ISOLATION, IDENTIFICATION AND FUNCTIONAL-SIGNIFICANCE OF [HYP(2)]MET-CALLATOSTATIN AND DES GLY-PRO MET-CALLATOSTATIN, 2 FURTHER POSTTRANSLATIONAL MODIFICATIONS OF THE BLOWFLY NEUROPEPTIDE MET-CALLATOSTATIN

Two post-translationally modified neuropeptides of the Met-callatostatin (Gly-Pro-Pro-Tyr-Asp-Phe-Gly-Met-NH2) family have been identified from head extracts of the blowfly Calliphora vomitoria. They are the octapeptide, [Hyp(2)]Met-callatostatin, (Gly-Hyp-Pro-Tyr-Asp-Phe-Gly-Met-NH2) and the truncated hexapeptide, des Gly-Pro Met-callatostatin (Pro-Tyr-Asp-Phe-Gly-Met-NH2). The existence of the [Hyp(2)]Met-callatostatin variant, in addition to the previously identified [Hyp(3)]Met-callatostatin peptide, suggests that the motif for prolyl hydroxylation in C. vomitoria is more variable than those known from mammalian and other invertebrate studies where, in those regulatory peptides containing a pair of adjacent prolyl residues so far studied, e.g., bradykinin, and the mosquito peptide Aea HP-I, only one of the pair (the second) is known to undergo hydroxylation. The truncated hexapeptide, des Gly-Pro Met-callatostatin could be produced as a result of the action of a dipeptidyl peptidase II type of enzyme which is known from mammalian studies to be unique in its ability to cleave between the two prolyl residues of an Xaa-Pro-Pro- sequence, where Xaa is any unprotected NH2-terminal amino acid. This enzyme is, however, considered unlikely to be able to cleave the Gly-Hyp-Prosequence, which would suggest a functional significance for such a post-translational modification. For this reason, it is of interest that [Hyp(2)]Met-callatostatin (and earlier, [Hyp(3)]Met-callatostatin) have been shown to be potent inhibitors of the spontaneous contractions of the hindgut of C. vomitoria (biphasic dose-response curve with IC50 values of 10(-14) M and 10(-7) M). The hexapeptide, although active, is less potent (IC50 = 10(-7) M, the same as that previously recorded for the unmodified 'parent' molecule, Met-callatostatin). The study emphasises the continuing need for peptide