immune complexes as well as locally in glomeruli. Incidence of IgAN varies greatly by geographical location; the disease is rare in central Africa but accounts for up to 40% of native-kidney biopsies in eastern Asia. Some of this variation may be explained by genetically determined influences on the pathogenesis of the disease. Genome-wide association studies to date have identified several loci associated with IgAN. Some of these loci are associated with the increased prevalence of IgAN, whereas others, such as deletion of complement factor H-related genes 1 and 3, are protective against the disease. Understanding the molecular mechanisms and genetic and biochemical factors involved in formation and activities of pathogenic IgA1-containing immune complexes will enable the development of future disease-specific therapies as well as identification of non-invasive disease-specific biomarkers.
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Affiliation(s)
- Barbora Knoppova
- Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL, USA
- Department of Immunology, Faculty of Medicine and Dentistry, Palacky University and University Hospital, Olomouc, Czech Republic
| | - Colin Reily
- Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA
| | - Nicolas Maillard
- Université Jean Monnet, Saint Etienne, France
- PRES Université de Lyon, Lyon, France
| | - Dana V. Rizk
- Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA
| | - Zina Moldoveanu
- Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL, USA
| | - Jiri Mestecky
- Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL, USA
| | - Milan Raska
- Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL, USA
- Department of Immunology, Faculty of Medicine and Dentistry, Palacky University and University Hospital, Olomouc, Czech Republic
| | - Matthew B. Renfrow
- Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL, USA
| | - Bruce A. Julian
- Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA
| | - Jan Novak
- Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL, USA
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Solecka BA, Weise C, Laffan MA, Kannicht C. Site-specific analysis of von Willebrand factor O-glycosylation. J Thromb Haemost 2016; 14:733-46. [PMID: 26784534 DOI: 10.1111/jth.13260] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2015] [Accepted: 12/30/2015] [Indexed: 02/06/2023]
Abstract
BACKGROUND O-glycosylation of von Willebrand factor (VWF) affects many of its functions; however, there is currently no information on the occupancy of the 10 putative O-glycosylation sites. OBJECTIVES The aim of this study was the site-specific analysis of VWF O-glycosylation. METHODS Tryptic VWF-O-glycopeptides were isolated by lectin affinity chromatography and/or by reverse-phase high-performance liquid chromatography. Subsequently, the purified glycopeptides were analyzed by glycosidase digestion and mass spectrometry. RESULTS We found that all 10 predicted O-glycosylation sites in VWF are occupied. The majority of the glycan structures on all glycosylation sites is represented by disialyl core 1 O-glycan. The presence of core 2 O-glycan was also confirmed; interestingly, this structure was not evenly distributed among all 10 glycosylation sites. Analysis of the glycopeptides flanking the A1 domain revealed that generally more core-2-type O-glycan was present on the C-terminal Cluster 2 glycopeptide (encompassing T(1468) , T(1477) , S(1486) and T(1487) ) compared with the N-terminal Cluster 1 glycopeptide (encompassing T(1248) , T(1255) , T(1256) and S(1263) ). Disialosyl motifs were present on both glycopeptides flanking the A1 domain and on the glycosylation site T(2298) in the C1 domain. In addition, we identify sulfation of core 2 O-glycans and the presence of the rare Tn antigen. CONCLUSIONS This is the first study to describe the qualitative and semi-quantitative distribution of O-glycan structures on all 10 O-glycosylation sites, which will provide a valuable starting point for further studies exploring the functional and structural implications of O-glycosylation in VWF.
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Affiliation(s)
- B A Solecka
- Molecular Biochemistry, Octapharma, Berlin, Germany
| | - C Weise
- Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany
| | - M A Laffan
- Department of Haematology, Faculty of Medicine, Hammersmith Hospital Campus, Imperial College, London, UK
| | - C Kannicht
- Molecular Biochemistry, Octapharma, Berlin, Germany
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Plomp R, Bondt A, de Haan N, Rombouts Y, Wuhrer M. Recent Advances in Clinical Glycoproteomics of Immunoglobulins (Igs). Mol Cell Proteomics 2016; 15:2217-28. [PMID: 27009965 PMCID: PMC4937499 DOI: 10.1074/mcp.o116.058503] [Citation(s) in RCA: 52] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2016] [Indexed: 11/06/2022] Open
Abstract
Antibody glycosylation analysis has seen methodological progress resulting in new findings with regard to antibody glycan structure and function in recent years. For example, antigen-specific IgG glycosylation analysis is now applicable for clinical samples because of the increased sensitivity of measurements, and this has led to new insights in the relationship between IgG glycosylation and various diseases. Furthermore, many new methods have been developed for the purification and analysis of IgG Fc glycopeptides, notably multiple reaction monitoring for high-throughput quantitative glycosylation analysis. In addition, new protocols for IgG Fab glycosylation analysis were established revealing autoimmune disease-associated changes. Functional analysis has shown that glycosylation of IgA and IgE is involved in transport across the intestinal epithelium and receptor binding, respectively.
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Affiliation(s)
- Rosina Plomp
- From the ‡Leiden University Medical Center, Center for Proteomics and Metabolomics, Leiden, The Netherlands
| | - Albert Bondt
- From the ‡Leiden University Medical Center, Center for Proteomics and Metabolomics, Leiden, The Netherlands; §Leiden University Medical Center, Department of Rheumatology, Leiden, The Netherlands
| | - Noortje de Haan
- From the ‡Leiden University Medical Center, Center for Proteomics and Metabolomics, Leiden, The Netherlands
| | - Yoann Rombouts
- ¶Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS, UPS, France
| | - Manfred Wuhrer
- From the ‡Leiden University Medical Center, Center for Proteomics and Metabolomics, Leiden, The Netherlands;
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Salvadori M, Rosso G. Update on immunoglobulin A nephropathy, Part I: Pathophysiology. World J Nephrol 2015; 4:455-467. [PMID: 26380197 PMCID: PMC4561843 DOI: 10.5527/wjn.v4.i4.455] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/23/2015] [Revised: 08/24/2015] [Accepted: 08/30/2015] [Indexed: 02/06/2023] Open
Abstract
Immunoglobulin A (IgA) nephropathy is one of the most common glomerulonephritis and its frequency is probably underestimated because in most patients the disease has an indolent course and the kidney biopsy is essential for the diagnosis. In the last years its pathogenesis has been better identified even if still now several questions remain to be answered. The genetic wide association studies have allowed to identifying the relevance of genetics and several putative genes have been identified. The genetics has also allowed explaining why some ancestral groups are affected with higher frequency. To date is clear that IgA nephropathy is related to auto antibodies against immunoglobulin A1 (IgA1) with poor O-glycosylation. The role of mucosal infections is confirmed, but which are the pathogens involved and which is the role of Toll-like receptor polymorphism is less clear. Similarly to date whether the disease is due to the circulating immunocomplexes deposition on the mesangium or whether the antigen is already present on the mesangial cell as a "lanthanic" deposition remains to be clarified. Finally also the link between the mesangial and the podocyte injury and the tubulointerstitial scarring, as well as the mechanisms involved need to be better clarified.
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Yasutake J, Suzuki Y, Suzuki H, Hiura N, Yanagawa H, Makita Y, Kaneko E, Tomino Y. Novel lectin-independent approach to detect galactose-deficient IgA1 in IgA nephropathy. Nephrol Dial Transplant 2015; 30:1315-21. [PMID: 26109484 PMCID: PMC4513896 DOI: 10.1093/ndt/gfv221] [Citation(s) in RCA: 107] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2015] [Accepted: 04/16/2015] [Indexed: 12/15/2022] Open
Abstract
Background Galactose-deficient IgA1 (Gd-IgA1) is a critical effector molecule in the pathogenesis of IgA nephropathy (IgAN). Although many researchers have measured serum levels of Gd-IgA1 using snail helix aspersa agglutinin (HAA) lectin-based assay, the lectin-dependent assay has some serious problems in robustness. In this study, we aimed to establish a more robust and stable enzyme-linked immunosorbent assay (ELISA) method that uses a specific monoclonal antibody to recognize a hinge region in human Gd-IgA1 (Gd-IgA1 ELISA). Methods Rats were immunized with human Gd-IgA1 hinge region peptide to obtain Gd-IgA1-specific monoclonal antibody KM55. Gd-IgA1 ELISA for specifically detecting serum Gd-IgA1 was consequently constructed. Serum Gd-IgA1 concentrations in human subjects were measured using KM55 ELISA assay. To further confirm specificity of the Gd-IgA1-specific antibody, KM55 was also applied for immunofluorescence staining of glomerular Gd-IgA1 in paraffin-embedded sections of renal biopsy specimens. Results Measurement of serum levels of Gd-IgA1 in human subjects by Gd-IgA1 ELISA revealed increased serum Gd-IgA1 level in patients with IgAN compared with patients with other renal diseases or non-renal diseases. Importantly, the results obtained from Gd-IgA1 ELISA positively correlated with those from the HAA lectin-based assay (R = 0.75). Immunofluorescence staining of renal biopsy specimens with KM55 detected glomerular co-localization of Gd-IgA1 and IgA. Conclusion This novel lectin-independent method with KM55 for measuring serum levels of Gd-IgA1 can pave the way for more convincing diagnosis and activity assessment of IgAN, and can expedite clinical research to better understand this difficult disease.
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Affiliation(s)
- Junichi Yasutake
- Division of Nephrology, Department of Internal Medicine, Juntendo University Faculty of Medicine, Tokyo, Japan Kyowa Hakko Kirin Co., Ltd, Tokyo Japan
| | - Yusuke Suzuki
- Division of Nephrology, Department of Internal Medicine, Juntendo University Faculty of Medicine, Tokyo, Japan
| | - Hitoshi Suzuki
- Division of Nephrology, Department of Internal Medicine, Juntendo University Faculty of Medicine, Tokyo, Japan
| | - Naoko Hiura
- Division of Nephrology, Department of Internal Medicine, Juntendo University Faculty of Medicine, Tokyo, Japan Kyowa Hakko Kirin Co., Ltd, Tokyo Japan
| | - Hiroyuki Yanagawa
- Division of Nephrology, Department of Internal Medicine, Juntendo University Faculty of Medicine, Tokyo, Japan
| | - Yuko Makita
- Division of Nephrology, Department of Internal Medicine, Juntendo University Faculty of Medicine, Tokyo, Japan
| | | | - Yasuhiko Tomino
- Division of Nephrology, Department of Internal Medicine, Juntendo University Faculty of Medicine, Tokyo, Japan
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Zhu Z, Desaire H. Carbohydrates on Proteins: Site-Specific Glycosylation Analysis by Mass Spectrometry. ANNUAL REVIEW OF ANALYTICAL CHEMISTRY (PALO ALTO, CALIF.) 2015; 8:463-483. [PMID: 26070719 DOI: 10.1146/annurev-anchem-071114-040240] [Citation(s) in RCA: 61] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/04/2023]
Abstract
Glycosylation on proteins adds complexity and versatility to these biologically vital macromolecules. To unveil the structure-function relationship of glycoproteins, glycopeptide-centric analysis using mass spectrometry (MS) has become a method of choice because the glycan is preserved on the glycosylation site and site-specific glycosylation profiles of proteins can be readily determined. However, glycopeptide analysis is still challenging given that glycopeptides are usually low in abundance and relatively difficult to detect and the resulting data require expertise to analyze. Viewing the urgent need to address these challenges, emerging methods and techniques are being developed with the goal of analyzing glycopeptides in a sensitive, comprehensive, and high-throughput manner. In this review, we discuss recent advances in glycoprotein and glycopeptide analysis, with topics covering sample preparation, analytical separation, MS and tandem MS techniques, as well as data interpretation and automation.
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Affiliation(s)
- Zhikai Zhu
- Ralph N. Adams Institute for Bioanalytical Chemistry, Department of Chemistry, University of Kansas, Lawrence, Kansas 66047;
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Novak J, Rizk D, Takahashi K, Zhang X, Bian Q, Ueda H, Ueda Y, Reily C, Lai LY, Hao C, Novak L, Huang ZQ, Renfrow MB, Suzuki H, Julian BA. New Insights into the Pathogenesis of IgA Nephropathy. KIDNEY DISEASES 2015; 1:8-18. [PMID: 26568951 DOI: 10.1159/000382134] [Citation(s) in RCA: 56] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
BACKGROUND IgA nephropathy, a frequent cause of end-stage renal disease, is an autoimmune disease wherein immune complexes consisting of IgA1 with galactose-deficient O-glycans (autoantigen) and anti-glycan autoantibodies deposit in glomeruli and induce renal injury. Multiple genetic loci associated with disease risk have been identified. The prevalence of risk alleles varies geographically, highest in eastern Asia and northern Europe, fewer in other parts of Europe and North America, and the least in Africa. IgA nephropathy is diagnosed from pathological assessment of a renal biopsy specimen. Currently, therapy is not disease-targeted but rather is focused on maintaining control of blood pressure and proteinuria, ideally with suppression of angiotensin II. Possible additional approaches differ between countries. Disease-specific therapy as well as new tools for diagnosis, prognosis, and assessment of responses to therapy are needed. SUMMARY Glycosylation pathways associated with aberrant O-glycosylation of IgA1 and, thus, production of autoantigen, have been identified. Furthermore, unique characteristics of the autoantibodies in IgA nephropathy have been uncovered. Many of these biochemical features are shared by patients with IgA nephropathy and Henoch-Schönlein purpura nephritis, suggesting that the two diseases may represent opposite ends of a spectrum of a disease process. Understanding the molecular mechanisms involved in formation of pathogenic IgA1-containing immune complexes will enable development of disease-specific therapies as well as diagnostic and prognostic biomarkers. KEY MESSAGES IgA nephropathy is an autoimmune disease caused by glomerular deposition of nephritogenic circulating immune complexes consisting of galactose-deficient IgA1 (autoantigen) bound by anti-glycan autoantibodies. A better understanding of the multi-step process of pathogenesis of IgA nephropathy and the genetic and environmental contributing factors will lead to development of biomarkers to identify patients with progressive disease who would benefit from a future disease-specific therapy.
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Affiliation(s)
- Jan Novak
- University of Alabama at Birmingham, Birmingham, AL, USA
| | - Dana Rizk
- University of Alabama at Birmingham, Birmingham, AL, USA
| | - Kazuo Takahashi
- University of Alabama at Birmingham, Birmingham, AL, USA ; School of Medicine, Fujita Health University, Toyoake, Japan
| | - XianWen Zhang
- University of Alabama at Birmingham, Birmingham, AL, USA ; Longhua Hospital, Shanghai University of TCM, Shanghai, China
| | - Qi Bian
- University of Alabama at Birmingham, Birmingham, AL, USA ; Changhai Hospital, Second Military Medical University, Shanghai, China
| | - Hirouki Ueda
- University of Alabama at Birmingham, Birmingham, AL, USA
| | - Yoshimi Ueda
- University of Alabama at Birmingham, Birmingham, AL, USA
| | - Colin Reily
- University of Alabama at Birmingham, Birmingham, AL, USA
| | - Ling-Yun Lai
- University of Alabama at Birmingham, Birmingham, AL, USA ; Fudan University Huashan Hospital, Shanghai, China
| | | | - Lea Novak
- University of Alabama at Birmingham, Birmingham, AL, USA
| | | | | | - Hitoshi Suzuki
- University of Alabama at Birmingham, Birmingham, AL, USA ; Juntendo University Faculty of Medicine, Tokyo, Japan
| | - Bruce A Julian
- University of Alabama at Birmingham, Birmingham, AL, USA
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Huang J, Guerrero A, Parker E, Strum JS, Smilowitz JT, German JB, Lebrilla CB. Site-specific glycosylation of secretory immunoglobulin A from human colostrum. J Proteome Res 2015; 14:1335-49. [PMID: 25629924 DOI: 10.1021/pr500826q] [Citation(s) in RCA: 62] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Secretory immunoglobulin A (sIgA) is a major glycoprotein in milk and plays a key role in mediating immune protection of the gut mucosa. Although it is a highly glycosylated protein, its site-specific glycosylation and associated glycan micro-heterogeneity have still not been fully elucidated. In this study, the site-specific glycosylation of sIgA isolated from human colostrum (n = 3) was analyzed using a combination of LC-MS and LC-MS/MS and in-house software (Glycopeptide Finder). The majority of the glycans found are biantennary structures with one or more acidic Neu5Ac residues; however, a large fraction belonged to truncated complex structures with terminal GlcNAc. Multiple glycosites were identified with nearly 30 glycan compositions located at seven sites on the secretory component, six compositions at a single site on the J chain, and 16 compositions at five sites on the IgA heavy (H) chain. Site-specific heterogeneity and relative quantitation of each composition and the extent of occupation at each site were determined using nonspecific proteases. Additionally, 54 O-linked glycan compositions located at the IgA1 hinge region (HR) were identified by comparison against a theoretical O-glycopeptide library. This represents the most comprehensive report to date detailing the complexity of glycan micro-heterogeneity with relative quantitation of glycoforms for each glycosylation site on milk sIgA. This strategy further provides a general method for determining site-specific glycosylation in large protein complexes.
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Affiliation(s)
- Jincui Huang
- Department of Chemistry, ‡Foods for Health Institute, §Department of Food Science and Technology, and ∥Department of Biochemistry and Molecular Medicine, University of California , Davis, California 95616, United States
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Suzuki Y, Suzuki H, Yasutake J, Tomino Y. Paradigm shift in activity assessment of IgA nephropathy – optimizing the next generation of diagnostic and therapeutic maneuvers via glycan targeting. Expert Opin Biol Ther 2015; 15:583-93. [DOI: 10.1517/14712598.2015.1006624] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
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Novak J, Raska M, Mestecky J, Julian BA. IgA Nephropathy and Related Diseases. Mucosal Immunol 2015. [DOI: 10.1016/b978-0-12-415847-4.00105-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/23/2022]
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Hwang VJ, Ulu A, van Hoorebeke J, Weiss RH. Biomarkers in IgA nephropathy. Biomark Med 2014; 8:1263-77. [DOI: 10.2217/bmm.14.92] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
IgA nephropathy is the most common primary glomerulonephritis and presents with gross hematuria and upper respiratory infection, with slow progression to end-stage renal disease in up to 50% of affected patients. Kidney biopsies are the gold standard method of diagnosis and frequently are not performed as the majority of individuals are asymptomatic. Thus, there is a need to discover and validate prognostic and predictive biomarkers that can be noninvasively obtained and are specific to this disease. Here we discuss the current state of research in this area and examine validated and clinically promising biofluid and tissue biomarkers of IgA nephropathy.
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Affiliation(s)
- Vicki J Hwang
- Division of Nephrology, Department of Internal Medicine, Genome & Biomedical Sciences Building, Room 6312, University of California, Davis, CA 95616, USA
- Integrative Genetics & Genomics Graduate Group, 227 Life Sciences, University of California, Davis, CA 95616, USA
| | - Arzu Ulu
- Division of Nephrology, Department of Internal Medicine, Genome & Biomedical Sciences Building, Room 6312, University of California, Davis, CA 95616, USA
| | - Justin van Hoorebeke
- Division of Nephrology, Department of Internal Medicine, Genome & Biomedical Sciences Building, Room 6312, University of California, Davis, CA 95616, USA
- Molecular, Cellular & Integrative Physiology, 227 Life Sciences, University of California, Davis, CA 95616, USA
| | - Robert H Weiss
- Division of Nephrology, Department of Internal Medicine, Genome & Biomedical Sciences Building, Room 6312, University of California, Davis, CA 95616, USA
- Integrative Genetics & Genomics Graduate Group, 227 Life Sciences, University of California, Davis, CA 95616, USA
- Molecular, Cellular & Integrative Physiology, 227 Life Sciences, University of California, Davis, CA 95616, USA
- Cancer Center, University of California, Davis, CA 95616, USA
- Medical Service, Mather VA Medical Center, Sacramento, CA, USA
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Stuchlova Horynova M, Vrablikova A, Stewart TJ, Takahashi K, Czernekova L, Yamada K, Suzuki H, Julian BA, Renfrow MB, Novak J, Raska M. N-acetylgalactosaminide α2,6-sialyltransferase II is a candidate enzyme for sialylation of galactose-deficient IgA1, the key autoantigen in IgA nephropathy. Nephrol Dial Transplant 2014; 30:234-8. [PMID: 25281698 DOI: 10.1093/ndt/gfu308] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
BACKGROUND Galactose-deficient O-glycans in the hinge region (HR) of immunoglobulin A1 (IgA1) play a key role in the pathogenesis of IgA nephropathy (IgAN). O-Glycans of circulatory IgA1 consist of N-acetylgalactosamine (GalNAc) with a β1,3-linked galactose; both sugars may be sialylated. In patients with IgAN, α2,6-sialylated GalNAc is a frequent form of the galactose-deficient O-glycans. Prior analyses of IgA1-producing cells had indicated that α2,6-sialyltransferase II (ST6GalNAc-II) is likely responsible for sialylation of GalNAc of galactose-deficient IgA1, but direct evidence is missing. METHODS We produced a secreted variant of recombinant human ST6GalNAc-II and an IgA1 fragment comprised of Cα1-HR-Cα2. This IgA1 fragment and a synthetic HR peptide with enzymatically attached GalNAc residues served as acceptors. ST6GalNAc-II activity was assessed in vitro and the attachment of sialic acid to these acceptors was detected by lectin blot and mass spectrometry. RESULTS ST6GalNAc-II was active with both acceptors. High-resolution mass spectrometry analysis revealed that up to three sialic acid residues were added to the GalNAc residues of the HR glycopeptide. CONCLUSIONS Our data provide direct evidence that ST6GalNAc-II can sialylate GalNAc of galactose-deficient IgA1. As serum levels of galactose-deficient IgA1 with sialylated glycoforms are increased in IgAN patients, our data explain the corresponding part of the biosynthetic pathway.
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Affiliation(s)
- Milada Stuchlova Horynova
- Department of Immunology, Faculty of Medicine and Dentistry, Palacky University, Olomouc 77515, Czech Republic Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA
| | - Alena Vrablikova
- Department of Immunology, Faculty of Medicine and Dentistry, Palacky University, Olomouc 77515, Czech Republic Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA
| | - Tyler J Stewart
- Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA
| | - Kazuo Takahashi
- Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA Department of Nephrology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan
| | - Lydie Czernekova
- Department of Immunology, Faculty of Medicine and Dentistry, Palacky University, Olomouc 77515, Czech Republic Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA
| | - Koshi Yamada
- Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA Division of Nephrology, Department of Internal Medicine, Juntendo University Faculty of Medicine, Tokyo 113-8421, Japan
| | - Hitoshi Suzuki
- Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA Division of Nephrology, Department of Internal Medicine, Juntendo University Faculty of Medicine, Tokyo 113-8421, Japan
| | - Bruce A Julian
- Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA
| | - Matthew B Renfrow
- Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, USA
| | - Jan Novak
- Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA
| | - Milan Raska
- Department of Immunology, Faculty of Medicine and Dentistry, Palacky University, Olomouc 77515, Czech Republic Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA
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Abstract
PURPOSE OF REVIEW In this article, we review recent findings on the pathogenesis and genetics of immunoglobulin A (IgA) nephropathy. RECENT FINDINGS During the past 2 years, the understanding of the pathogenesis of IgA nephropathy has evolved as a result of progress in technology and new tools that have been developed. Since 1968, when IgA nephropathy was described as an IgA-IgG immune-complex disease, the knowledge base expanded to allow definition of IgA nephropathy as an autoimmune disease with a multihit pathogenetic process. Specifically, galactose-deficient immunoglobulin A1 (IgA1) is recognized by unique autoantibodies, resulting in the formation of pathogenic immune complexes that ultimately deposit in the glomerular mesangium and induce renal injury. New approaches using high-resolution mass spectrometry have provided unique insight at the molecular level into IgA1 O-glycosylation. Cutting-edge genome-wide association studies revealed multiple disease-associated risk loci and have mapped their geographic and racial distribution. SUMMARY Recent studies of molecular and genetic defects operating in IgA nephropathy can define new biomarkers specific for the disease that can be developed into clinical assays to aid in the diagnosis, assessment of prognosis, and monitoring of disease progression. Moreover, disease-specific targets are being discovered that may lead to development of new approaches for treatment.
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Lehoux S, Mi R, Aryal RP, Wang Y, Schjoldager KTBG, Clausen H, van Die I, Han Y, Chapman AB, Cummings RD, Ju T. Identification of distinct glycoforms of IgA1 in plasma from patients with immunoglobulin A (IgA) nephropathy and healthy individuals. Mol Cell Proteomics 2014; 13:3097-113. [PMID: 25071157 DOI: 10.1074/mcp.m114.039693] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Immunoglobulin A nephropathy (IgAN) is the most common form of glomerulonephritis worldwide and is histologically characterized by the deposition of IgA1 and consequent inflammation in the glomerular mesangium. Prior studies suggested that serum IgA1 from IgAN patients contains aberrant, undergalactosylated O-glycans, for example, Tn antigen and its sialylated version, SialylTn (STn), but the mechanisms underlying aberrant O-glycosylation are not well understood. Here we have used serial lectin separation technologies, Western blot, enzymatic modifications, and mass spectrometry to explore whether there are different glycoforms of IgA1 in plasma from patients with IgAN and healthy individuals. Although total plasma IgA in IgAN patients was elevated ∼ 1.6-fold compared with that in healthy donors, IgA1 in all samples was unexpectedly separable into two distinct glycoforms: one with core 1 based O-glycans, and the other exclusively containing Tn/STn structures. Importantly, Tn antigen present on IgA1 from IgAN patients and controls was convertible into the core 1 structure in vitro by recombinant T-synthase. Our results demonstrate that undergalactosylation of O-glycans in IgA1 is not restricted to IgAN and suggest that in vivo inefficiency of T-synthase toward IgA1 in a subpopulation of B or plasma cells, as well as overall elevation of IgA, may contribute to IgAN pathogenesis.
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Affiliation(s)
| | | | | | | | - Katrine T-B G Schjoldager
- §Department of Cellular and Molecular Medicine, Copenhagen Center for Glycomics, Copenhagen, Denmark
| | - Henrik Clausen
- §Department of Cellular and Molecular Medicine, Copenhagen Center for Glycomics, Copenhagen, Denmark
| | - Irma van Die
- ¶Department of Molecular Cell Biology & Immunology, VU University Medical Center, Amsterdam, The Netherlands
| | - Yoosun Han
- ‖Medicine, Emory University School of Medicine, Atlanta, GA 30322
| | - Arlene B Chapman
- ‖Medicine, Emory University School of Medicine, Atlanta, GA 30322
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Takahashi K, Raska M, Stuchlova Horynova M, Hall SD, Poulsen K, Kilian M, Hiki Y, Yuzawa Y, Moldoveanu Z, Julian BA, Renfrow MB, Novak J. Enzymatic sialylation of IgA1 O-glycans: implications for studies of IgA nephropathy. PLoS One 2014; 9:e99026. [PMID: 24918438 PMCID: PMC4053367 DOI: 10.1371/journal.pone.0099026] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2014] [Accepted: 04/23/2014] [Indexed: 11/18/2022] Open
Abstract
Patients with IgA nephropathy (IgAN) have elevated circulating levels of IgA1 with some O-glycans consisting of galactose (Gal)-deficient N-acetylgalactosamine (GalNAc) with or without N-acetylneuraminic acid (NeuAc). We have analyzed O-glycosylation heterogeneity of naturally asialo-IgA1 (Ale) myeloma protein that mimics Gal-deficient IgA1 (Gd-IgA1) of patients with IgAN, except that IgA1 O-glycans of IgAN patients are frequently sialylated. Specifically, serum IgA1 of healthy controls has more α2,3-sialylated O-glycans (NeuAc attached to Gal) than α2,6-sialylated O-glycans (NeuAc attached to GalNAc). As IgA1-producing cells from IgAN patients have an increased activity of α2,6-sialyltransferase (ST6GalNAc), we hypothesize that such activity may promote premature sialylation of GalNAc and, thus, production of Gd-IgA1, as sialylation of GalNAc prevents subsequent Gal attachment. Distribution of NeuAc in IgA1 O-glycans may play an important role in the pathogenesis of IgAN. To better understand biological functions of NeuAc in IgA1, we established protocols for enzymatic sialylation leading to α2,3- or α2,6-sialylation of IgA1 O-glycans. Sialylation of Gal-deficient asialo-IgA1 (Ale) myeloma protein by an ST6GalNAc enzyme generated sialylated IgA1 that mimics the Gal-deficient IgA1 glycoforms in patients with IgAN, characterized by α2,6-sialylated Gal-deficient GalNAc. In contrast, sialylation of the same myeloma protein by an α2,3-sialyltransferase yielded IgA1 typical for healthy controls, characterized by α2,3-sialylated Gal. The GalNAc-specific lectin from Helix aspersa (HAA) is used to measure levels of Gd-IgA1. We assessed HAA binding to IgA1 sialylated at Gal or GalNAc. As expected, α2,6-sialylation of IgA1 markedly decreased reactivity with HAA. Notably, α2,3-sialylation also decreased reactivity with HAA. Neuraminidase treatment recovered the original HAA reactivity in both instances. These results suggest that binding of a GalNAc-specific lectin is modulated by sialylation of GalNAc as well as Gal in the clustered IgA1 O-glycans. Thus, enzymatic sialylation offers a useful model to test the role of NeuAc in reactivities of the clustered O-glycans with lectins.
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Affiliation(s)
- Kazuo Takahashi
- Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America
- Department of Nephrology, Fujita Health University School of Medicine, Toyoake, Japan
| | - Milan Raska
- Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America
- Faculty of Medicine and Dentistry, Department of Immunology, Palacky University in Olomouc, Olomouc, Czech Republic
| | - Milada Stuchlova Horynova
- Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America
- Faculty of Medicine and Dentistry, Department of Immunology, Palacky University in Olomouc, Olomouc, Czech Republic
| | - Stacy D. Hall
- Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America
| | - Knud Poulsen
- Department of Biomedicine, Aarhus University, Aarhus, Denmark
| | - Mogens Kilian
- Department of Biomedicine, Aarhus University, Aarhus, Denmark
| | - Yoshiyuki Hiki
- Fujita Health University School of Health Sciences, Toyoake, Japan
| | - Yukio Yuzawa
- Department of Nephrology, Fujita Health University School of Medicine, Toyoake, Japan
| | - Zina Moldoveanu
- Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America
| | - Bruce A. Julian
- Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America
- Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, United States of America
| | - Matthew B. Renfrow
- UAB Biomedical FT-ICR MS Laboratory, Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama, United States of America
| | - Jan Novak
- Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America
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