Caricain: A basis for enzyme therapy for coeliac disease

  • Hugh J. Cornell School of Applied Science, RMIT University
  • Teodor Stelmasiak Glutagen Pty Ltd
Keywords: caricain, gliadin, coeliac disease, enzyme therapy, lysosomes


Gliadin, a glycoprotein present in wheat and other grass cereals, is a causative agent in coeliac disease. It is therefore important to find methods for the detoxification of gliadin. Lysosomal integrity is lost in patients with active coeliac disease but restored when gliadin is removed from the diet. We employed a rat liver lysosome assay to monitor the extent of detoxification of a gliadin digest by caricain, a protein enzyme found in papaya. Pre-incubating the gliadin digest for different durations with caricain allowed the kinetics of the detoxification process to be studied. A significant degree of protection (80%) of the lysosomes was achieved with 1.7% w/w of caricain on substrate after incubation for 2 h at 37 °C. The detoxification followed first-order kinetics with a rate constant of 1.7 x 10-4/s. The enzyme was strongly inhibited by imidazole, but weakly by phenylmethyl sulphonyl fluoride, as was also a caricain-enriched fraction from ion-exchange chromatography of papaya oleo-resin. The value of caricain in the detoxification of gliadin was confirmed in the present studies and this enzyme shows promise for enzyme therapy in coeliac disease.


1. Cornell HJ, Stelmasiak T. A unified hypothesis of coeliac disease with implications for management of patients. Amino Acids. 2007;33:43–49. doi:10.1007/s00726-006-0420-0, PMid:17013762

2. Bronstein HD, Haeffner LJ, Kowlessar OD. Enzymatic digestion of gliadin: The effect of the resultant peptides in adult coeliac disease. Clin Chim Acta. 1966;14:141–155. doi:10.1016/0009-8981(66)90080-5

3. Kocna P, Mothes T, Krchnak V, Fric P. Relationship between gliadin peptide structure and their effect on the fetal chick duodenum. Zeitschrift fur Lebensmitteluntersuchung und-Forschung. 1991;192:116–119. doi:10.1007/BF01202623

4.Cornell HJ, Mothes T. The activity of wheat gliadin peptides in in vitro assays for coeliac disease. Biochim Biophys Acta. 1993;1181:169–173. PMid:8481406

5. Cornell HJ, Macrae FA, Melny J, et al. Enzyme therapy for management of coeliac disease. Scand J Gastroent. 2005;40:1304-1312. doi:10.1080/00365520510023855, PMid:16243716

6. Cornell HJ, Doherty W, Stelmasiak T. Papaya latex enzymes capable of detoxification of gliadin. Amino Acids. 2010;38:155–165. doi:10.1007/s00726-008-0223-6, PMid:19156482

7. Dubey VK, Pande M, Singh BK, Jagannadham MV. Papain-like proteases: Applications of their inhibitors. Afr J Biotechnol. 2007;6:1077–1086.

8. Zerhouni S, Amrani A, Nijs M, et al. Purification and characterization of papaya glutamine cyclotransferase, a plant enzyme highly resistant to chemical, acid and thermal denaturation. Biochim Biophys Acta. 1998;1387:275–290. doi:10.1016/S0167-4838(98)00140-X

9. Azarkan M, El Moussaoui A, Van Wuytswinkel D, Dehon G, Looze Y. Fractionation and purification of the enzymes stored in the latex of Carica papaya. J Chromatogr B Analyt Technol Biomed Life Sci. 2003;790:229–238. doi:10.1016/S1570-0232(03)00084-9

10. Riecken EO, Stewart JS, Booth CC, Pearse AGE. A histochemical study of the role of lysosomal enzymes in idiopathic steatorrhoea before and during a gluten- free diet. Gut. 1966;7:317–332. doi:10.1136/gut.7.4.317, PMid:591741, PMCid:1552434

11. Williams VR, Williams HB. Basic physical chemistry for the life sciences. San Francisco: WH Freeman and Company, 1973; p. 277–313.

12. Laidler KJ. Physical chemistry with biological applications. Menlo Park, CA: Benjamin Cummings, 1978; p. 427–451.

13. Cornell HJ, Townley RRW. Investigating possible intestinal peptidase deficiency in coeliac disease. Clin Chim Acta. 1973;43:113–125. doi:10.1016/00098981(73)90126-5

14. Terrell DA. The isolation of native glutaminyl cyclase from Saccharomyces cerevisiae. Honours thesis, San Marcos, Texas State University, 2006.

15. De Ritis G, Auricchio S, Jones HW, Lew EJ-L, Bernardin JE, Kasarda DD. In vitro (organ culture) studies of the toxicity of specific A-gliadin peptides in coeliac disease. Gastroenterology. 1988;94:41–47. PMid:3335296

16. Cornell HJ. The aetiology of coeliac disease and its significance for therapy. Curr Topics Peptide Protein Res. 2005;7:17–22.

17. Ensari A, Marsh MN, Moriarty KJ, Moore CM, Fido RJ, Tatham AS. Studies in vivo of w-gliadins in gluten sensitivity (coeliac sprue disease). Clin Sci. 1998;95:419–424. doi:10.1042/CS19980129, PMid:9748417

18. Cornell HJ, Rivett DE. In vitro mucosal digestion of synthetic gliadin-derived peptides in coeliac disease. J Protein Chem. 1995;14:335–339. doi:10.1007/BF01886790, PMid:8590601

19. Sturgess R, Day P, Ellis HJ, et al. Wheat peptide challenge in coeliac disease. Lancet. 1994;343:758–761. doi:10.1016/S0140-6736(94)91837-6

20. McLachlan A, Cullis PG, Cornell HJ. The use of extended motifs for focussing on toxic peptides in coeliac disease. J Biochem Mol Biol Biophys. 2002;6:319–324. doi:10.1080/1025814021000003238, PMid:12385967

21. Szwajcer-Dey E, Rasmussen J, Meldal M, Breddam K. Proline-specific endopeptidases from microbial sources: Isolation of an enzyme from Xanthomonas sp. JBacteriol. 1992;174:2454–2459. PMid:1556065, PMCid:205881

22. Matysiak-Budnik T, Candalh C, Cellier C, et al. Limited efficiency of prolyl-endopeptidase in the detoxification of gliadin peptides in coeliac disease. Gastroenterology. 2005;129:786–796. doi:10.1053/j.gastro.2005.06.016, PMid:16143118

23. Donlon J, Stevens FM. No lack of prolyl oligopeptidase (POP) in the coeliac mucosa. Proceedings of the 11th International Symposium on Coeliac Disease; 2004 April 28 – May 1; Belfast, Ireland. Belfast: The Coeliac Society of Ireland; 2004. p. 19. 24. Arnon R. The cysteine proteases: Papain. Methods Enzymol. 1970;19:226–244. doi:10.1016/0076-6879(70)19017-3

25. Messer M, Anderson CM, Hubbard L. Studies on the mechanism of destruction of the toxic action of wheat gluten in coeliac disease by crude papain. Gut. 1964;5:295–303. doi:10.1136/gut.5.4.295, PMid:14209911, PMCid:1413471

26. Stepniak D, Spaenij-Dekking L, Mitea C, et al. Highly efficient gluten degradation with a newly identified prolyl endopeptidase: Implications for coeliac disease. Am J Physiol Gastrointest Liver Physiol. 2006;291:G621–G629. doi:10.1152/ajpgi.00034.2006, PMid:16690904

27. Cornell HJ, Stelmasiak T. Commentary – Strategies for improved outcomes for those with coeliac disease. In: Edwards MA, editor. Coeliac disease – Etiology, diagnosis and treatment. New York: Nova Science, 2009; p. 207–211.