ZHENG, J; CHOI, C.Y.; WATSON, R.D.*; University of AL at Birmingham: Molecular Cloning of cDNAs Encoding Two Crustacean Hyperglycemic Hormone Isoforms From the Blue Crab (Callinectes sapidus)

Crustacean hyperglycemic hormone (CHH) is a peptide hormone first purified from eyestalk neural tissue. CHH has been implicated in regulation of several physiological processes, including carbohydrate metabolism, ecdysis, water and ion balance, and reproduction. We report here the molecular cloning of cDNAs encoding two putative CHH preprohormones from the blue crab Callinectes sapidus. Using a PCR based cloning strategy (RT PCR followed by 5′ and 3′ RACE), we cloned from blue crab eyestalk ganglia a cDNA (CsCHH-1) encoding a putative CHH. Sequence analysis of CsCHH-1 revealed a 417 bp open reading frame encoding a 139 residue peptide (CsCHH-1). The deduced amino acid sequence of CsCHH-1 includes a 26 residue signal peptide, a 38 residue CHH precursor related peptide (CPRP), a 72 residue mature CHH, and a C-terminal protease processing site. The mature CHH-1 showed high sequence homology to known brachyuran CHHs. RT-PCR revealed the Cs-CHH-1 transcript was present in eyestalk ganglia, but undetectable in thoracic ganglion, ventral nerve cord, brain, muscle, or gill. An amplified band similar in size to CsCHH-1 was present in thoracic ganglion, ventral nerve cord, brain, and Y-organ, but was undetectable in eyestalk ganglia. Cloning and sequence analysis of this second cDNA (CsCHH-2) revealed a 408 bp open reading frame encoding a second putative CHH isoform (CsCHH-2). The deduced amino acid sequence of CsCHH-2 includes a 26 residue signal peptide, a 38 residue CPRP, and a 71 residue mature CHH. The signal peptide and CPRP are identical to those of CsCHH-1. The 71 residue mature CHH-2 shows 62.7% sequence identity to the mature CHH-1 isoform. The physiological significance of the two CHH isoforms is unknown. Grant sponsor: National Science Foundation, IBN-0213047 (RDW).