Published online Jun 28, 2024. doi: 10.3748/wjg.v30.i24.3044
Revised: May 10, 2024
Accepted: May 27, 2024
Published online: June 28, 2024
Processing time: 98 Days and 0.8 Hours
We comment here on the article by Stefanolo et al entitled “Effect of Aspergillus niger prolyl endopeptidase in patients with celiac disease on a long-term gluten-free diet”, published in the World Journal of Gastroenterology. Celiac disease is a well-recognized systemic autoimmune disorder. In genetically susceptible people, the most evident damage is located in the small intestine, and is caused and worsened by the ingestion of gluten. For that reason, celiac patients adopt a gluten-free diet (GFD), but it has some limitations, and it does not prevent re-exposure to gluten. Research aims to develop adjuvant therapies, and one of the most studied alternatives is supplementation with Aspergillus niger prolyl endopeptidase protease (AN-PEP), which is able to degrade gluten in the stomach, reducing its concentration in the small intestine. The study found a high adherence to the GFD, but did not address AN-PEP as a gluten immunogenic peptide reducer, as it was only tested in patients following a GFD and not in gluten-exposing conditions. This study opens up new research perspectives in this area and shows that further study is needed to clarify the points that are still in doubt.
Core Tip: Involuntary or voluntary exposure to gluten can lead to severe and persistent symptoms in celiac patients on long-term gluten-free diets. Aspergillus niger prolyl endopeptidase metabolizes gluten before it reaches the small intestine, reducing the celiac disease-specific symptoms. That was demonstrated by the decrease in the concentration of gluten immunogenic peptide.
- Citation: Colella M, Cafiero C, Palmirotta R. Aspergillus niger prolyl endopeptidase in celiac disease. World J Gastroenterol 2024; 30(24): 3044-3047
- URL: https://www.wjgnet.com/1007-9327/full/v30/i24/3044.htm
- DOI: https://dx.doi.org/10.3748/wjg.v30.i24.3044
Celiac disease (CeD) is a systemic autoimmune disorder that occurs in patients with a genetic susceptibility. The introduction of gluten into the diet of one of these individuals results in the activation of specific T-cells that recognize the specific epitopes, characterizing the disease. This response can damage the gastrointestinal tissues and cause the characteristic symptoms. Most of the damage occurs in the small intestine. and it is caused by involuntary or voluntary exposure to gluten. A gluten-free diet (GFD) is recommended in these patients, even if it is a difficult, time-consuming and expensive process, but it represents the only way to avoid the appearance of symptoms. Indeed, after exposure to gluten, these patients can experience enteropathy and persistent symptoms due to intestinal mucosal damage. For this reason, there is a need for novel therapies that can be which can be integrated into a GFD. Mammalian enzymes cannot degrade gluten because of its proline and glutamine-rich sequences. The result is the exposure of the intestinal mucosa is exposed to gluten immunogenic peptides (GIPs), which is necessary in CeD to reactivate gluten T-cells and the disease. Several probiotics and microbial proteases have been studied for the purpose of degrading more gluten, but the most efficient at this time is Aspergillus niger prolyl endopeptidase (AN-PEP)[1-3].
Under optimal conditions Aspergillus niger prolyl endoprotease efficiently cleaves proteins. AN-PEP is active at a low pH, is resistant to pepsin, sand is active in the stomach, reducing the GIP before reaching the small intestine. This oligopeptidase digests gluten into nontoxic fragments, and its use in production of gluten-free food has been proposed recently[3]. For example, in late 2018, Vega K proposed the GRAS claim for a recombinant strain of Aspergillus niger that has shown a high efficacy for the production of gluten-reduced foods, and currently is also used in beer production (https://www.fda.gov/media/134876/download).
A study by Stefanolo et al[4] examined the effects of AN-PEP in celiac patients following a GFD and the prevention of CeD symptoms during involuntary exposure to gluten. This was an exploratory, placebo-controlled trial. The first phase was a 4-wk run-in period followed by a 4-wk period during which patients were randomized to test (two AN-PEP capsules per meal) and control (placebo) arms. To check the effects of the trial, the authors assayed the stool GIP, CeD-specific serology, and assessed the celiac symptom index (CSI) and quality of life (QoL) using the SF-36 questionnaire. Thirty-seven patients were randomized and 628/640 stool samples were collected. Results show that GIP in 65.6% of total samples (GIP < 0.08 μg/g), was > 0.32 μg/g in 0.5%, which potentially causes mucosal damage, and lower than the run-in period in 44.7%. This was a pilot, double-blind, prospective, randomized, placebo-controlled study. The aim was to explore whether the oral administration of AN-PEP can be used to prevent the effects of exposure to gluten in CeD patients. However the small number of participants (only 37 patients participated in the study) make it inconclusive. The results require confirmation by large cohort studies.
Adult patients with CeD on a long-term GFD (more than 2 years) were enrolled between October 2020 and July 2022. The criteria for enrolling patients were: Histological and serological diagnosis of CeD, GFD for more than 2 years, at least one bowel movement per day, ability to provide serum samples, collection of stool samples every Tuesday and Friday, and the ability to answer questionnaires. The exclusion criteria were: Metabolic disorders, refractory CeD, use of drugs that increase stool GIP excretion (laxatives, probiotics). A total of 37 patients were enrolled in this study, as they satisfied all the criteria. The clinical trial was organized in two different phases. In the first phase, all patients followed a run-in period, which was important to stabilize dietary adherence. In the second phase, they were randomized in two arms. Seventeen patients were treated with GliadinX, 2 cp/meal (6 cp/d); 325 mg/cp, made up of 70% AN-PEP (obtained from a genetically modified Aspergillus niger), 30% maltodextrin, and citric acid. The remaining 20 patients were in the control group, which ingested a placebo (maltodextrin, citric acid, and microcrystalline cellulose).
Every patient had to collect stool samples every Tuesday and Friday (both in the run-in and the second phase) for subsequent processing with an ELISA kit (iVYLISA GIP-Stool; Biomedal S.L., Sevilla, Spain) to quantify the presence of GIP. A total of 640 stool samples were expected but only 628 arrived and were processed in the laboratory following current protocols[5-7]. The results show that 65.6% of all samples (both run-in and treatment period) had undetectable GIP (< 0.08 μg/g), indicating the effectiveness of GFD, and none of these patients had a GIP > 0.64 μg/g. In the AN-PEP arm, GIP. Decreased in more than 50% of the samples, indicating the effectiveness of AN-PEP. The statistical analysis included comparison of the two arms with the Mann-Whitney or Wilcoxon test (comparison between the beginning and the end of treatment), proportion (χ² test), and comparison of the proportion (McNemar test). The analysis found no statistically significant differences between the results that were compared. However, this study examined only lack of gluten as the experimental condition and did not investigate the condition of exposure to gluten.
Serum samples were assayed for IgA tissue transglutaminase antibodies (tTG IgA ELISA kit, QUANTA LiteTM, h-tTG IgA; Inova Diagnostic Inc., San Diego, CA, United States), and IgA antibodies reacting with deaminated gliadin-derived peptides (IgA DGP ELISA kit, QUANTA LiteTM DGP IgA, Inova Diagnostic Inc.). The assay results showed no statistically significant differences between the placebo arm and the AN-PEP arm.
In addition, questionnaires were distributed to the patients before randomization and at the end of the trial. The CSI consisted of 36 items about specific and general symptoms affecting CeD patients, and the SF-36 QoL questionnaire consisted of eight subsections about physical functioning, general health, and mental health. In the CSI, the answer to each question ranges from 1 to 5, where 1 means no symptoms and 5 means symptoms with the highest intensity. A lower score was seen in the AN-PEP arm compared with the placebo, but the difference was not statistically significant. The SF-36 score ranges from 0 to 100, where 0 means poorest health and 100 optimal health status. At the end of the study, the general health score was better in the placebo arm and the vitality and the abdominal pain scores were higher in the AN-PEP arm.
According to current literature, AN-PEP seems to have a positive impact on CeD symptoms because it is able to degrade gluten before it reaches the small intestine, so the mucosa is exposed to a lower gluten load[8,9]. However, the results are not concordant and no consensus exists until now about this issue[10,11].
The study did not explore the efficiency of AN-PEP during an exposure to gluten. For this reason, more research is needed to better understand its full potential. In this pilot study, AN-PEP treatment did not significantly decrease the GIP stool concentration. Although this is a pilot study, the results support further investigation of other adjuvant therapies with GFD.
1. | Shetty R, Heiner Bang-Berthelsen C, Ciurkot KW, Vestergaard M, Hägglund PM, Prakash HS, Hobley TJ. Characterization of gluten-degrading prolyl endoprotease from Thermococcus kodakarensis. FEMS Microbiol Lett. 2022;368. [PubMed] [DOI] [Cited in This Article: ] [Reference Citation Analysis (0)] |
2. | Ballini A, Charitos IA, Cantore S, Topi S, Bottalico L, Santacroce L. About Functional Foods: The Probiotics and Prebiotics State of Art. Antibiotics (Basel). 2023;12. [PubMed] [DOI] [Cited in This Article: ] [Cited by in F6Publishing: 31] [Reference Citation Analysis (0)] |
3. | Moreno Amador ML, Arévalo-Rodríguez M, Durán EM, Martínez Reyes JC, Sousa Martín C. A new microbial gluten-degrading prolyl endopeptidase: Potential application in celiac disease to reduce gluten immunogenic peptides. PLoS One. 2019;14:e0218346. [PubMed] [DOI] [Cited in This Article: ] [Cited by in Crossref: 10] [Cited by in F6Publishing: 16] [Article Influence: 3.2] [Reference Citation Analysis (0)] |
4. | Stefanolo JP, Segura V, Grizzuti M, Heredia A, Comino I, Costa AF, Puebla R, Temprano MP, Niveloni SI, de Diego G, Oregui ME, Smecuol EG, de Marzi MC, Verdú EF, Sousa C, Bai JC. Effect of Aspergillus niger prolyl endopeptidase in patients with celiac disease on a long-term gluten-free diet. World J Gastroenterol. 2024;30:1545-1555. [PubMed] [DOI] [Cited in This Article: ] [Cited by in CrossRef: 6] [Reference Citation Analysis (1)] |
5. | Dahlbom I, Nyberg BI, Berntson L, Hansson T. Simultaneous detection of IgA and IgG antibodies against tissue transglutaminase: The preferred pre-biopsy test in childhood celiac disease. Scand J Clin Lab Invest. 2016;76:208-216. [PubMed] [DOI] [Cited in This Article: ] [Cited by in Crossref: 7] [Cited by in F6Publishing: 9] [Article Influence: 1.1] [Reference Citation Analysis (0)] |
6. | Joukar F, Yeganeh S, Shafaghi A, Mahjoub-Jalali MR, Hassanipour S, Santacroce L, Mavaddati S, Mansour-Ghanaei F. The seroprevalence of celiac disease in patients with symptoms of irritable bowel syndrome: A cross-sectional study in the north of Iran. Hum Antibodies. 2022;30:97-103. [PubMed] [DOI] [Cited in This Article: ] [Cited by in Crossref: 1] [Cited by in F6Publishing: 1] [Article Influence: 0.5] [Reference Citation Analysis (0)] |
7. | Bolia R, Thapar N. Celiac Disease in Children: A 2023 Update. Indian J Pediatr. 2024;91:481-489. [PubMed] [DOI] [Cited in This Article: ] [Cited by in Crossref: 3] [Cited by in F6Publishing: 3] [Article Influence: 3.0] [Reference Citation Analysis (0)] |
8. | Xiao B, Zhang C, Song X, Wu M, Mao J, Yu R, Zheng Y. Rationally engineered prolyl endopeptidases from Sphingomonas capsulata with improved hydrolytic activity towards pathogenic peptides of celiac diseases. Eur J Med Chem. 2020;202:112499. [PubMed] [DOI] [Cited in This Article: ] [Cited by in Crossref: 4] [Cited by in F6Publishing: 8] [Article Influence: 2.0] [Reference Citation Analysis (0)] |
9. | Tack GJ, van de Water JM, Bruins MJ, Kooy-Winkelaar EM, van Bergen J, Bonnet P, Vreugdenhil AC, Korponay-Szabo I, Edens L, von Blomberg BM, Schreurs MW, Mulder CJ, Koning F. Consumption of gluten with gluten-degrading enzyme by celiac patients: a pilot-study. World J Gastroenterol. 2013;19:5837-5847. [PubMed] [DOI] [Cited in This Article: ] [Cited by in CrossRef: 91] [Cited by in F6Publishing: 95] [Article Influence: 8.6] [Reference Citation Analysis (0)] |
10. | Janssen G, Christis C, Kooy-Winkelaar Y, Edens L, Smith D, van Veelen P, Koning F. Ineffective degradation of immunogenic gluten epitopes by currently available digestive enzyme supplements. PLoS One. 2015;10:e0128065. [PubMed] [DOI] [Cited in This Article: ] [Cited by in Crossref: 34] [Cited by in F6Publishing: 35] [Article Influence: 3.9] [Reference Citation Analysis (0)] |
11. | Szaflarska-Popławska A. Non-dietary methods in the treatment of celiac disease. Prz Gastroenterol. 2015;10:12-17. [PubMed] [DOI] [Cited in This Article: ] [Cited by in Crossref: 1] [Cited by in F6Publishing: 4] [Article Influence: 0.4] [Reference Citation Analysis (0)] |