Structural specificities and significance for coeliac disease of wheat gliadin peptides able to agglutinate or to prevent agglutination of K562(S) cells

Abstract

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.

Introduction

The involvement of prolamine fractions of bread wheat, rye, barley and probably oats in eliciting coeliac disease symptoms upon digestion in susceptible individuals has been demonstrated for many years (Van de Kramer et al., 1953, Cornell and Townley, 1974, Baker and Read, 1976). A number of studies have also shown that the agglutinating activity of K 562 (S) cells of prolamine peptic–tryptic (PT) digests is highly correlated with the toxicity of these prolamines in coeliac disease (Auricchio et al., 1984, Auricchio et al., 1987, Auricchio et al., 1990a, Auricchio et al., 1990b, De Ritis et al., 1987, Cornell et al., 1988, De Vincenzi et al., 1994a, De Vincenzi et al., 1994b, De Vincenzi et al., 1995, Maiuri et al., 1996). Furthermore, two peptide fragments (31–43 and 44–55) of A-gliadin (a highly purified α-gliadin protein with high toxicity in coeliac disease) have been found to be very active in damaging the intestinal mucosa, both in vivo and in vitro (Marsh et al., 1995, Maiuri et al., 1996) and to agglutinate K 562 (S) cells at very low concentrations (De Vincenzi et al., 1994a).

Gliadin PT digests from durum wheats have been demonstrated to be unable to agglutinate K 562 (S) cells and to have a much lower adverse effect on coeliac intestinal specimens; moreover, the durum wheat gliadin digests, in contrast to bread wheat ones, did not show any effect on the in vitro differentiation of fetal rat intestine or on the growth of undifferentiated cell cultures (Auricchio et al., 1982, Rocca et al., 1983, Stammati Paganuzzi et al., 1985, De Vincenzi et al., 1995). Further investigation showed the presence in these digests of a peptide fraction, also present in the PT digests of bread wheat gliadins, with cell agglutinating activity, as well as of another peptide fraction, absent in the PT digests of bread wheat, which was able to interfere with cell agglutination induced by the former fraction (De Vincenzi et al., 1995).

The agglutination-inhibiting fraction from gliadin PT digest of one variety of durum wheat was further fractionated and characterized, thus showing that most of its activity was associated with a peptide of 1157.5 Da molecular mass whose sequence is shown in Table 1 (De Vincenzi et al., 1997).

In the light of the interest of the biological activity of this peptide for the clarification of the etiology of coeliac disease and in view of some possible practical applications, the authors considered it useful to undertake a structure–activity relationship study on this peptide by synthesizing a number of peptides structurally related to the 1157.5-Da peptide and investigating their ability to protect K 562 (S) from agglutination induced by a number of cereal prolamine peptides. Moreover, the authors have also investigated cell agglutinating activities of two small peptides, one derived from peptide 31–43 and the other from peptide 44–55.