The Sulfakinins of the Blowfly Calliphora Vomitoria. Peptide Isolation, Gene Cloning and Expression Studies

Abstract

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.

Peptidomic analysis of the larval Drosophila melanogaster central nervous system by two‐dimensional capillary liquid chromatography quadrupole time‐of‐flight mass spectrometry

“…Sulfakinins have been identified in various insects including the cockroaches Leucophaea maderae 11,12 and Periplaneta americana, 13 the locust Locusta migratoria 14 and the flies Neobellieria bullata 15 and Calliphora vomitoria. 16 Similar to CCK in vertebrates, sulfakinins reduce food intake in locusts and stimulate contraction in visceral muscles. 17 CCK-like peptides and insect sulfakinins share a sulfated tyrosine residue, which is essential for their activity.…”

Identification and cardiotropic actions of sulfakinin peptides in the American lobster Homarus americanus

“…translated nucleotide sequence versus protein sequence (Altschul et al, 1997)] and annotated accordingly. A single EST (NCBI accession number CN952349) with significant homology to a sulfakinin precursor from the blowfly Calliphora vomitoria (NCBI accession number Q7M3V5) (Duve et al, 1995) was identified during this analysis.…”

“…translated nucleotide sequence versus protein sequence), a H. americanus EST (accession number CN952349) with homology to blowfly C. vomitoria prepro-sulfakinin (NCBI accession number Q7M3V5) (Duve et al, 1995) was identified. Using a combination of vector-and insert-specific forward-and reverse-sequencing primers (Table·2), a 1523 base pair (bp), putative full-length cDNA was sequenced (NCBI accession number EF418605).…”

“…First using biochemical methods, and more recently via molecular techniques, a number of sulfakinin isoforms have been identified from insects (Nachman et al, 1986a;Nachman et al, 1986b;Nichols et al, 1988;Veenstra, 1989;Schoofs et al, 1990;Fonagy et al, 1992;Nichols, 1992;Duve et al, 1995;Maestro et al, 2001) (NCBI accession number AY341429; NCBI accession number AY758365) (Table·1). In contrast, sulfakinin isoforms have thus far been biochemically isolated and characterized from just two crustaceans, the penaeid shrimp Penaeus monodon and Litopenaeus vannamei (Johnsen et al, 2000;Torfs et al, 2002) (Table·1).…”

“…1 This study; 2 (Johnsen et al, 2000); 3 (Torfs et al, 2002); 4 (Nachman et al, 1986a); 5 (Nachman et al, 1986b); 6 (Veenstra, 1989); 7 (Maestro et al, 2001); 8 (Nichols et al, 1988); 9 (Nichols, 1992); 10 (Fonagy et al, 1992); 11 (Duve et al, 1995); 12 NCBI accession number AY341429; 13 NCBI accession number AY758365; 14 (Schoofs et al, 1990); 15 (Johnsen and Rehfeld, 1990). The origin of the N-terminal pyro-residue in the short SK isoforms of shrimp was originally given as being derived from glutamine rather than glutamic acid (as our molecular analysis has shown is the case for the Hoa-SK I), though there is no data to prove this assignment currently.…”