Degradation of coeliac disease-inducing rye secalin by germinating cereal enzymes: diminishing toxic effects in intestinal epithelial cells

Abstract

Currently the only treatment for coeliac disease is a lifelong gluten-free diet excluding food products containing wheat, rye and barley. There is, however, only scarce evidence as to harmful effects of rye in coeliac disease. To confirm the assumption that rye should be excluded from the coeliac patient's diet, we now sought to establish whether rye secalin activates toxic reactions in vitro in intestinal epithelial cell models as extensively as wheat gliadin. Further, we investigated the efficacy of germinating cereal enzymes from oat, wheat and barley to hydrolyse secalin into short fragments and whether secalin-induced harmful effects can be reduced by such pretreatment. In the current study, secalin elicited toxic reactions in intestinal Caco-2 epithelial cells similarly to gliadin: it induced epithelial cell layer permeability, tight junctional protein occludin and ZO-1 distortion and actin reorganization. In high-performance liquid chromatography and mass spectroscopy (HPLC-MS), germinating barley enzymes provided the most efficient degradation of secalin and gliadin peptides and was thus selected for further in vitro analysis. After germinating barley enzyme pretreatment, all toxic reactions induced by secalin were ameliorated. We conclude that germinating enzymes from barley are particularly efficient in the degradation of rye secalin. In future, these enzymes might be utilized as a novel medical treatment for coeliac disease or in food processing in order to develop high-quality coeliac-safe food products.

Fig. 1

Enzymatic degradation of gliadin and secalin analysed by high-performance liquid chromatography and mass spectroscopy (HPLC-MS) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). HPLC-MS elution profiles of gliadin (a) and secalin (b) (both 6 mg/ml) incubated with 0·3 mg/ml of oat, wheat and barley enzyme preparations. Full range m/z = 480–2000. (c) SDS-PAGE analysis of secalin (1 mg/ml) degraded by germinated grain preparations (1 µg/ml). Lane 1 = size standard, lane 2 = secalin, lane 3 = secalin + germinating oat enzymes, lane 4 = secalin + germinating wheat enzymes, lane 5 = secalin + germinating barley enzymes, lane 6 = secalin + heat inactivated germinating barley enzymes. (d) HPLC-MS elution profiles of pepsin–trypsin (PT)-digested secalin (black line), PT–secalin pretreated with germinating barley enzymes (red line) and germinating barley enzymes + PT (blue line). Full range m/z = 480–2000. Retention times of the standard peptides 12-mer (16·5 min) and 33-mer (23·3 min) are shown by arrows.

Fig. 2

Transepithelial resistance (TER) in Caco-2 cells cultured with medium only or challenged with pepsin–trypsin-digested bovine serum albumin (PT–BSA), pepsin–trypsin-digested gliadin (PT–G), pepsin–trypsin-digested secalin (PT–S) or germinating barley enzyme-pretreated PT–S (PT–S + enzymes) at different time-points. Data are given as mean percentages of baseline TER values ± standard error of the mean derived from six independent experiments performed in duplicate. Statistical analyses by two-tailed Mann–Whitney U-test, where P < 0·05 (*) was considered statistically significant.

Fig. 3

The appearance of tight junctions in Caco-2 cells cultured with medium only, pepsin–trypsin-digested bovine serum albumin (PT–BSA), pepsin–trypsin-digested gliadin (PT–G), pepsin–trypsin-digested secalin (PT–S) or enzymatically pretreated pepsin–trypsin-digested secalin (PT–S + enzymes). Immunofluorescent staining of occludin (a) and ZO-1 (b). Magnification 100×. Pictures are representative images from experiments performed in duplicate three independent times.

Fig. 4

Actin cytoskeleton rearrangement as membrane ruffling in Caco-2 cells. (a) Representative images of actin membrane ruffling (arrows) in Caco-2 cells cultured with medium only, pepsin–trypsin-digested bovine serum albumin (PT–BSA), pepsin–trypsin-digested gliadin (PT–G), pepsin–trypsin-digested secalin (PT–S) or germinating barley enzyme-pretreated PT–S (PT–S + enzymes). Magnification 20×. (b) Quantitation of ruffle formation was measured as percentage of total cell layer edge length. Data are given as mean values ± standard error of the mean derived from three independent experiments performed in duplicate. Statistical analyses by two-tailed Mann–Whitney U-test, where P < 0·05 was considered statistically significant; n.s.: not significant.