gastrins and drosulfakinin-1

Tyrosine O-sulfation is a posttranslational modification of secretory and membrane proteins transported through the Golgi apparatus, which is widespread among higher eukaryotes. O-Sulfated tyrosines are not immediately identified during sequencing of peptides and proteins, because the sulfate ester is acid labile and rapidly hydrolyses to tyrosine in strong acidic solutions. Little is known about the hydrolysis at mildly acidic solutions, which are used during several protein purification and analysis procedures. We have examined the stability of tyrosine sulfate using sulfated gastrin-17, caerulein, and drosulfokinin as models for tyrosine O-sulfated peptides. The peptides were incubated in acidic solutions in a pH range of 1 to 3 at different temperatures and time spans. Only marginal hydrolysis of gastrin-17 was observed in triflouroacetic acid at room temperature or below. Comparison of the acid hydrolysis of the three peptides showed that hydrolysis rate depends mainly on the primary amino acid composition of the peptide. The activation energy (E(a)) for the hydrolysis of sulfated gastrin-17 was found to be E(a)=98.7+/-5 kJ mol(-1). This study serves as a general reference for handling tyrosine sulfated peptides in aqueous acidic solutions. We conclude that tyrosine sulfate is more stable under normal protein purification conditions than previously assumed.

Topics: Ceruletide; Gastrins; Hydrogen-Ion Concentration; Hydrolysis; Neuropeptides; Oligopeptides; Peptide Fragments; Protein Processing, Post-Translational; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Temperature; Time Factors; Tyrosine

The nonapeptide, Phe-Asp-Asp-Tyr(SO3)-Gly-His-Met-Arg-Phe-NH2 was isolated from heads of the blowfly Calliphora vomitoria. Designated callisulfakinin I, the peptide is identical to the earlier known drosulfakinin I of Drosophila melanogaster and to neosulfakinin I of Neobellieria bullata. It belongs to the sulfakinin family, all known members of which (from flies, cockroaches and locusts) have the C-terminal heptapeptide sequence Asp-Tyr(SO3)-Gly-His-Met-Arg-Phe-NH2. The callisulfakinin gene of C. vomitoria was cloned and sequenced. In addition to callisulfakinin I, the DNA revealed a coding sequence for the putative tetradecapeptide. Gly-Gly-Glu-Glu-Gln-Phe-Asp-Asp-Tyr-Gly-His- Met-Arg-Phe-NH2, callisulfakinin II. However, this peptide was not identified in the fly head extracts. Confocal laser scanning immunocytochemical studies with antisera raised against the synthetic undecapeptide C-terminal fragment of drosulfakinin II from D. melanogaster, Asp-Gln-Phe-Asp-Asp-Tyr(SO3)- Gly-His-Met-Arg-Phe-NH2, revealed only four pairs of sulfakinin neurones in the brain of C. vomitoria and no others anywhere else in the neural, endocrine or gut tissues. In situ hybridisation studies with a digoxigenin-labelled sulfakinin gene probe (from the blowfly Lucilia cuprina) also revealed only four pairs of neurones in the brain. The perikarya of two pairs of cells are situated medially in the caudo-dorsal region, close to the roots of the ocellar nerve. The other perikarya are slightly more posterior and lateral. Although it has been suggested by several authors that the insect sulfakinins are homologous to the vertebrate peptides gastrin and cholecystokinin, such arguments (based essentially on C-terminal structural similarities) do not take account of important differences in the C-terminal tetrapeptide. His-Met-Arg-Phe-NH2 in the sulfakinins, compared with Trp-Met-Asp-Phe-NH2 in gastrin and cholecystokinin. Furthermore, whereas the sulfakinin neurons of C. vomitoria are small in number and have a very specialised location, a greater number of cells throughout the nervous system react positively to gastrin/cholecystokinin antisera. Chromatographic profiles of the present study also revealed peaks of gastrin/cholecystokinin-immunoreactive material separate from the sulfakinin peptides. This evidence suggests that the insect and vertebrate peptides may not necessarily be homologous.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cholecystokinin; Cloning, Molecular; Conserved Sequence; Diptera; DNA; Gastrins; Gene Expression; Genes, Insect; Immunohistochemistry; In Situ Hybridization; Molecular Sequence Data; Neuropeptides; Oligopeptides; Sequence Homology, Amino Acid

Homologues to the cholecystokinin (CCK)-gastrin peptide family have been cloned from Drosophila. The CCK-like precursor found in Drosophila has been designated drosulfakinin (DSK). Genomic and cDNA clones corresponding to the Drosophila neuropeptide precursor encode for three putative peptides. The three peptides (DSK-0, Asn-Gln-Lys-Thr-Met-Ser-Phe-Gly; DSK-I, Phe-Asp-Asp-Tyr-Gly-His-Met-Arg-Phe-Gly; DSK-II, Gly-Gly-Asp-Asp-Gln-Phe-Asp-Asp-Tyr-Gly-His-Met-Arg-Phe-Gly) are flanked by prohormone processing sites and contain C-terminal glycyl residues, a potential amidation site. Two of the peptides, DSK-I and DSK-II, are homologous to CCK-gastrin peptides. Each of the two homologues include a CCK-gastrin-like C-terminal pentapeptide and a conserved sequence corresponding to the sulfated tyrosine in bioactive CCK. The third peptide encoded by the drosulfakinin precursor represents a novel peptide. In situ tissue hybridization indicates the presence of the transcript in the adult head. Chromosomal localization maps the gene to the third chromosome near 81F.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cholecystokinin; Deoxyribonuclease EcoRI; DNA; DNA Restriction Enzymes; DNA, Recombinant; Drosophila melanogaster; Gastrins; Intercellular Signaling Peptides and Proteins; Molecular Sequence Data; Neuropeptides; Nucleic Acid Hybridization; Oligopeptides; Peptides; Protein Precursors; RNA, Messenger; Sequence Homology, Nucleic Acid