Comparative peptidomics of the endocrine pancreas: islet hormones from the clawed frog Xenopus laevis and the red-bellied newt Cynops pyrrhogaster

Abstract

Electrospray mass spectrometry coupled with reversephase HPLC was used to identify peptides in the molecular mass range 3000-6000 Da in extracts of the pancreata of the clawed frog Xenopus laevis (Anura: Pipidae) and the red-bellied newt Cynops pyrrhogaster (Caudata: Salamandridae). Amino acid sequences of insulins, peptides derived from the post-translational processing of proglucagons and pancreatic polypeptide were determined by automated Edman degradation. Three molecular forms of insulin were isolated from the tetraploid organism X. laevis that represent insulin-1 and insulin-2, as deduced from the nucleotide sequences of previously characterized cDNAs, and a third form which differed from insulin-2 by the single amino acid substitution Asp 21 <Glu in the B-chain. The amino acid sequence of Xenopus preproglucagons (genes 1 and 2 ) may be deduced from the nucleotide sequences of cDNAs but the pathways of post-translation processing of the precursors are not known. Two molecular forms of glucagon with 36 amino acids, derived from genes 1 and 2 and representing glucagon-29 extended from its C terminus by different heptapeptides, and five molecular forms of glucagon-like peptide 1 (GLP-1) were isolated. The GLPs represent proglucagon-(77-113), -(122-158) and -(160-191) from gene 1, and proglucagon-(77-113) and -(160-191) from gene 2. A single molecular form of insulin, glucagon-36, a C-terminally -amidated GLP-1 with 30 amino acid residues, a 33 amino acid residue GLP-2 and pancreatic polypeptide were isolated from the pancreatic extract of the diploid organism C. pyrrhogaster. This study has illustrated the power of electrospray mass spectrometry for the rapid and reliable identification of peptides in chromatographic fractions without the need to use radioimmunoassay, radioreceptor assay or bioassay.

Using Evolutionary Information and Ancestral Sequences to Understand the Sequence–Function Relationship in GLP-1 Agonists

“…Tetrapod GLP-1 sequences were collected from databases and the literature; Agkistrodon piscivorus (GI:45272441), Amphiuma tridactylum (three-toed salamander), 56 Bufo marinus (giant toad), 21 Ceratophrys ornata (ornate horned frog), 57 Cynops pyrrhogaster (Japanese firebelly newt), 32 Gallus gallus (common chiken), 58 Hoplobatrachus rugulosus (Taiwanese frog), 23 Heloderma Suspectum (Gila monster), 24 Homo sapiens (human), 59 Pyxicephalus adspersus (African bull frog), 60 Pipa pipa (suriname toad), 61 Rana catesbeiana (bull frog), 62 Rana pipiens (northern leopard frog), 22 Xenopus leavis (African clawed frog), 32 Xenopus tropicalis (western clawed frog) (GI:55742076), and H. suspectum exendin-4. 25 Cleavage positions were in most cases determined from sequencing of the peptide itself; otherwise they were transferred from the closest homolog.…”

Identification and Characterization of a Glucagon Receptor from the Goldfish Carassius auratus: Implications for the Evolution of the Ligand Specificity of Glucagon Receptors in Vertebrates

“…3,7). Glucagon and GLP-1 have been found in different species of fish and other nonmammalian vertebrates, including cyclostomes (lamprey (8,9), holocephalans (ratfish) (10), teleosts (catfish) (11), salmon (12), trout (13), goldfish (14), frogs (15)(16)(17)(18), and chicken (13).…”

Peptidomics

“…Instead of homogenizing the tissue in water with 0.25% acetic acid [28], we used an extraction mixture containing 90% methanol, 9% water and 1% acetic acid, which will denature and inactivate any proteases immediately. A similar extraction method was used in the past for classical neuropeptide purifications [30] as well as in peptidomics [9,14,15,82].…”