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Amundson DE, Shah US, de Necochea-Campion R, Jacobs M, LaRosa SP, Fisher CJ. Removal of COVID-19 Spike Protein, Whole Virus, Exosomes, and Exosomal MicroRNAs by the Hemopurifier® Lectin-Affinity Cartridge in Critically Ill Patients With COVID-19 Infection. Front Med (Lausanne) 2021; 8:744141. [PMID: 34692735 PMCID: PMC8531584 DOI: 10.3389/fmed.2021.744141] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2021] [Accepted: 09/08/2021] [Indexed: 12/23/2022] Open
Abstract
Coronavirus−19 (COVID-19) has rapidly spread throughout the world resulting in a significant amount of morbidity and mortality. Despite advances in therapy, social distancing, masks, and vaccination many places in the world continue to see an increase in the number of cases and deaths. Viremia is commonly present in severely ill patients with COVID-19 infections and is associated with organ dysfunction and poor outcomes. Exosomes released by activated cells have been implicated in the pathogenesis of COVID-19 infection. We report the experience of two cases of critically ill COVID-19 patients treated with the Hemopurifier; a lectin affinity cartridge designed to remove mannosylated viruses and exosomes. Both patients tolerated the Hemopurifier sessions without adverse effects. In the first patient removal of exosomes and exosomal microRNAs was associated with improved coagulopathy, oxygenation, and clinical recovery, while in a second patient removal of COVID-19 by the Hemopurifier cartridge was observed. The Hemopurifier is currently under further investigation in up to 40-patients in a safety and feasibility study in ICU patients with COVID-19 infection.
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Affiliation(s)
- Dennis E Amundson
- Department of Critical Care, Scripps Mercy Hospital Chula Vista, Chula Vista, CA, United States
| | - Usman S Shah
- Department of Critical Care, Hoag Hospital Newport Beach, Newport Beach, CA, United States
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Luo YD, Zhang QL, Yao SJ, Lin DQ. Evaluation of adsorption selectivity of immunoglobulins M, A and G and purification of immunoglobulin M with mixed-mode resins. J Chromatogr A 2018; 1533:77-86. [DOI: 10.1016/j.chroma.2017.12.018] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2017] [Revised: 12/07/2017] [Accepted: 12/08/2017] [Indexed: 12/13/2022]
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3
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Alba A, Marcet R, Otero O, Hernández HM, Figueredo M, Sarracent J. Isolation of 1E4 IgM Anti-Fasciola hepatica Rediae Monoclonal Antibody from Ascites: Comparison of Two Purification Protocols. Monoclon Antib Immunodiagn Immunother 2016; 35:52-6. [PMID: 26828226 DOI: 10.1089/mab.2015.0050] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Purification of immunoglobulin M (IgM) antibodies could be challenging, and is often characterized by the optimization of the purification protocol to best suit the particular features of the molecule. Here, two different schemes were compared to purify, from ascites, the 1E4 IgM monoclonal antibody (mAb) previously raised against the stage of redia of the trematode Fasciola hepatica. This immunoglobulin is used as capture antibody in an immunoenzymatic assay to detect parasite ongoing infection in its intermediate hosts. The first purification protocol of the 1E4 mAb involved two chromatographic steps: an affinity chromatography on a Concanavalin A matrix followed by size exclusion chromatography. An immunoaffinity chromatography was selected as the second protocol for one-step purification of the antibody using the crude extract of adult parasites coupled to a commercial matrix. Immunoreactivity of the fractions during purification schemes was assessed by indirect immunoenzymatic assays against the crude extract of F. hepatica rediae, while purity was estimated by protein electrophoresis. Losses on the recovery of the antibody isolated by the first purification protocol occurred due to protein precipitation during the concentration of the sample and to low resolution of the size exclusion molecular chromatography step regarding this particular immunoglobulin. The immunoaffinity chromatography using F. hepatica antigens as ligands proved to be the most suitable protocol yielding a pure and immunoreactive antibody. The purification protocols used are discussed regarding efficiency and difficulties.
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Affiliation(s)
- Annia Alba
- 1 Laboratorio de Anticuerpos Monoclonales, Departamento de Parasitología, Instituto de Medicina Tropical "Pedro Kourí," La Habana , Cuba
| | - Ricardo Marcet
- 1 Laboratorio de Anticuerpos Monoclonales, Departamento de Parasitología, Instituto de Medicina Tropical "Pedro Kourí," La Habana , Cuba
| | - Oscar Otero
- 2 Laboratorio de Anticuerpos Monoclonales, Vicepresidencia de Investigaciones , Instituto Finlay, La Habana, Cuba
| | - Hilda M Hernández
- 1 Laboratorio de Anticuerpos Monoclonales, Departamento de Parasitología, Instituto de Medicina Tropical "Pedro Kourí," La Habana , Cuba
| | - Mabel Figueredo
- 1 Laboratorio de Anticuerpos Monoclonales, Departamento de Parasitología, Instituto de Medicina Tropical "Pedro Kourí," La Habana , Cuba
| | - Jorge Sarracent
- 1 Laboratorio de Anticuerpos Monoclonales, Departamento de Parasitología, Instituto de Medicina Tropical "Pedro Kourí," La Habana , Cuba
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Natsuka S, Masuda M, Sumiyoshi W, Nakakita SI. Improved method for drawing of a glycan map, and the first page of glycan atlas, which is a compilation of glycan maps for a whole organism. PLoS One 2014; 9:e102219. [PMID: 25006806 PMCID: PMC4090225 DOI: 10.1371/journal.pone.0102219] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2014] [Accepted: 06/16/2014] [Indexed: 11/19/2022] Open
Abstract
Glycan Atlas is a set of glycan maps over the whole body of an organism. The glycan map that includes data of glycan structure and quantity displays micro-heterogeneity of the glycans in a tissue, an organ, or cells. The two-dimensional glycan mapping is widely used for structure analysis of N-linked oligosaccharides on glycoproteins. In this study we developed a comprehensive method for the mapping of both N- and O-glycans with and without sialic acid. The mapping data of 150 standard pyridylaminated glycans were collected. The empirical additivity rule which was proposed in former reports was able to adapt for this extended glycan map. The adapted rule is that the elution time of pyridylamino glycans on high performance liquid chromatography (HPLC) is expected to be the simple sum of the partial elution times assigned to each monosaccharide residue. The comprehensive mapping method developed in this study is a powerful tool for describing the micro-heterogeneity of the glycans. Furthermore, we prepared 42 pyridylamino (PA-) glycans from human serum and were able to draw the map of human serum N- and O-glycans as an initial step of Glycan Atlas editing.
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Affiliation(s)
- Shunji Natsuka
- Department of Biology, Faculty of Science, Niigata University, Niigata, Japan
- * E-mail:
| | - Mayumi Masuda
- Department of Biology, Faculty of Science, Niigata University, Niigata, Japan
| | - Wataru Sumiyoshi
- Life Science Research Center, Kagawa University, Takamatsu, Kagawa, Japan
| | - Shin-ichi Nakakita
- Life Science Research Center, Kagawa University, Takamatsu, Kagawa, Japan
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Li N, Xu H, Fan K, Liu X, Qi J, Zhao C, Yin P, Wang L, Li Z, Zha X. Altered β1,6-GlcNAc branched N-glycans impair TGF-β-mediated epithelial-to-mesenchymal transition through Smad signalling pathway in human lung cancer. J Cell Mol Med 2014; 18:1975-91. [PMID: 24913443 PMCID: PMC4244013 DOI: 10.1111/jcmm.12331] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2014] [Accepted: 04/15/2014] [Indexed: 12/21/2022] Open
Abstract
The change of oligosaccharide structure has been revealed to be crucial for glycoproteins' biological functions and cell biological characteristics. N-acetylglucosaminy transferase V (GnT-V), a key enzyme catalysing the reaction of adding β1, 6-N-acetylglucosamine (GlcNAc) on asparagine-linked oligosaccharides of cell proteins, has been implicated to a metastastic-promoting oncoprotein in some carcinomas. However, this correlation might not be subjected to all types of cancers, for example, in non-small cell lung cancers, low level of GnT-V expression is associated with relatively short survival time and poor prognosis. To explain the role of GnT-V in lung cancer progression, we studied the association of GnT-V expression with lung cancer EMT behaviour. We found that GnT-V expression was correlated with epithelial marker positively and mesenchymal marker negatively. GnT-V levels, as well as β1,6-GlcNAc branched N-glycans, were strongly reduced in TGF-β1-induced EMT of human lung adenocarcinoma A549 cells. Further studies showed that suppression of β1,6-GlcNAc branched N-glycans by inhibitor or GnT-V silencing in A549 cells could promote TGF-β1-induced EMT-like changes, cell migration and invasion. Meanwhile, overexpression of GnT-V impaired TGF-β1-induced EMT, migration and invasion. It suggests that GnT-V suppresses the EMT process of lung cancer cells through inhibiting the TGF-β/Smad signalling and its downstream transcription factors in a GnT-V catalytic activity–dependent manner. Taken together, the present study reveals a novel mechanism of GnT-V as a suppressor of both EMT and invasion in human lung cancer cells, which may be useful for fully understanding N-glycan's biological roles in lung cancer progression.
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Affiliation(s)
- Na Li
- Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai, China; Key Laboratory of Glycoconjugate Research, Ministry of Health, Shanghai, China; Key Laboratory of Molecular Medicine, Ministry of Education, Shanghai, China; Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences/Shanghai Jiao Tong University School of Medicine, Shanghai, China
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7
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Affinity chromatographic purification of human immunoglobulin M from human B lymphocyte cell culture supernatant. Biochem Eng J 2013. [DOI: 10.1016/j.bej.2012.10.003] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
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Gautam S, Loh KC. Human pIgR mimetic peptidic ligand for affinity purification of IgM Part II: Ligand binding characteristics. Sep Purif Technol 2013. [DOI: 10.1016/j.seppur.2012.09.024] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
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9
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Gautam S, Loh KC. Immunoglobulin-M purification — Challenges and perspectives. Biotechnol Adv 2011; 29:840-9. [DOI: 10.1016/j.biotechadv.2011.07.001] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2010] [Revised: 06/28/2011] [Accepted: 06/29/2011] [Indexed: 11/15/2022]
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10
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Gupta G, Surolia A, Sampathkumar SG. Lectin microarrays for glycomic analysis. OMICS-A JOURNAL OF INTEGRATIVE BIOLOGY 2010; 14:419-36. [PMID: 20726799 DOI: 10.1089/omi.2009.0150] [Citation(s) in RCA: 80] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Abstract
Glycomics is the study of comprehensive structural elucidation and characterization of all glycoforms found in nature and their dynamic spatiotemporal changes that are associated with biological processes. Glycocalyx of mammalian cells actively participate in cell-cell, cell-matrix, and cell-pathogen interactions, which impact embryogenesis, growth and development, homeostasis, infection and immunity, signaling, malignancy, and metabolic disorders. Relative to genomics and proteomics, glycomics is just growing out of infancy with great potential in biomedicine for biomarker discovery, diagnosis, and treatment. However, the immense diversity and complexity of glycan structures and their multiple modes of interactions with proteins pose great challenges for development of analytical tools for delineating structure function relationships and understanding glyco-code. Several tools are being developed for glycan profiling based on chromatography, mass spectrometry, glycan microarrays, and glyco-informatics. Lectins, which have long been used in glyco-immunology, printed on a microarray provide a versatile platform for rapid high throughput analysis of glycoforms of biological samples. Herein, we summarize technological advances in lectin microarrays and critically review their impact on glycomics analysis. Challenges remain in terms of expansion to include nonplant derived lectins, standardization for routine clinical use, development of recombinant lectins, and exploration of plant kingdom for discovery of novel lectins.
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Affiliation(s)
- Garima Gupta
- Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India
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Ding J, Bao J, Zhu D, Zhang Y, Wang DC. Crystal structures of a novel anti-HIV mannose-binding lectin from Polygonatum cyrtonema Hua with unique ligand-binding property and super-structure. J Struct Biol 2010; 171:309-17. [DOI: 10.1016/j.jsb.2010.05.009] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2010] [Revised: 04/29/2010] [Accepted: 05/18/2010] [Indexed: 10/19/2022]
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Paiva M, Soeiro M, Barbosa H, Meirelles M, Delain E, Araújo-Jorge T. Glycosylation patterns of human alpha2-macroglobulin: Analysis of lectin binding by electron microscopy. Micron 2010; 41:666-73. [DOI: 10.1016/j.micron.2010.02.015] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2010] [Accepted: 02/26/2010] [Indexed: 10/19/2022]
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Tscheliessnig A, Ong D, Lee J, Pan S, Satianegara G, Schriebl K, Choo A, Jungbauer A. Engineering of a two-step purification strategy for a panel of monoclonal immunoglobulin M directed against undifferentiated human embryonic stem cells. J Chromatogr A 2009; 1216:7851-64. [DOI: 10.1016/j.chroma.2009.09.059] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2009] [Revised: 09/17/2009] [Accepted: 09/23/2009] [Indexed: 11/29/2022]
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Mahassni SH, Klapper DG, Hiskey RG. Purification of a Murine IgM Monoclonal Antibody. Hybridoma (Larchmt) 2009; 28:189-97. [DOI: 10.1089/hyb.2008.0087] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Affiliation(s)
- Sawsan H. Mahassni
- Department of Biochemistry, King Abdulaziz University, Jeddah, Saudi Arabia
| | - David G. Klapper
- Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, North Carolina
| | - Richard G. Hiskey
- Department of Chemistry, University of North Carolina at Chapel Hill, North Carolina
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Kaur A, Singh R, Singh Kamb S, Singh J, J. Kotwal G. In vitro Antiviral Activity of Crinum latifolium
Lectin Against Poxvirus Replication. ACTA ACUST UNITED AC 2008. [DOI: 10.3923/jbs.2008.1236.1240] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
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Alonso E, Gómez L, Madrid JF, Sáez FJ. Identification of mannose moieties in N- and O-linked oligosaccharides of the primordial germ cells of Xenopus embryos. Microsc Res Tech 2006; 69:595-9. [PMID: 16721733 DOI: 10.1002/jemt.20318] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
The presence of mannose (Man) in the glycoconjugates of primordial germ cells (PGCs) of Xenopus embryos was elucidated by lectin histochemistry with Concanavalin A (Con A) and snowdrop (Galanthus nivalis) bulb lectin (GNA), in combination with deglycosylative pretreatments: beta-elimination, which removes O-linked oligosaccharides, and incubation with Peptide N glycosidase F (PNGase F), which removes N-linked glycan chains. In addition, histochemistry with Con A, which binds to Man and glucose (Glc), was also performed after glucose-oxidase incubation, which converts Glc into gluconic acid, and GNA was carried out after acid hydrolysis, which removes terminal sialic acid (NeuAc) moieties. PGCs were analyzed during their migration over the mesentery until the genital ridge, and after colonization of this gonad anlage. The results showed that for both lectins: (1) the PGCs and other surrounding tissue showed a similar binding pattern, and (2) the staining in the PGCs was similar in the developmental stages studied. Labeling with Con A was due to Man, and not to Glc, as shown after incubation with glucose-oxidase, and it was assumed that Man was in N-linked oligosaccharides. However, GNA labeling was mainly due to O-linked oligosaccharides, because the pretreatment of beta-elimination turned cells negative. Moreover, acid hydrolysis pretreatment gave rise to a stronger GNA-staining, suggesting that either Man was also in subterminal position to NeuAc or some Man-containing glycans were unmasked after removal of NeuAc from other oligosaccharide chains.
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Affiliation(s)
- Edurne Alonso
- Departamento de Biología Celular e Histología, Facultad de Ciencia y Tecnología, Universidad del País Vasco/Euskal Herriko Unibertsitatea, B Sarriena s/n, E 48940 Leioa (Vizcaya), Spain
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Yamamoto K, Ito S, Yasukawa F, Konami Y, Matsumoto N. Measurement of the carbohydrate-binding specificity of lectins by a multiplexed bead-based flow cytometric assay. Anal Biochem 2005; 336:28-38. [PMID: 15582555 DOI: 10.1016/j.ab.2004.09.030] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2004] [Indexed: 11/23/2022]
Abstract
Carbohydrate binding underlies many cell recognition events. Here, we describe a multiplexed glyco-bead array method for determining the carbohydrate-binding specificities of plant lectins using a bead-based flow cytometric analysis. N-glycans including high mannose, hybrid, and complex types and O-glycans from glycoproteins were immobilized on multiplexed beads, and the specificities of 13 kinds of sugar chains were monitored within 2 h in a single reaction. This strategy is easy, rapid, reproducible, and suitable for small samples and allows the reliable and simultaneous elucidation of sugar-binding properties under identical conditions.
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Affiliation(s)
- Kazuo Yamamoto
- Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, 277-8562 Chiba, Japan.
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Ooi LSM, Sun SSM, Wang H, Ooi VEC. New mannose-binding lectin isolated from the rhizome of Sarsaparilla Smilax glabra Roxb. (Liliaceae). JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 2004; 52:6091-6095. [PMID: 15453671 DOI: 10.1021/jf030837o] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/24/2023]
Abstract
A new mannose-binding lectin, designated SGM2, was isolated from the rhizome of a Chinese medicinal herb Smilax glabra (also known as sarsaparilla in general) by saline extraction, ammonium sulfate precipitation and fractionation, and affinity chromatography on fetuin- and mannose-agarose. SGM2 is shown to have a molecular mass of 37 kDa on gel filtration and 12.5 kDa on SDS-PAGE, indicating that it is a trimeric protein composed of three identical subunits. When the first 30 amino acid residues at the N-terminal were compared, SGM2 had approximately 40% homology with those of some other monocots. SGM2 had the property of hemagglutinating activity toward rabbit erythrocytes, which could be reversed by mannose and mannose polymers. SGM2 exhibited antiviral activities against both herpes simplex virus type 1 (HSV-1) and respiratory syncytial virus (RSV) with the same EC(50) of 8.1 microM.
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Affiliation(s)
- Linda S M Ooi
- Department of Biology, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong
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Zhu LX, Liu J, Li YC, Kong YY, Staib C, Sutter G, Wang Y, Li GD. Full-length core sequence dependent complex-type glycosylation of hepatitis C virus E2 glycoprotein. World J Gastroenterol 2002; 8:499-504. [PMID: 12046079 PMCID: PMC4656430 DOI: 10.3748/wjg.v8.i3.499] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/05/2001] [Revised: 12/23/2001] [Accepted: 01/23/2002] [Indexed: 02/06/2023] Open
Abstract
AIM To study HCV polyprotein processing is important for the understanding of the natural history of HCV and the design of vaccines against HCV. The purpose of this study is to investigate the affection of context sequences on hepatitis C virus (HCV) E2 processing. METHODS HCV genes of different lengths were expressed and compared in vaccinia virus/T7 system with homologous patient serum S94 and mouse anti-serum M( E2116) raised against E.coli -derived E2 peptide, respectively. Deglycosylation analysis and GNA ( Galanthus nivalus ) lectin binding assay were performed to study the post-translational processing of the expressed products. RESULTS E2 glycoproteins with different molecular weights (-75 kDa and -60 kDa) were detected using S94 and M( E2116), respectively. Deglycosylation analysis showed that this difference was mainly due to different glycosylation. Endo H resistance and its failure to bind to GNA lectin demonstrated that the higher molecular weight form (75 kDa) of E2 was complex-type glycosylated, which was readily recognized by homologous patient serum S94. Expression of complex-type glycosylated E2 could not be detected in all of the core-truncated constructs tested, but readily detected in constructs encoding full-length core sequences. CONCLUSION The upstream conserved full-length core coding sequence was required for the production of E2 glycoproteins carrying complex-type N-glycans which reacted strongly with homologous patient serum and therefore possibly represented more mature forms of E2. As complex-type N-glycans indicated modification by Golgi enzymes, the results suggest that the presence of full-length core might be critical for E1/E2 complex to leave ER. Our data may contribute to a better understanding of the processing of HCV structural proteins as well as HCV morphogenesis.
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Affiliation(s)
- Li-Xin Zhu
- Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China
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Koppel R, Solomon B. IgM detection via selective recognition by mannose-binding protein. JOURNAL OF BIOCHEMICAL AND BIOPHYSICAL METHODS 2001; 49:641-7. [PMID: 11694307 DOI: 10.1016/s0165-022x(01)00225-1] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
Ca(2+)-dependent mannose-binding proteins (MBPs) belong to the family of animal lectins isolated from the liver and serum of rabbits, humans and rodents. They perform in vivo as defense molecules that act as opsonins by enhancing the clearance of mannose-rich pathogens and have been used in vitro for the purification of immunoglobulin M (IgM). In this study, we used MBPs as a sensitive and specific reagent for the detection of IgM due to their high specificity for mannose found only in IgM carbohydrate regions. MBP performed as a sensitive alternative to the usually used anti-IgM, where very low concentrations of IgM should be detected. IgM plays a central role in the initial response of the immune system to the invasion of foreign pathogens, as the early detection of the appearance of pathogenic IgM in biological fluids is of great significance in the diagnosis and treatment of many acute pathological cases. The development of a highly sensitive and reliable assay for the detection of low concentrations of IgM based on covalent binding on epoxy film-coated surfaces and selective recognition of IgM by MBP may be of clinical importance.
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Affiliation(s)
- R Koppel
- Department of Molecular Microbiology and Biotechnology, Faculty of Life Sciences, Tel-Aviv University, Ramat Aviv, Tel-Aviv 69978, Israel
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Boschetti E, Jungbauer A. 15 Separation of antibodies by liquid chromatography. HANDBOOK OF BIOSEPARATIONS 2000. [DOI: 10.1016/s0149-6395(00)80062-8] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
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Palombo G, Verdoliva A, Fassina G. Affinity purification of immunoglobulin M using a novel synthetic ligand. JOURNAL OF CHROMATOGRAPHY. B, BIOMEDICAL SCIENCES AND APPLICATIONS 1998; 715:137-45. [PMID: 9792505 DOI: 10.1016/s0378-4347(98)00104-2] [Citation(s) in RCA: 46] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
While monoclonal antibodies of the G class can be conveniently purified by affinity chromatography using immobilized protein A or G, even on a large scale, scaling up IgM purification still presents several problems, since specific and cost-effective ligands for IgM are not available. A synthetic peptide (TG19318), deduced from the screening of a combinatorial peptide library, was characterized previously by our group for its binding properties for immunoglobulins of the G class and its applicability as a synthetic ligand for polyclonal and monoclonal IgG purification, from sera or cell culture supernatants. In this study, we have examined the ligand recognition properties for IgM, immobilizing the synthetic peptide on different affinity supports and examining its ability to purify IgMs from serum, ascitic fluid and cell culture supernatants. TG19318 affinity columns proved useful for a very convenient one-step purification of monoclonal IgMs directly from crude sources, loading the samples on the columns equilibrated with saline buffers at pH values ranging from 5 to 7, and eluting adsorbed IgM by a buffer change to 0.1 M acetic acid or 0.05-0.1 M sodium bicarbonate, pH 9.0. Antibody purity after affinity purification was very high, close to 85-95%, as determined by densitometric scanning of sodium dodecyl sulfate-polyacrylamide gels of purified fractions, and by gel permeation analysis. Antibody activity was fully recovered after purification, as determined by immunoassays. Column capacity was related to the type of support used for ligand immobilization, and ranged from 2 to 8 mg of IgM/ml of support.
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Affiliation(s)
- G Palombo
- Tecnogen S.C.p.A., Parco Scientifico, Piana di Monte Verna (CE), Italy
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23
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Bertrand O, Cochet S, Cartron JP. Expanded bed chromatography for one-step purification of mannose binding lectin from tulip bulbs using mannose immobilized on DEAE Streamline. J Chromatogr A 1998. [DOI: 10.1016/s0021-9673(98)00575-5] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
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Dam TK, Bachhawat K, Rani PG, Surolia A. Garlic (Allium sativum) lectins bind to high mannose oligosaccharide chains. J Biol Chem 1998; 273:5528-35. [PMID: 9488677 DOI: 10.1074/jbc.273.10.5528] [Citation(s) in RCA: 52] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Two mannose-binding lectins, Allium sativum agglutinin (ASA) I (25 kDa) and ASAIII (48 kDa), from garlic bulbs have been purified by affinity chromatography followed by gel filtration. The subunit structures of these lectins are different, but they display similar sugar specificities. Both ASAI and ASAIII are made up of 12.5- and 11.5-kDa subunits. In addition, a complex (136 kDa) comprising a polypeptide chain of 54 +/- 4 kDa and the subunits of ASAI and ASAIII elutes earlier than these lectins on gel filtration. The 54-kDa subunit is proven to be alliinase, which is known to form a complex with garlic lectins. Constituent subunits of ASAI and ASAIII exhibit the same sequence at their amino termini. ASAI and ASAIII recognize monosaccharides in mannosyl configuration. The potencies of the ligands for ASAs increase in the following order: mannobiose (Manalpha1-3Man) < mannotriose (Manalpha1-6Manalpha1-3Man) approximately mannopentaose << Man9-oligosaccharide. The addition of two GlcNAc residues at the reducing end of mannotriose or mannopentaose enhances their potencies significantly, whereas substitution of both alpha1-3- and alpha1-6-mannosyl residues of mannotriose with GlcNAc at the nonreducing end increases their activity only marginally. The best manno-oligosaccharide ligand is Man9GlcNAc2Asn, which bears several alpha1-2-linked mannose residues. Interaction with glycoproteins suggests that these lectins recognize internal mannose as well as bind to the core pentasaccharide of N-linked glycans even when it is sialylated. The strongest inhibitors are the high mannose-containing glycoproteins, which carry larger glycan chains. Indeed, invertase, which contains 85% of its mannose residues in species larger than Man20GlcNAc, exhibited the highest binding affinity. No other mannose- or mannose/glucose-binding lectin has been shown to display such a specificity.
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Affiliation(s)
- T K Dam
- Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India
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25
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Abstract
Erythrocyte agglutination by lectins from Allium sativum was inhibited only by mannose of the sugars tested. However, asialofetuin was more effective inhibitor of agglutination as compared to mannose. This led to the use of an asialofetuin-silica affinity column to isolate agglutinins of 110 and 25 kDa (ASA110 and ASA25). While ASA25 is a dimeric protein comprising of subunits of 12.5 and 13.0 kDa, ASA110 is a glycoprotein of two identical subunits of 47 kDa. ASA110 revealed to have a high content of aspartic acid, glycine, leucine and serine but low content of cysteine and methionine. It contains 14 residues of neutral sugars in addition to 43 residues of hexosamines per mole of lectin and requires metal ions for its functional conformation. Serological cross-reactions with other species showed some common epitopes of ASA110 and ASA25 present in A. porrum, A. ascalonicum, Narcissus alba, PHA and Con A but not in A. cepa. ASA110 with CHO cells indicated it to be weakly cytotoxic with LD50 of 160 microg/ml.
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Affiliation(s)
- A Gupta
- Department of Molecular Biology and Biochemistry, Guru Nanak Dev University, Amritsar, India
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27
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Debbage PL. A systematic histochemical investigation in mammals of the dense glycocalyx glycosylations common to all cells bordering the interstitial fluid compartment of the brain. Acta Histochem 1996; 98:9-28. [PMID: 9054194 DOI: 10.1016/s0065-1281(96)80046-8] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Abstract
Microanatomical evidence is presented that the intercellular fluid (ICF) compartment of the central nervous tissue is lined entirely and exclusively by heavily glycosylated cells, with glycoconjugates exposed primarily at the apical cell surface, fronting the CSF or blood. On both common ependymal cells and on those specialised to form the choroid plexus epithelium, oligosaccharides coat the cilia and microvilli at the apical surface, and also the smoother lateral and basal cell surfaces. In the ependyma, folded and wrinkled structures seem especially associated with freely exposed carbohydrates. On cerebral endothelial cells, oligosaccharides coat the luminal surface densely and the basal surface lightly. The patterns of carbohydrate distribution thus vary from one cell type to another, but the different cell types all bear essentially the same set of oligosaccharides, variations being due largely to degree of terminal sialylation. Furthermore, the same set of oligosaccharides borders the brain in a broad spectrum of mammals, including pouched and placental mammals. In both epithelia and endothelia, the lectin binding sites visualised in fixed and embedded preparations were shown to be exposed likewise at the cell surfaces in unfixed tissues and so able to bind molecules present in the fluid (CSF or blood) bathing the cells in vivo. This phylogenetically ancient enclosure of the ICF compartment in a "ring of sugars" is suggested to relate to regulation of the central neuronal microenvironment.
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Affiliation(s)
- P L Debbage
- Anatomy Department, Ludwig-Maximilians-University, Munich, Germany
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28
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Hester G, Kaku H, Goldstein IJ, Wright CS. Structure of mannose-specific snowdrop (Galanthus nivalis) lectin is representative of a new plant lectin family. NATURE STRUCTURAL BIOLOGY 1995; 2:472-9. [PMID: 7664110 DOI: 10.1038/nsb0695-472] [Citation(s) in RCA: 155] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
Abstract
Tetrameric Galanthus nivalis agglutinin (50,000 M(r)) belongs to a super-family of alpha-D-mannose-specific plant bulb lectins known to be potent inhibitors of retroviruses. The 2.3 A crystal structure of this lectin complexed with methyl alpha-D-mannose reveals a novel three-fold symmetric beta-sheet polypeptide fold. Three antiparallel four-stranded beta-sheets, each with a conserved mannose-binding site, are arranged as a 12-stranded beta-barrel. The tetramer displays 222 symmetry. Pairs of monomers form stable dimers through C-terminal strand exchange. The so formed hybrid beta-sheets are the sites for high affinity mannose binding in the dimer interface. Occupancy observed at corresponding sites in other beta-sheets suggests a potential for twelve sites per tetramer.
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Affiliation(s)
- G Hester
- Department of Biochemistry, Virginia Commonwealth University, Richmond 23298, USA
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29
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Bartoszewicz ZP, Noronha AB, Fujita N, Sato S, Bö L, Trapp BD, Quarles RH. Abnormal expression and glycosylation of the large and small isoforms of myelin-associated glycoprotein in dysmyelinating quaking mutants. J Neurosci Res 1995; 41:27-38. [PMID: 7545761 DOI: 10.1002/jnr.490410105] [Citation(s) in RCA: 21] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
The relative expression of large (L) and small (S) isoforms of the myelin-associated glycoprotein (MAG) and their glycosylation were compared in developing spinal cord of quaking and control mice. Using antisera specific for L- and S-MAG, respectively, it was shown that S-MAG is the principal isoform in quaking mice at all ages between 13 and 72 days, although L-MAG was just detectable by western blotting at the early ages. Both L- and S-MAG have higher apparent molecular weights in quaking mice than in controls. Experiments involving lectin binding and glycosidase treatment demonstrated that the higher molecular weight of MAG in the quaking mutant was due to a higher content of N-acetylneuraminic acid residues linked alpha 2-3 to galactose as well as to more branching of oligosaccharide moieties indicated by a higher content of subterminal galactose residues. The total sialic acid measured by HPAE-chromatography in purified quaking MAG was 40% higher than in control MAG. By contrast, quaking MAG contained less of the adhesion-related, HNK-1 carbohydrate epitope. Another difference was that a lower molecular weight form of MAG with predominantly high mannose oligosaccharides was prominent in young quaking mice, but not in controls. The abnormalities of MAG expression related to splicing of its mRNA and glycosylation may contribute to the myelin pathology in quaking mutants.
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Affiliation(s)
- Z P Bartoszewicz
- Myelin and Brain Development Section, NINDS, National Institutes of Health, Bethesda, Maryland, USA
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30
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Novales-Li P. Purification of murine immunoglobulin M from spent tissue culture supernatant by one-step gel filtration chromatography procedure. Biomed Chromatogr 1995; 9:42-7. [PMID: 7734933 DOI: 10.1002/bmc.1130090109] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
Abstract
Optimal purification of immunoglobulin M (IgM) grown as spent tissue culture supernatant can be achieved by a straightforward size-exclusion chromatography procedure. Preparative isolation of IgM was achieved by precipitation with saturated ammonium sulphate to achieve a 45% solution. IgM was then purified by gel filtration chromatography with a solution of 100 mM Tris + 150 mM NaCl at pH 8. Denaturing electrophoresis and Western immunoblotting revealed two prominent bands at 80 and 25 kDa, indicative of the heavy and light chains of IgM respectively. The specific immunoreactivity of this IgM was assessed by a modified enzyme antigen capture assay. In contrast to affinity and ion-exchange chromatographies, gel filtration allows retention of IgM immunoreactivity.
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Affiliation(s)
- P Novales-Li
- Department of Pharmacology, University of Oxford, UK
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31
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Smeets K, Van Damme EJ, Peumans WJ. Comparative study of the post-translational processing of the mannose-binding lectins in the bulbs of garlic (Allium sativum L.) and ramsons (Allium ursinum L.). Glycoconj J 1994; 11:309-20. [PMID: 7873927 DOI: 10.1007/bf00731204] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
The biosynthesis and processing of the homodimeric and heterodimeric lectins from the bulbs of garlic (Allium sativum) and ramsons (wild garlic; Allium ursinum) were studied using pulse and pulse-chase labelling experiments on developing bulbs. By combining the results of the in vivo biosynthesis studies and the cDNA cloning of the respective lectins, the sequence of events leading from the primary translation products into the mature lectin polypeptides could be reconstructed. From this it is demonstrated that garlic and ramsons use different schemes of post-translational modifications in order to synthesize apparently similar lectins from totally different precursors. Both the homomeric garlic lectin (ASAII) and its homologue in ramsons (AUAII) are synthesized on the endoplasmic reticulum (ER) as nonglycosylated 13.5 kDa precursors, which, after their transport out of the ER are converted into the mature 12.0 kDa lectin polypeptides by the cleavage of a C-terminal peptide. The heterodimeric garlic lectin ASAI is synthesized on the ER as a single glycosylated precursor of 38 kDa, which after its transport out of the ER undergoes a complex processing which gives rise to two mature lectin subunits of 11.5 and 12.5 kDa. In contrast, both subunits of the heterodimeric ramsons lectin AUAI are synthesized separately on the ER as glycosylated precursors, which after their transport out of the ER are deglycosylated and further processed into the mature lectin polypeptides by the cleavage of a C-terminal peptide.
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Affiliation(s)
- K Smeets
- Laboratory for Phytopathology and Plant Protection, Katholieke Universiteit Leuven, Belgium
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32
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Van Damme JM, Smeets K, Torrekens S, Van Leuven F, Peumans WJ. Characterization and molecular cloning of mannose-binding lectins from the Orchidaceae species Listera ovata, Epipactis helleborine and Cymbidium hybrid. EUROPEAN JOURNAL OF BIOCHEMISTRY 1994; 221:769-77. [PMID: 8174556 DOI: 10.1111/j.1432-1033.1994.tb18790.x] [Citation(s) in RCA: 56] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
Mannose-binding lectins were purified from the leaves of three Orchidaceae species, namely Listera ovata (twayblade), Epipactis helleborine (broad-leaved helleborine) and Cymbidium hybrid, using affinity chromatography on Mannose - Sepharose-4B. Apparently, the Orchidaceae lectins are dimeric proteins composed of lectin subunits of 12-13 kDa. All of the isolated lectins exhibit exclusive specificity towards mannose. A cDNA library constructed from poly(A) rich RNA isolated from leaves of L. ovata was screened for cDNA clones encoding the lectin using colony hybridization. Since N-terminal sequence analysis of the twayblade lectin revealed some sequence similarity to the previously cloned mannose-binding lectin Hippeastrum hybrid (amaryllis) ovaries, the amaryllis lectin cDNA clone was used as a probe to screen the L. ovata library. Subsequently, the cDNA clone encoding the L. ovata lectin was used to screen the cDNA libraries from the taxonomically related orchid species Cymbidium hybrid and E. helleborine. Sequence analysis of the lectin cDNA clones from different Orchidaceae species revealed approximately 50% sequence similarity both at the nucleotide and amino acid level. The Orchidaceae lectins are apparently translated from mRNAs consisting of approximately 800 nucleotides. The primary translation products are preproproteins which are converted into the mature lectins following post-translational modifications. Southern blot analysis of genomic DNA has shown that the lectins are most probably encoded by a family of closely related genes which is in good agreement with the sequence heterogeneity found between different lectin cDNA clones of one species.
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Affiliation(s)
- J M Van Damme
- Laboratory for Phytopathology and Plant Protection, Katholieke Universiteit Leuven, Belgium
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33
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Van Damme EJ, Smeets K, Engelborghs I, Aelbers H, Balzarini J, Pusztai A, van Leuven F, Goldstein IJ, Peumans WJ. Cloning and characterization of the lectin cDNA clones from onion, shallot and leek. PLANT MOLECULAR BIOLOGY 1993; 23:365-376. [PMID: 8106012 DOI: 10.1007/bf00029011] [Citation(s) in RCA: 28] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/22/2023]
Abstract
Characterization of the lectins from onion (Allium cepa), shallot (A. ascalonicum) and leek (A. porrum) has shown that these lectins differ from previously isolated Alliaceae lectins not only in their molecular structure but also in their ability to inhibit retrovirus infection of target cells. cDNA libraries constructed from poly(A)-rich RNA isolated from young shoots of onion, shallot and leek were screened for lectin cDNA clones using colony hybridization. Sequence analysis of the lectin cDNA clones from these three species revealed a high degree of sequence similarity both at the nucleotide and at the amino acid level. Apparently the onion, shallot and leek lectins are translated from mRNAs of ca. 800 nucleotides. The primary translation products are preproproteins (ca. 19 kDa) which are converted into the mature lectin polypeptides (12.5-13 kDa) after post-translational modifications. Southern blot analysis of genomic DNA has shown that the lectins are most probably encoded by a family of closely related genes which is in good agreement with the sequence heterogeneity found between different lectin cDNA clones of one species.
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Affiliation(s)
- E J Van Damme
- Laboratory for Phytopathology and Plant Protection, Katholieke Universiteit Leuven, Belgium
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34
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Van Damme JM, Smeets K, Torrekens S, Van Leuven F, Peumans WJ. The mannose-specific lectins from ramsons (Allium ursinum L.) are encoded by three sets of genes. EUROPEAN JOURNAL OF BIOCHEMISTRY 1993; 217:123-9. [PMID: 8223549 DOI: 10.1111/j.1432-1033.1993.tb18226.x] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
Lectin cDNA clones encoding the two mannose-binding lectins from ramsons (allium ursinum L.) bulbs, AUAI and AUAII (AUA, Allium ursinum agglutinin), were isolated and characterized. Sequence comparison of the different cDNA clones isolated revealed three types of lectin clones called LECAUAG0, LECAUAG1 and LECAUAG2, which besides the obvious differences in their sequences also differ from each other in the number of potential glycosylation sites within the C-terminal peptide of the lectin precursor. In vivo biosynthesis studies of the ramson lectins have shown that glycosylated lectin precursors occur in the organelle fraction of radioactively labeled ramson bulbs. Despite the similarities between the A. ursinum and the A. sativum (garlic) lectins at the protein level, molecular cloning of the two ramson lectins has shown that the lectin genes in A. ursinum are organized differently. Whereas in A. sativum the lectin polypeptides of the heterodimeric ASAI are encoded by one large precursor, those of the heterodimeric AUAI lectin are derived from two different precursors. These results are confirmed by Northern blot hybridization of A. ursinum RNA which, after hybridization with a labeled lectin cDNA, reveals only one band of 800 nucleotides in contrast to A. sativum RNA which yields two bands of 1400 and 800 nucleotides. Furthermore it is shown that the two mannose-binding lectins are differentially expressed.
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Affiliation(s)
- J M Van Damme
- Laboratory for Phytopathology and Plant Protection, Catholic University of Leuven, Belgium
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35
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Takagaki M, Knibbs RN, Roth J, Goldstein IJ. Monoclonal antibodies that recognize the trisaccharide epitope Gal alpha 1-3Gal beta 1-4GlcNAc present on Ehrlich tumor cell membrane glycoproteins. HISTOCHEMISTRY 1993; 100:139-47. [PMID: 7503969 DOI: 10.1007/bf00572900] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
Monoclonal antibodies were prepared against the trisaccharide Gal alpha 1-3Gal beta 1-4GlcNAc, a sequence which occurs on the surface of Ehrlich ascites tumor cells as well as in thyroglobulin, laminin and a variety of other proteins. This was accomplished by immunizing BALB/c mice with the fraction of Ehrlich cell membrane glycoproteins obtained by affinity chromatography on a Griffonia simplicifolia I (GS I) column which selectively binds alpha-D-galactosyl-terminated structures. Detection of Gal alpha 1-3Gal beta 1-4GlcNAc-specific antibodies was accomplished by employing glycoproteins containing the trisaccharide sequence; fusion with spleen cells from an immunized mouse was accomplished in the presence of polyethylene glycol (PEG1500). An enzyme-linked immunosorbent assay (ELISA) system was used to identify two clones (2.10G and 6.8E), which recognized the desired trisaccharide conjugate. These clones also recognized a thyroglobulin fraction isolated by GS I affinity chromatography and murine laminin, both of which possess the Gal alpha 1-3Gal beta 1-4GlcNAc sequence. Inhibition of antibody-trisaccharide reactivity, examined employing an ELISA assay, revealed that two trisaccharides, Gal alpha 1-3Gal beta 1-4GlcNAc/Glc, were the best inhibitory haptens; Gal beta 1-4GlcNAc (LacNAc), Gal alpha 1-3Gal and Gal beta 1-4Glc (lactose) were poor inhibitors. Indirect immunofluorescence staining of unfixed Ehrlich cells using the monoclonal antibody at 4 degrees C revealed fluorescence over the entire cell surface. Indirect immunogold labeling of semithin and ultrathin sections of aldehyde fixed and Lowicryl K4M-embedded Ehrlich cells resulted in specific labeling of the cell surface and internal structure.(ABSTRACT TRUNCATED AT 250 WORDS)
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Affiliation(s)
- M Takagaki
- Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-0624
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36
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Gilljam G. Envelope glycoproteins of HIV-1, HIV-2, and SIV purified with Galanthus nivalis agglutinin induce strong immune responses. AIDS Res Hum Retroviruses 1993; 9:431-8. [PMID: 8318269 DOI: 10.1089/aid.1993.9.431] [Citation(s) in RCA: 59] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023] Open
Abstract
Lectin affinity chromatography was used to purify in a single step the envelope glycoproteins of HIV-1, HIV-2, and SIV. Envelope glycoproteins carry the major determinants essential for protection by the humoral immune response. The purification of these proteins has previously been a laborious procedure. The glycoproteins were purified by a one-step procedure to a high level of purity by using Galanthus nivalis agglutinin (GNA). The purified glycoprotein had CD4-binding and antigenic reactivities. Strong immune responses to envelope proteins and peptides were seen in mice and primates after immunization with these preparations.
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Affiliation(s)
- G Gilljam
- Department of Virology, National Bacteriological Laboratory, Stockholm, Sweden
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37
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Van Leuven F, Torrekens S, Van Damme E, Peumans W, Van den Berghe H. Mannose-specific lectins bind alpha-2-macroglobulin and an unknown protein from human plasma. Protein Sci 1993; 2:255-63. [PMID: 7680268 PMCID: PMC2142346 DOI: 10.1002/pro.5560020214] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
Abstract
GNA, the mannose-specific lectin from Galanthus nivalis was confirmed to bind alpha-2-macroglobulin (A2M) but another protein was copurified with A2M from total human plasma. A total of 23 other lectins with diverse specificities were tested for reaction with human A2M and with three other members of the A2M family. NPA, a mannose-specific lectin isolated from Narcissus pseudonarcissus bulbs, and RSA, the Rhizoctonia solani agglutinin, were selected for further testing. For isolation of A2M, immobilized NPA was superior to GNA because its binding capacity was an order of magnitude higher. The specificity of these lectins must be very similar however, because the same unknown plasma protein was also bound by NPA. A2M and the unknown protein must share a unique mannose carbohydrate structure not present in any other human plasma protein. The copurified protein subunit size of 185 kDa is very similar to that of A2M, but the native molecular mass of 350 kDa indicated a noncovalent homodimer structure. Together with the acid isoelectric point this is not typical for any known plasma protein nor for any unidentified spot on the two-dimensional map of human plasma proteins. No immunological reaction with available antisera was evident. A specific antiserum raised to the unknown protein demonstrated its presence in all human plasma samples examined. The N-terminal residue was blocked, whereas internal protein sequences obtained after CNBr fragmentation and proteolysis were not homologous to any known protein sequence. These data demonstrate that this protein is unknown and not a proteinase inhibitor of the A2M family.
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Affiliation(s)
- F Van Leuven
- Center of Human Genetics, Katholieke Universiteit van Leuven, Belgium
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38
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Burd RS, Raymond CS, Ratz CA, Dunn DL. A rapid procedure for purifying IgM monoclonal antibodies from murine ascites using a DEAE-disk. Hybridoma (Larchmt) 1993; 12:135-42. [PMID: 8454300 DOI: 10.1089/hyb.1993.12.135] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023]
Abstract
A method for purifying IgM monoclonal antibodies (mAbs) from murine ascites using a DEAE-disk is described. After ammonium sulfate precipitation, ascites proteins are redissolved and loaded onto a DEAE-disk. MAb then is eluted from the disk using a stepwise NaCl gradient. IgM mAb produced by this procedure was > 95% pure as assessed by reducing SDS-PAGE analysis and was free of significant IgG contamination as determined by double radial immunodiffusion analysis. Yield of IgM mAb was approximately 2% of total ascites protein and approximately 10% of the amount of IgM contained in crude ascites. MAb retained immunoreactivity as assessed by ELISA, and the affinity index was evaluated by thiocyanate elution and remained unchanged. This two step technique for purifying IgM mAb from murine ascites is rapid, simple, and yields mAb of sufficient purity and immunoreactivity for the majority of mAb applications.
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Affiliation(s)
- R S Burd
- Department of Surgery, University of Minnesota, Minneapolis 55455
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39
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Mahmood N, Hay AJ. An ELISA utilizing immobilised snowdrop lectin GNA for the detection of envelope glycoproteins of HIV and SIV. J Immunol Methods 1992; 151:9-13. [PMID: 1629622 DOI: 10.1016/0022-1759(92)90101-x] [Citation(s) in RCA: 42] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
A simple, reliable ELISA for the quantitative detection of the envelope glycoproteins of both HIV and SIV is described. It incorporates the snowdrop lectin GNA to capture the glycoprotein antigens and combines the high selectivity of GNA binding with its broad reactivity with the glycoproteins of HIV-1, HIV-2 and SIV.
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Affiliation(s)
- N Mahmood
- MRC Collaborative Centre, Mill Hill, London, UK
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40
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Vanderpuye OA, Labarrere CA, McIntyre JA. Glycosylation of membrane cofactor protein (CD46) in human trophoblast, kidney and platelets. BIOCHIMICA ET BIOPHYSICA ACTA 1992; 1121:301-8. [PMID: 1627608 DOI: 10.1016/0167-4838(92)90161-6] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
Many cell surface glycoconjugates are differentiation markers and are involved in cell-cell and intermolecular interactions in development, immunity and cancer. Membrane cofactor protein (MCP) comprises structurally related 65 and 55 kDa glycoproteins that bear O- and N-linked glycans. MCP prevents amplification of autologous complement action on human cells. We used immunoblotting with MCP-specific monoclonal antibody TRA-2-10 to determine lectin-binding properties and glycosidase sensitivities of MCP in a study of cell-specific variation in glycosylation of this protein. The results showed that N-linked glycans on placental syncytiotrophoblast and cytotrophoblast, kidney and platelet MCP are similar in binding to concanavalin A and Lens culinaris lectins, but are not bound by leucophytohemagglutinin. Lectin binding prior to and after neuraminidase digestion indicates that MCP from these sources is highly sialylated. 65 kDa MCP was confirmed to contain more O-linked glycans than 55 kDa MCP. A fraction of platelet 65 kDa MCP is distinct, however, in bearing peripheral fucose residues. Syncytiotrophoblast is unique in containing a 110 kDa form of MCP in non-reducing SDS-PAGE that resembles 65 kDa MCP in glycosylation. Chorion laeve MCP in 4 of 8 preparations was unusually heterogeneous and differed from syncytiotrophoblast MCP after neuraminidase digestion in the forms bound to peanut agglutinin and WGA. The results indicated for the first time, differences in O-linked glycosylation of MCP in chorion laeve cytotrophoblast relative to syncytiotrophoblast, platelet and kidney MCP. We conclude that structures of MCP glycans can differ between trophoblasts and other cell types.
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Affiliation(s)
- O A Vanderpuye
- Center for Reproduction and Transplantation Immunology, Methodist Hospital of Indiana, Indianapolis 46202
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Kaku H, Goldstein IJ, Van Damme EJ, Peumans WJ. New mannose-specific lectins from garlic (Allium sativum) and ramsons (Allium ursinum) bulbs. Carbohydr Res 1992; 229:347-53. [PMID: 1394291 DOI: 10.1016/s0008-6215(00)90580-9] [Citation(s) in RCA: 44] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
Two new mannose-binding lectins were isolated from garlic (Allium sativum, ASA) and ramsons (Allium ursinum, AUA) bulbs, of the family Alliaceae, by affinity chromatography on immobilized mannose. The carbohydrate-binding specificity of these two lectins was studied by quantitative precipitation and hapten-inhibition assay. ASA reacted strongly with a synthetic linear (1----3)-alpha-D-mannan and S. cerevisiae mannan, weakly with a synthetic (1----6)-alpha-D-mannan, and failed to precipitate with galactomannans from T. gropengiesseri and T. lactis-condensi, a linear mannopentaose, and murine IgM. On the other hand, AUA gave a strong reaction of precipitation with murine IgM, and good reactions with S. cerevisiae mannan and both synthetic linear mannans, suggesting that the two lectins have somewhat different binding specificities for alpha-D-mannosyl units. Of the saccharides tested as inhibitors of precipitation, those with alpha-(1----3)-linked mannosyl units were the best inhibitors of ASA, the alpha-(1----2)-, alpha-(1----4)-, and alpha-(1----6)-linked mannobioses and biosides having less than one eighth the affinity of the alpha-(1----3)-linked compounds. The N-terminal amino acid sequence of ASA exhibits 79% homology with that of AUA, and moderately high homology (53%) with that of snowdrop bulb lectin, also an alpha-D-mannosyl-binding lectin.
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Affiliation(s)
- H Kaku
- Department of Biological Chemistry, University of Michigan, Ann Arbor 48109
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Van Damme EJ, Kaku H, Perini F, Goldstein IJ, Peeters B, Yagi F, Decock B, Peumans WJ. Biosynthesis, primary structure and molecular cloning of snowdrop (Galanthus nivalis L.) lectin. EUROPEAN JOURNAL OF BIOCHEMISTRY 1991; 202:23-30. [PMID: 1718752 DOI: 10.1111/j.1432-1033.1991.tb16339.x] [Citation(s) in RCA: 98] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
Poly(A)-rich RNA isolated from ripening ovaries of snowdrop (Galanthus nivalis L.) yielded a single 17-kDa lectin polypeptide upon translation in a wheat-germ cell-free system. This lectin was purified by affinity chromatography. Translation of the same RNA in Xenopus leavis oocytes revealed a lectin polypeptide which was about 2 kDa smaller than the in vitro synthesized precursor, suggesting that the oocyte system had removed a 2-kDa signal peptide. A second post-translational processing step was likely to be involved since both the in vivo precursor and the Xenopus translation products were about 2 kDa larger than the mature lectin polypeptide. This hypothesis was confirmed by the structural analysis of the amino acid sequence of the mature protein and the cloned mRNA. Edman degradation and carboxypeptidase Y digestion of the mature protein, and structural analysis of the peptides obtained after chemical cleavage and modification, allowed determination of the complete 105 amino acid sequence of the snowdrop lectin polypeptide. Comparison of this sequence with the deduced amino acid sequence of a lectin cDNA clone revealed that besides the mature lectin polypeptide, the lectin mRNA also encoded a 23 amino acid signal-sequence and a C-terminal extension of 29 amino acids, which confirms the results from in vitro translation experiments.
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Affiliation(s)
- E J Van Damme
- Laboratory for Phytopathology and Plant Protection, Catholic University of Leuven, Belgium
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Knutson VP, Buck RA, Moreno RM. Purification of a murine monoclonal antibody of the IgM class. J Immunol Methods 1991; 136:151-7. [PMID: 1999647 DOI: 10.1016/0022-1759(91)90001-v] [Citation(s) in RCA: 24] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
A simple method is described for the purification of murine monoclonal antibodies of the IgM class. Ascites fluid was subjected to ammonium sulfate precipitation. The precipitate was redissolved and dialyzed and subsequently subjected to gel filtration chromatography on Ultrogel AcA22 and finally ion exchange chromatography on DEAE-Sepharose. The purity of the antibody was assessed by reducing SDS gel electrophoresis, and estimated to be greater than 85% pure after gel filtration, and greater than 95% pure after the ion exchange chromatography. The immuno-activity of the antibody was assessed throughout the purification scheme by the ability of the antibody to bind to immunogen immobilized to nitrocellulose. Approximately 50% antibody activity was recovered from this purification scheme after gel filtration, but only 10% of the initial activity could be recovered after ion exchange chromatography. This result with ion exchange chromatography underscores the lability of IgM antibodies to immobilization. Therefore, if the recovery of immunoreactive IgM antibody is a goal, purification schemes of IgM antibodies should avoid ion exchange or affinity chromatography.
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Affiliation(s)
- V P Knutson
- University of Texas Medical School, Houston, Department of Pharmacology 77225
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Van Damme EJ, Peumans WJ. Developmental changes and tissue distribution of lectin inGalanthus nivalis L. andNarcissus cv. Carlton. PLANTA 1990; 182:605-609. [PMID: 24197383 DOI: 10.1007/bf02341038] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Accepted: 06/12/1990] [Indexed: 06/02/2023]
Abstract
A sensitive immunosorbent assay was developed to quantify the lectin in different tissues ofGalanthus nivalis (snowdrop) andNarcissus cv. Carlton (daffodil) and follow the distribution of the lectin during the life cycle of the plants. The lectin in snowdrops and daffodils occurs in almost all plant tissues. Moreover, in many tissues the lectin is the most prominent protein. High lectin concentrations are found in the bulb where the lectin accounts for up to 15% of the total protein during the resting period. However, as the shoot grows and the plant turns on to flowering the lectin content rapidly decreases. Soon after flowering the lectin accumulates in the new bulb units. Whereas in daffodil the lectin concentration in the aerial plant parts is about one order of magnitude lower than in the bulb, lectin concentrations in the upper parts of snowdrop are similar to those in the bulb. The lectin in the former tissues is already present before the sprout emerges. As the shoot starts to grow lectin concentrations in leaves, stems and flower parts gradually decrease so that at flowering time virtually all lectin has disappeared from the aerial parts. The highest lectin concentrations are found in the ovary and increase, initially, as the sprout emerges from the bulb.
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Affiliation(s)
- E J Van Damme
- Laboratorium voor Fytopathologie en Plantenbescherming, Katholieke Universiteit Leuven, Willem de Croylaan 42, B-3030, Leuven, Belgium
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Kaku H, Van Damme EJ, Peumans WJ, Goldstein IJ. Carbohydrate-binding specificity of the daffodil (Narcissus pseudonarcissus) and amaryllis (Hippeastrum hybr.) bulb lectins. Arch Biochem Biophys 1990; 279:298-304. [PMID: 2350177 DOI: 10.1016/0003-9861(90)90495-k] [Citation(s) in RCA: 86] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
The carbohydrate binding specificity of the daffodil (Narcissus pseudonarcissus; NPA) and amaryllis (Hippeastrum hybr.; HHA) lectins, isolated from extracts of their bulbs by affinity chromatography on immobilized mannose, was studied by quantitative precipitation, sugar hapten inhibition, and affinity chromatography on the immobilized lectins. These lectins gave strong precipitation reactions with several yeast mannans, but did not precipitate with alpha-D-glucans (e.g., dextrans and glycogen). Interestingly, both lectins reacted strongly with yeast galactomannans having multiple nonreducing terminal alpha-D-galactosyl groups, a synthetic linear alpha-1,6-mannan, and an alpha-1,3-mannan (DP = 30). Treatment of the linear alpha-1,3-mannan with periodate, resulting in oxidation of the terminal, nonreducing mannosyl group, did not reduce its reactivity with NPA or HHA. Taken together, these observations suggest that NPA and HHA react not only with terminal but also with internal alpha-D-mannosyl residues. Sugar hapten inhibition studies showed these lectins to possess the greatest specific activity for alpha-D-mannosyl units whereas D-Glc and D-GlcNAc did not inhibit either lectin precipitation system. Of the oligosaccharides tested, the best inhibitor of NPA interaction was alpha-1,6-linked mannotriose, which was twice as good an inhibitor as Man alpha 1,6Man alpha-O-Me and 10 times better than methyl alpha-D-mannoside. On the other hand, oligosaccharides containing either 1,3- or 1,6-linked mannosyl units were good inhibitors of the HHA-mannan precipitation system (6- to 20-fold more active than D-Man). These results indicate that both lectins appear to possess an extended binding site(s) complementary to at least three 1,6-linked alpha-mannosyl units. Various glycosylasparagine glycopeptides which contain alpha-1,6-Man units were retarded on the immobilized NPA column. On the other hand, those containing either alpha-1,3- or alpha-1,6-mannosyl residues were retarded on the immobilized HHA column; Man5-GlcNAc2-Asn [containing two Man alpha 1,3(Man alpha 1,6) units] bound to the HHA column. On the contrary, glycopeptides with hybrid type glycan chains were not retarded on either column. These two new lectins which differ in their fine sugar binding specificity from each other, and also from the snowdrop lectin, should prove to be useful probes for the detection and preliminary characterization of glycoconjugates on cell surfaces and in solution.
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Affiliation(s)
- H Kaku
- Department of Biological Chemistry, University of Michigan, Ann Arbor 48109
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