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Recent experimental evidence suggests that enterocyte apoptosis is greater than hitherto assumed and may be responsible for villous atrophy in coeliac disease. We have previously demonstrated that a small peptide (M.W. 1157.5 Da), identified as the sequence H(2)N-gln-gln-pro-gln-asp-ala-val-gln-pro-phe-COOH from durum wheat gliadin, is able to prevent K 562 (S) cell agglutination induced by the peptic-tryptic digests (PT) of prolamin fractions from the cereals which are not tolerated in coeliac disease (i.e. bread wheat, rye, barley and possibly oats), and toxic A-gliadin peptides in coeliac disease. In the present study we have investigated the effects of the bread wheat gliadin digest (PT) on apoptosis of Caco-2 cells and whether the '1157.5' Da peptide may in any way interfere with them. We evaluated both earlier biochemical and later morphological nuclear apoptotic events in the human colon adenocarcinoma cell line Caco-2. After 48 h exposure to the PT gliadin digest and the '1157.5' Da peptide, apoptosis was detected both for the early-stage apoptotic cells (adherent cells) and the late-stage apoptotic ones (detached cells which were floating in the culture medium). Exposure to the PT gliadin digest resulted in a high percentage of adherent cells that underwent cell death by apoptosis (about 30%), independent of the concentration range used; while the presence in the culture medium of peptide '1157.5' Da determined complete inhibition of cell death. On the other hand, morphological nuclear modifications observed in the floating cells showed a difference in the rate of the apoptosis dependent on the PT concentration, with partial protection in the presence of the peptide. These findings show an action of bread wheat gliadin peptides leading to cell death by apoptosis in the Caco-2 cell line and that the '1157.5' Da peptide is capable of preventing such an effect.

Experience gained through investigations on coeliac disease makes it possible to propose a screening method based on agglutination of isolated K562(S) cells to evaluate the occurrence in food protein of amino acid sequences that are able to adversely affect coeliac and related gluten-sensitive patients. The method consists of in vitro sequential peptic and tryptic digestion of food protein fractions under optimal pH, temperature and time conditions and in vitro incubation of the digest with K562(S) cells; the toxic potential is detected as an agglutination of K 562 (S) cells after a short incubation. Other in vitro test systems, including atrophic coeliac intestinal mucosa and rat fetal intestine, can be used to confirm the results obtained with the isolated cells. A fractionation step of the proteolytic digest on a sepharose-mannan column before exposure of the in vitro systems to the separated peptide fractions adds to the sensitivity of the method. This screening method is not only very useful to investigate action mechanisms in coeliac disease, but also to assess the safety of genetically-modified plant foods and novel foods for gluten-sensitive patients.

Two peptides corresponding to bread wheat A-gliadin fragments 31-43 and 44-55, well known for their ability to damage the coeliac disease intestinal mucosa both in vitro and in vivo, have been confirmed to be very active in inducing in vitro agglutination of K 562 (S) cells. Removal of six amino acid residues from the carboxy-terminal end of the 31-43 peptide, or of five amino acid residues from the amino terminal end of the 44-55 peptide, resulted in a lower, but still very significant, cell agglutination activity. The peptide consisting of ten amino acid residues with a molecular mass of 1157.5 Da, isolated from durum wheat gliadin, was able to prevent agglutination of K 562 (S) cells induced not only by prolamine peptic-tryptic digests from all the cereals toxic in coeliac disease (i.e. bread wheat, rye, barley and oats), but also by the 31-43 and 44-55 peptides. The ability to protect K 562 (S) cells from agglutination was exhibited to the fullest extent also by all the peptides derived from the 1157.5-Da peptide by five progressive deletions of the terminal carboxylic residue, whereas the sixth consecutive deletion yielded a completely inactive peptide. A similar total loss of activity was observed upon addition of a glycine residue to the amino terminal residue of the 1157.5-Da peptide and all the above-mentioned active peptides derived from it. The remarkable sequence homologies existing between peptides able to induce [Gln-Gln-Gln-Pro and -Pro-Ser-Gln-Gln-] or to prevent [H2N-Gln-Gln-Pro-Gln-Asp-COOH] induction of cell agglutination strongly suggest that all these peptides compete for identical or structurally related binding sites on the cell surface.

A peptide (m.w. 1157.5 Da) able to prevent the agglutination of K562(S) cells induced by the peptic-tryptic prolamine digests of the cereals toxic in coeliac disease (i.e. bread wheat, rye, barley and oat) was characterized as one of the components of the peptic-tryptic digest of durum wheat gliadin. This peptide was synthesized in a high degree of purity with the solid phase method with the Applied Biosystem 431A. An amino acid sequence was identified in the 1157.5 Da peptide as being related to the largest common sequences previously detected in a series of bread wheat toxic peptides by other authors.

The K 562 (S) cell agglutinating activity of peptides obtained from in vitro digestion of bread wheat gliadins has been shown to be associated with a small fraction (coded as Fraction C), that can be easily separated by affinity chromatography of the whole digest on a sepharose 6-B-mannan or sepharose 6-B-oligomers of N-acetyl-glucosamine. Although the whole gliadin digests from 12 durum wheat varieties were unable to agglutinate K 562 (S) cells, all these digests were found to contain an active Fraction C. The lack of agglutinating activity of the whole durum wheat gliadin digests has been shown to be associated with the presence in these digests of another peptide fraction (coded as Fraction B) that is eluted much earlier from the sepharose 6-B-mannan column and is able to inhibit the cell agglutinating activity of Fraction C. Such an active Fraction B is not present in bread wheat gliadin peptides, although peptides with the same elution profile as Fraction B have been detected.