Characterization of a cDNA clone encoding molluscan insulin‐related peptide II of Lymnaea stagnalis

Abstract

A cDNA clone encoding molluscan insulin-related peptide (MIP) I1 was isolated from a cDNA library of the central nervous system (CNS) of the freshwater snail, Lymnaea stagnalis, using a heterologous screening with a previously identified MIP cDNA (renamed MIP-I cDNA). The MIP-I1 cDNA encodes a preprohormone resembling the organization of preproinsulin, with a putative signal sequence, and A and B chains; however, in this case connected by two distinct C peptides, Ca and CP, instead of a single C peptide, a phenomenon which represents a new development in the prohormone organization of peptides belonging to the insulin superfamily. The A and B chains of MIP I1 and I differ remarkably in primary structure; in contrast, the Ca peptide domains are fully identical. MIP I1 has only limited sequence similarity with insulins and related peptides. Both MIP I1 and I exhibit structural features, which make them a unique class of the insulin superfamily. The MIP I and I1 genes are expressed in a single type of neuron: the growth-controlling neuroendocrine light green cells of the Lymnaea CNS. Note. The novel nucleotide sequence data published here have been submitted to the EMBL, GENBANK and BOBJ sequence data bank(s) and are available under accession number(s) X59302. tion, with the exception of an extra C-peptide domain, which is present in preproMIP 11. Although MIP I and I1 differ substantially, they have structural features in common that are absent in other insulins and related peptides. This makes them a unique class of the insulin superfamily. MATERIALS AND METHODS Screening of the Lymnaea cDNA libraryWe screened 20000 clones of an amplified cDNA library in Lgtl0 of the CNS of L. stagnalis [16], using Hybond-N membranes (Amersham International Corporation) at a density of 5000 plaque-forming units/l35-mm filter. Clones were purified by rescreening at a lower plaque density. Plaque lifts were carried out as recommended by the manufacturer. Hybridization of the membranes was performed in 6 x NaCl/Cit (NaCl/Cit: 0.15 M NaCl and 15 mM sodium citrate) for 16 h at 65°C. The filters were washed in NaCl/Cit for 45 min at 65°C. Autoradiography was done on preflashed Kodak X-OMAT using an intensifying screen. MIP-I cDNA radiolabelingRadiolabeled cDNA was synthesized by primer extension on a single-stranded M13 MIP-I cDNA clone [8] using the Klenow fragment of DNA polymerase I. The M13-specific primer 5'-TGACCGGCAGCAAAATG-3' was used. [a-32P]dATP was incorporated during the reaction. A mixture of 0.1 pmol M13 single-stranded DNA and 0.5 pmol primer were heated to 95°C and allowed to cool to 20°C. To the mixture, 10 pCi [E~'P]~ATP and 7 U Klenow enzyme (Boehringer Mannheim) were added and the reaction was carried out for 20 min at 20°C. A chase reaction was performed by adding 0.2 mM dATP for 10 min at 20°C. Next,

Isolation and chemical characterization of a novel insulin‐related neuropeptide from the freshwater snail, Lymnaea stagnalis

“…The residues at A5 and A15 of MIP 111 that could not be detected (Table 2) very likely represent modified glutamic acid residues. This is based on studies (K. W. Li, unpublished results) showing that predictcd glutamic acid residues of MIP I and I1 [5,6] at identical positions could also not be detected. In all three MIP, both the A and B chains are extended at the N-terminus and two extra cysteine residues are present in the N-terminal parts (at A4 and B-6), indicating that a third interchain disulphide bridge may be present.…”

“…These cells are present as four clusters of altogether approximately 150 identifiable giant neurones, which use the periphery of the two median lip nerves as their neurohaemal area. Endocrinological experiments have shown that the secretions of the LGC control various different processes, such as body growth [2, 31, carbohydrate metabolism [3] and reproductive activities [4].Molecular genetic studies have demonstrated that the LGC express two genes coding for the distinct, though related, molluscan insulin-related peptides (MIP) I and I1 [5,6]. Here, we present evidence that the LGC express yet another MIP, designated MIP 111, which has only approximately N 50% similarity with MIP I and 11.…”

Molecular cloning of a cDNA encoding insulin-like androgenic gland factor from the kuruma prawn Marsupenaeus japonicus and analysis of its expression

“…5). Insulin family peptides also have the six Cys residues [19][20][21][22][23]. The six Cys residues in the insulin family peptides form two interchain disulfide bonds between the B chain and the A chain, and an intrachain disulfide bond in the A chain.…”

Biosynthesis of Insulin

“…Proinsulin-like molecules in molluscs and insects have C peptides roughly similar in size to those in vertebrates. These also are excised by cleavage at basic residue pairs (138,271).…”