Title

Structure and function of mandibular organ inhibiting hormones from the crustacean sinus gland

Date of Completion

January 1997

Keywords

Biology, Molecular|Biology, Cell

Degree

Ph.D.

Abstract

It has been known that factors, so-called mandibular organ inhibiting hormones (MOIH), from the crustacean sinus gland (SG) inhibit the production of juvenile hormone-like compound, methylfarnesoate from the mandibular organ (MO). These factors were isolated and characterized. Their functions other than MO inhibition were also investigated. Studies on a newly discovered neuropeptide family from SG using an in vitro MO bioassay on the crayfish Procambarus indicated that MOIHs are members of this neuropeptide family, which includes crustacean hyperglycemic hormone (CHH), molting inhibiting hormone (MIH) and vitellogenesis inhibiting hormone (VIH). Using two-step reverse phase HPLC, three peptides with MOIH activity, namely P21, P22, and P25 were isolated and purified from the spider crab Libinia emarginata. SDS-PAGE showed that all three peptides had the same molecular weight. The Molecular mass was determined to be 8,439 for P25, 8,474 for P22, and 8,398 for P21 by mass spectrometry. All three peptides have similar amino acid compositions and contain 72-76 residues. These MOIHs caused significant hyperglycemia when injected into the fiddler crab Uca pugilator. The primary structure of MOIH P22 has been determined. It has 72 amino acid residues (deduced molecular mass 8490.5 Da) with pyroglutamic acid at the N-terminus and NH$\sb2$ at the C-terminus. It shares a high percentage of sequence identity with other members of the aforementioned CHH neuropeptides family. A cDNA library was constructed using enriched polyA(+) RNA from the eyestalks of the crab Libinia emarginata and lZiplox cDNA cloning system (BRL). A 137 bp probe was generated by RT-PCR using degenerated primers derived from the amino acid sequence of the MOIH. Screening of 500,000 clones from the cDNA library with the 137 bp probe resulted in six positive clones. One of the positive clones contained a full length cDNA sequence of 972 bp encoding the MOIH P22. This cDNA sequence encodes a preprohormone peptide with 137 amino acid residues, including a 26-amino acid long signal peptide, a 34-amino acid long precursor peptide, a dibasic peptide, the full length of 72-amino acid long MOIH P22, and a tri-peptide Gly-Lys-Lys which designates the potential amidation site at the C-terminus of the mature peptide. ^