Serum zonulin as a marker of intestinal mucosal barrier function: May not be what it seems
Mary Ajamian, David Steer, Gennaro Rosella, Peter R Gibson, Mary Ajamian, David Steer, Gennaro Rosella, Peter R Gibson
Abstract
The protein, zonulin, has emerged as a popular serological marker to assess the integrity of the intestinal mucosal barrier. However, there is limited information on the utility of serum zonulin to indicate gastrointestinal disease and the validity of zonulin detection in widely-used commercial assays. The current study reports differences in zonulin levels across patient groups with gastrointestinal dysfunction compared with healthy individuals, though methodological inconsistencies indicated that actual zonulin protein was not detected by the commercial assays applied. The nature of the assays' detected antigen was investigated using immunoprecipitation followed by mass spectrometric analysis and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by protein staining. Top matches of the assays' detected antigen included haptoglobin and complement C3 for the assay manufactured by CUSABIO (Wuhan, China) and complement C3 for the assay manufactured by Immundiagnostik AG (Bensheim, Germany). These findings confirm that current commercial zonulin assays are not detecting the actual protein as prehaptoglobin-2. Until assay methodology is improved, we advise the greater scientific and medical community to exercise caution in considering the measurement of serum zonulin as a marker of mucosal barrier integrity.
Conflict of interest statement
The authors have declared that no competing interests exist.
Figures
![Fig 1. Haptoglobin phenotyping analysis by immunoblot.](https://www.ncbi.nlm.nih.gov/pmc/articles/instance/6331146/bin/pone.0210728.g001.jpg)
![Fig 2. Purported serum zonulin levels (ng/mL)…](https://www.ncbi.nlm.nih.gov/pmc/articles/instance/6331146/bin/pone.0210728.g002.jpg)
Fig 3. Comparison of purported serum zonulin…
Fig 3. Comparison of purported serum zonulin levels (ng/mL) between CUSABIO and Immundiagnostik ELISA assays.
Fig 4. Visualisation of immunoprecipitated protein products…
Fig 4. Visualisation of immunoprecipitated protein products and standards.
(A) 5 μg of human complement…
![Fig 3. Comparison of purported serum zonulin…](https://www.ncbi.nlm.nih.gov/pmc/articles/instance/6331146/bin/pone.0210728.g003.jpg)
![Fig 4. Visualisation of immunoprecipitated protein products…](https://www.ncbi.nlm.nih.gov/pmc/articles/instance/6331146/bin/pone.0210728.g004.jpg)
References
- Odenwald MA, Turner JR. Intestinal permeability defects: is it time to treat? Clinical gastroenterology and hepatology: the official clinical practice journal of the American Gastroenterological Association. 2013;11(9):1075–83. 10.1016/j.cgh.2013.07.001
- Fasano A. Zonulin and its regulation of intestinal barrier function: the biological door to inflammation, autoimmunity, and cancer. Physiological reviews. 2011;91(1):151–75. 10.1152/physrev.00003.2008 .
- Turner JR. Intestinal mucosal barrier function in health and disease. Nature reviews Immunology. 2009;9(11):799–809. 10.1038/nri2653 .
- Fasano A, Shea-Donohue T. Mechanisms of disease: the role of intestinal barrier function in the pathogenesis of gastrointestinal autoimmune diseases. Nature clinical practice Gastroenterology & hepatology. 2005;2(9):416–22. 10.1038/ncpgasthep0259 .
- Tripathi A, Lammers KM, Goldblum S, Shea-Donohue T, Netzel-Arnett S, Buzza MS, et al. Identification of human zonulin, a physiological modulator of tight junctions, as prehaptoglobin-2. Proceedings of the National Academy of Sciences of the United States of America. 2009;106(39):16799–804. 10.1073/pnas.0906773106
- Wang W, Uzzau S, Goldblum SE, Fasano A. Human zonulin, a potential modulator of intestinal tight junctions. Journal of cell science. 2000;113 Pt 24:4435–40. .
- Fasano A, Baudry B, Pumplin DW, Wasserman SS, Tall BD, Ketley JM, et al. Vibrio cholerae produces a second enterotoxin, which affects intestinal tight junctions. Proceedings of the National Academy of Sciences of the United States of America. 1991;88(12):5242–6.
- Cenac N, Chin AC, Garcia-Villar R, Salvador-Cartier C, Ferrier L, Vergnolle N, et al. PAR2 activation alters colonic paracellular permeability in mice via IFN-gamma-dependent and -independent pathways. The Journal of physiology. 2004;558(Pt 3):913–25. 10.1113/jphysiol.2004.061721
- Fasano A, Fiorentini C, Donelli G, Uzzau S, Kaper JB, Margaretten K, et al. Zonula occludens toxin modulates tight junctions through protein kinase C-dependent actin reorganization, in vitro. The Journal of clinical investigation. 1995;96(2):710–20. 10.1172/JCI118114
- Gutteridge JM. The antioxidant activity of haptoglobin towards haemoglobin-stimulated lipid peroxidation. Biochimica et biophysica acta. 1987;917(2):219–23. .
- Langlois MR, Delanghe JR. Biological and clinical significance of haptoglobin polymorphism in humans. Clin Chem. 1996;42(10):1589–600. .
- Wicher KB, Fries E. Prohaptoglobin is proteolytically cleaved in the endoplasmic reticulum by the complement C1r-like protein. Proceedings of the National Academy of Sciences of the United States of America. 2004;101(40):14390–5. 10.1073/pnas.0405692101
- Fasano A. Leaky gut and autoimmune diseases. Clin Rev Allergy Immunol. 2012;42(1):71–8. 10.1007/s12016-011-8291-x .
- Fasano A., Zonulin regulation of tight junctions, and autoimmune diseases. Annals of the New York Academy of Sciences. 2012;1258:25–33. 10.1111/j.1749-6632.2012.06538.x
- Hollon J, Puppa EL, Greenwald B, Goldberg E, Guerrerio A, Fasano A. Effect of gliadin on permeability of intestinal biopsy explants from celiac disease patients and patients with non-celiac gluten sensitivity. Nutrients. 2015;7(3):1565–76. 10.3390/nu7031565
- Barbaro MR CC, Caio G, Bellacosa L, De Giorgio R, Volta U, Stanghellini V, Barbara G. Increased Zonulin Serum Levels and Correlation with Symptoms in Non-Celiac Gluten Sensitivity and Irritable Bowel Syndrome with Diarrhea. United European Gastroenterology Journal. 2014;2:Supplement 1.
- Biesiekierski JR, Peters SL, Newnham ED, Rosella O, Muir JG, Gibson PR. No effects of gluten in patients with self-reported non-celiac gluten sensitivity after dietary reduction of fermentable, poorly absorbed, short-chain carbohydrates. Gastroenterology. 2013;145(2):320–8 e1-3. 10.1053/j.gastro.2013.04.051 .
- Newnham E, Srikanth J, Hosking P, Shepherd S, Gibson P, editors. Adjunctive induction therapy with oral effervescent budesonide in newly diagnosed celiac disease: Results of a pilot, randomized, double-blind, placebo-controlled trial. JOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY; 2017: WILEY 111 RIVER ST, HOBOKEN 07030–5774, NJ USA.
- Beswick L, Rosella O, Rosella G, Headon B, Sparrow MP, Gibson PR, et al. Exploration of predictive biomarkers of early infliximab response in acute severe colitis: A prospective pilot study. J Crohns Colitis. 2017. 10.1093/ecco-jcc/jjx146 .
- Scheffler L, Crane A, Heyne HO, Toenjes A, Schleinitz D, Ihling CH, et al. Widely used commercial ELISA for human Zonulin reacts with Complement C3 rather than pre-Haptoglobin 2. bioRxiv. 2017. 10.1101/157578
- Scheffler L, Crane A, Heyne H, Tonjes A, Schleinitz D, Ihling CH, et al. Widely Used Commercial ELISA Does Not Detect Precursor of Haptoglobin2, but Recognizes Properdin as a Potential Second Member of the Zonulin Family. Frontiers in endocrinology. 2018;9:22 10.3389/fendo.2018.00022
- Koch W, Latz W, Eichinger M, Roguin A, Levy AP, Schomig A, et al. Genotyping of the common haptoglobin Hp 1/2 polymorphism based on PCR. Clin Chem. 2002;48(9):1377–82. .
- Di Pierro M, Lu R, Uzzau S, Wang W, Margaretten K, Pazzani C, et al. Zonula occludens toxin structure-function analysis. Identification of the fragment biologically active on tight junctions and of the zonulin receptor binding domain. The Journal of biological chemistry. 2001;276(22):19160–5. 10.1074/jbc.M009674200 .
- Khaleghi S, Ju JM, Lamba A, Murray JA. The potential utility of tight junction regulation in celiac disease: focus on larazotide acetate. Therapeutic advances in gastroenterology. 2016;9(1):37–49. 10.1177/1756283X15616576
- Tosi M, Duponchel C, Meo T, Couture-Tosi E. Complement genes C1r and C1s feature an intronless serine protease domain closely related to haptoglobin. J Mol Biol. 1989;208(4):709–14. .
- Wicher KB, Fries E. Haptoglobin, a hemoglobin-binding plasma protein, is present in bony fish and mammals but not in frog and chicken. Proceedings of the National Academy of Sciences of the United States of America. 2006;103(11):4168–73. 10.1073/pnas.0508723103
- Kuo S-J, Wang F-S, Sheen J-M, Yu H-R, Wu S-L, Ko J-Y. Complement component C3: Serologic signature for osteogenesis imperfecta. Analysis of a comparative proteomic study. Journal of the Formosan Medical Association. 2015;114(10):943–9. 10.1016/j.jfma.2014.01.016
- Lehtinen MJ, Meri S, Jokiranta TS. Interdomain contact regions and angles between adjacent short consensus repeat domains. J Mol Biol. 2004;344(5):1385–96. 10.1016/j.jmb.2004.10.017 .
- Sunderhauf A, Skibbe K, Preisker S, Ebbert K, Verschoor A, Karsten CM, et al. Regulation of epithelial cell expressed C3 in the intestine—Relevance for the pathophysiology of inflammatory bowel disease? Mol Immunol. 2017;90:227–38. 10.1016/j.molimm.2017.08.003 .
- Sina C, Kemper C, Derer S. The intestinal complement system in inflammatory bowel disease: Shaping intestinal barrier function. Semin Immunol. 2018. 10.1016/j.smim.2018.02.008 .
Source: PubMed