Characterization of insulin and atypically processed proglucagon-
derived peptides from the Surinam toad Pipa pipa (Anura:Pipidae)
Beverly Matutte, J. Michael Conlon*
Regulatory Peptide Center, Department of Biomedical Sciences, Creighton University Medical School, Omaha, NE 68178-0405, USA
Received 5 May 2000; accepted 19 June 2000
Abstract
Electrospray mass spectrometry was used to identify insulin, glucagon and two peptides related to glucagon-like peptide-1 (GLP-1) in an extract
of the pancreas of the Surinam toad, Pipa pipa, a species belonging to the same family as the African clawed frog, Xenopus laevis. Purification
and characterization of the peptides established the primary structure of Pipa insulin as A-chain: GIVEQCCHSS
10
CTLLQLETYC
20
N and
B-Chain: FSNQR LCGSH
10
LVEALHLVCG
20
DRGFFYYPKA
30
. This amino acid sequence contains several substitutions (B5 His 3 Arg, B16
Tyr 3 His, A12 Ser 3 Thr, A14 Tyr3 Leu, A18Asn 3 Thr) of residues that have otherwise been quite strongly conserved during vertebrate
evolution. Pipa glucagon comprises 37 amino acid residues (HSQGTFTSDY
10
SKYLDSRRAQ
20
DFVQWLMNTK
30
QSGGLSS) and the 29
amino-acid-residue peptide was not identified in the extract. In Xenopus and mammalian preproglucagons, the glucagon-29 sequence is
followed by Lys-Arg which functions as a recognition site for a prohormone convertase. We propose that a point mutation in the gene
encoding Pipa preproglucagon has transformed the Lys
30
-Arg
31
processing site into Lys-Gln with the result that the site in no longer
recognized by the processing enzyme. In contrast, Pipa GLP-32 and GLP-37 are of the same molecular size as the corresponding peptides
from Xenopus. © 2000 Elsevier Science Inc. All rights reserved.
Keywords: Insulin; Glucagon; GLP; Posttranslational processing
1. Introduction
The structure and expression of the glucagon gene and
the pathways of post-translational processing of preproglu-
cagon in vertebrates are complex and both species- and
tissue-dependent [19,24]. Nucleotide sequence analysis of
cDNAs encoding preproglucagons from several mammalian
species have shown that glucagon is cosynthesized with two
structurally related peptides, termed glucagon-like pep-
tide-1 (GLP-1) and glucagon-like peptide-2 (GLP-2) [19].
In mammals, these peptides are secreted separately from the
small intestine but not from the pancreas [22]. In the case of
teleost fish, analysis of cloned DNA complementary to
preproglucagon mRNA from the pancreatic islets of the
anglerfish, Lophius americanus showed that neither of the
two preproglucagons from this species contains a region
corresponding to mammalian GLP-2 [21]. However, anal-
ysis of cDNAs encoding preproglucagons isolated from the
trout intestine demonstrated that the precursor contained the
GLP-2 sequence and it was proposed that an alternative
RNA splicing mechanism generates a preproglucagon
mRNA in the pancreas that lacks the region encoding the
GLP-2 sequence whereas the intestinal precursor encodes
both GLP-1 and GLP-2 [18].
The situation in Amphibia is further complicated by the
observation that in some species, the proglucagon contains
multiple copies of the GLP-1 sequence in addition to
GLP-2. It has been shown that a cloned cDNA encoding
preproglucagon from the African clawed toad Xenopus lae-
vis (Pipidae) contains the sequence of three peptides (GLP-
1A, -1B and -1C) with structural similarity to human GLP-1
in addition to sequences corresponding to mammalian glu-
cagon and GLP-2 [17]. Similarly, two peptides with 32- and
37-amino acid residues that resembled GLP-1 in both struc-
ture and insulinotropic activity were isolated from the pan-
creas of the cane toad, Bufo marinus (Bufonidae) [5]. In
contrast, only a single molecular form of GLP-1 was iso-
lated from the pancreata of the American bullfrog Rana
catesbeiana (Ranidae) [25], the African bullfrog Pyxicepha-
lus adspersus (Ranidae) [10], the S. American horned frog
* Corresponding author. Tel.: ϩ1-402-280-1733; fax: ϩ1-402-280-
2690.
E-mail address: jmconlon@creighton.edu (J.M. Conlon).
Peptides 21 (2000) 1355–1360
0196-9781/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved.
PII: S0196-9781(00)00278-3