Abstract
Tetraploidization, the duplication of the entire genome of an organism, results in twice the number of each gene present in the original diploid genome. Over time, the multiple copies of a single gene may accumulate different mutations and produce proteins or peptides with differences in their primary structures. The isolation of multiple forms of regulatory peptides from a single species is helpful in determining the ploidy status of the species, and comparisons of the primary structures of regulatory peptides from closely related species is useful in the determination of phylogenetic relationships among species. Identification of well-conserved amino acids in regulatory peptides from different species also provides insight into structure-activity relationships.|This study involves the purification and structural characterization of hormonal/ neurohormonal peptides from two non-mammalian vertebrate species that are believed to possess tetraploid genomes. Insulin, glucagon, two forms of somatostatin, three forms of peptide tyrosine-tyrosine (PYY), and two forms of vasoactive intestinal peptide (VIP) were isolated from gastroenteropancreatic (GEP) tissue of the pallid sturgeon, Scaphirhynchus albus. Two forms of gastrin-releasing peptide (GRP), neuromedin C, two forms of PYY, glucagon-like peptide IB (GLP-1B), and VIP were isolated from gastrointestinal extracts from the African clawed frog, Xenopus laevis. Peptides were purified by reversed-phase HPLC and characterized by electrospray ionization mass spectroscopy (ESI-MS) and automated Edman degradation.The single form of insulin isolated from the pallid sturgeon is identical to insulin previously isolated from the Russian sturgeon, Acipenser gueldenstaedti; insulin-2 isolated from the kaluga sturgeon, Huso dauricus; and insulin-2 from the paddlefish, Polyodon spathula. Pallid sturgeon insulin is approximately twofold less potent than human insulin in inhibiting the binding of [3-[125I] iodotyrosine-A14] human insulin to the soluble human insulin receptor. The mean concentrations of pallid sturgeon insulin and human insulin causing 50% inhibition of binding were 76 and 31 pM, respectively. The single form of glucagon is identical to glucagon-5 previously isolated from the kaluga sturgeon. Like kaluga sturgeon glucagon-3, glucagon-4, and glucagon-5, and the two forms of paddlefish glucagon, pallid sturgeon glucagon is 31 rather than 29 amino acid residues in length, which is indicative of an alternative pathway of posttranslational processing of proglucagon. Somatostatin-14 isolated from the pallid sturgeon is identical to human somatostatin-14, and was also isolated in a second molecular form with an oxidized Trp8 residue. [Pro2]somatostatin-14, previously isolated from the pituitary of the Russian sturgeon, was not detected in the pallid sturgeon GEP extract. Pallid sturgeon PYY (FPPKP5 EHPGD10 DAPAE15 DVAKY20 YTALR25 HYINL30 ITRQR35 Y.NH2) was also isolated in two other isoforms, one containing the substitution Phe'->Ala and the other containing Ala18—»Val. The three different forms of pallid sturgeon PYY are indicative of at least two duplications of the PYY gene in this species. One form of pallid sturgeon VIP is identical to VIP previously isolated from the bowfin, Amia calva, and the rainbow trout, Oncorhynchus mykiss. The second form of pallid sturgeon VIP differs by a single substitution (Ala4—»Ser). The presence of two forms of pallid sturgeon VIP is indicative of at least one VIP gene duplication in this species.|The primary structure of A laevis GRP-1 (APTSQ5 QHTEQ10 LSRSN15 INTRG20 SHWAV23 GHLM.NH2) differs from that of GRP-2 by a single amino acid substitution (Asn15—>Thr). X. laevis neuromedin C is identical to GRP20"29. The COOH-terminal domain of GRP has been highly conserved throughout vertebrate evolution except for the substitution Ser21—>Asn in some species. Synthetic GRP-1 produced concentration- dependent contractions of longitudinal smooth muscle strips from the stomach of X. laevis {pD2 = 8.93 + 0.32; n = 6). This response was unaffected by the presence of tetrodotoxin (1 pM), atropine (10 pM), or methysergide (10 pM), which suggests that the peptide acts directly with receptors at the smooth muscle surface. Synthetic GRP-1 produced a concentration-dependent relaxation of circular smooth muscle strips from the stomach of X. laevis (pD2 = 8.96 + 0.21; n = 8) after precontraction of the strips with carbachol (5 pM). This response was decreased (71 + 24% decrease in maximum response; n = 6) by indomethacin (10 pM), indicating the involvement of prostanoids. Porcine GRP, which differs from A laevis GRP-1 by 13 amino acids and contains two deletions in the N-terminal region, produced identical responses in these tissues, which demonstrates the importance of the highly conserved COOH-terminal region of GRP for the activity of the peptide. X. laevis PYY-1 (YPTKP5 ENPGN10 DASPE15 EMTKY20 LIALR25 HYINL30 VTRQR35 Y.NH2) differs from PYY-2 by six amino acid substitutions (Glu15-»Gln, Thr18—>Ala, Leu21->Met, Ile22->Thr, Ile28^Val, Val3I^Ile). These differences indicate that at least two PYY gene duplications have occurred within the X.laevis genome, and that at least one copy of the gene has undergone an accelerated rate of mutation. GLP-1B (HAEGT5 YTNDV10 TEYLE15 EKAAK20 EFIEW25 LIKGK30 PKKIR35 YS) isolated from A! laevis stomach and intestine is identical to the GLP-1B primary structure predicted from the nucleotide sequence of a cDNA, but with the site of posttranslational processing correctly identified. X. laevis VIP is identical to VIP previously isolated from the European green frog, Rana ridibunda, and the American alligator, Alligator mississippiensis.