Purification and sequencing of molluscan insulin-related peptide I (MIP I) from the neuroendocrine light green cells of Lymnaea stagnalis

Abstract

The body growth controlling cerebral neuroendocrine light green cells of the freshwater snail, Lymnaea stagnalis, express various members of a gene family encoding different though related prepromolluscan insulin-related peptides. In the present study, molluscan insulin-related peptide I (MIP I) together with the corresponding connecting peptide, C alpha peptide, have been isolated and structurally identified. MIP I is a heterodimer of A and B chains bonded by disulphide bridges. Two isoforms of MIP I could be discerned. Mass spectrometry revealed that of one form both the A and B chains have N-terminal pyroglutamyl residues, whereas of the other form only the B chain has such residues. After removal of the pyroglutamyl residues with pyroglutamate aminopeptidase, followed by disulphide bond cleavage and pyridylethylation of cysteine residues, the sequences of MIP I have been determined using Edman degradation as: A chain: (p)QGTTNIVCECCMKPCTLSELRQYCP; B chain: pQPSACNINDRPHRRGVCGSALADLVDPACSSSNGPA. The C alpha peptide has also been isolated and its sequence was determined as NAETDLDDPLRNIKLSSESALTYLY. These sequences are in agreement with those predicted by a cDNA sequence encoding preproMIP I, with the exception that the two C-terminal amino acids of the B chain are posttranslationally removed.