Cloning and sequence analysis of cDNA for precursor of a crustacean hyperglycemic hormone

Abstract

Crustacean hyperglycemic hormone (CHH) from Curcinus maenus, a 72 amino acid neuropeptide, originates in neurosecretory perikarya in the eyestalk ganglia. Poly (A)RNA was isolated from these perikarya and a cDNA library was prepared. Screening of 20000 clones with a 26-mer oligonucleotide, corresponding to a partial sequence of CHH, yielded one positive clone with an insert of approximately 2000 bp, which contained the complete coding sequence for a pre-pro CHH. This precursor consists of a putative 26 amino acid signal sequence, a 38 amino acid peptide of unknown function (Peptide C), and the CHH sequence at the carbofyl end. The CHH-sequence is flanked N-terminally by a Lys-Arg cleavage site and C-terminally by the tetrapeptide Gly-Arg-Lys-Lys which is followed by the stop codon.

Midgut epithelial endocrine cells are a rich source of the neuropeptides APSGFLGMRamide (Cancer borealis tachykinin-related peptide Ia) and GYRKPPFNGSIFamide (Gly1-SIFamide) in the crabs Cancer borealis, Cancer magister and Cancer productus

“…Before this study, only two crustacean brain-gut peptides had been identified: crustacean hyperglycemic hormone and crustacean hyperglycemic hormone precursor-related peptide, both of which are found in the nervous system and in the foregut and midgut of the crab C. maenas (Kegel et al, 1989;Weidemann et al, 1989;Tensen et al, 1991;Chung et al, 1999;Webster et al, 2000;Dircksen et al, 2001). Our identification of Gly 1 -SIFamide and CabTRP Ia in the Cancer midgut now brings to four the number of fully characterized brain-gut peptides in decapod species, and suggests the possibility that there may be a myriad of such peptides in the midgut epithelium of decapod crustaceans, as there are in insects.…”

Molecular cloning of crustacean putative molt‐inhibiting hormone (MIH) precursor

“…Both peptides share 28% of identical amino acid residues. The cDNA structure of the CHH precursor has previously been established in our laboratory [9]. Comparison of CHH and MIH precursors reveals striking and interesting differences.…”

“…To confirm this, it would be highly interesting to know the structure of the VIH precursor. Although CHH precursors other than that of Carcinus [9] have not yet been published, there is indirect evidence that they share structural characteristics, in particular the presence of CPRPs. Such peptides have been isolated and sequenced from the sinus glands of four species [17].…”

“…To amplify cDNA encoding MIH, DNA of a previously generated library from MTXO neurosecretory cells of Carcinus maenas [9] served as template for the PCR. Two degenerated oligonucleotides, deduced from the amino acid sequence of Carcinus MIH [6] were used as primers (MI: …”

“…We have previously elucidated the complete structure of the Carcinus CHH precursor [9] and describe here the complete MIH precursor of this species by isolation of a full length clone from the same cDNA-library.…”