Gonad-inhibiting hormone of the Norway lobster (Nephrops norvegicus): cDNA cloning, expression, recombinant protein production, and immunolocalization.
The gonad-inhibiting hormone (GIH) belongs to a neuropeptide family synthesized and released in a neurohemal complex of crustacean eyestalks. The GIH is involved in gonad maturation and plays a more complex role in the control of reproduction and molting. With a combination of reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends approaches we determined the cDNA sequence of the Norway lobster Nephrops norvegicus prepro GIH. The open reading frame of 339 bp codes for a polypeptide of 112 amino acids showing 96% identity with the other known GIH of Homarus americanus. The precursor peptide consists of a putative signal peptide of 31 amino acids and a putative mature peptide region of 81. RT-PCR analysis shows that GIH mRNA is expressed mainly in eyestalks, both in female and male; the expression of GIH mRNA also in supraesophageal ganglia suggests the existence of additional GIH-producing neurons besides those of eyestalks. A specific polyclonal antibody was raised against a portion of the mature peptide region obtained through expression in Escherichia coli fused to glutathione-S-transferase. Immunocytochemical studies were carried out by using this antibody in N. norvegicus and in other crustaceans, Munida rugosa and Squilla mantis; these locate GIH in superficial axon terminals of the releasing organ, the sinus gland. The identification of a second GIH sequence in crustaceans allows to hypothesize the occurrence, within the neuropeptide family, of three subfamilies probably involved in different functions: crustacean hyperglycemic hormones, GIHs and molt-inhibiting hormones/mandibular organ-inhibiting hormones.
Edomi
P
P
BRAIN Center, Department of Biology, University of Trieste, via Giorgieri 7, I-34127, Trieste, Italy. edomi@univ.trieste.it
Azzoni
E
E
Mettulio
R
R
Pandolfelli
N
N
Ferrero
E A
EA
Giulianini
P G
PG
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D005260:Female; D015870:Gene Expression; D007150:Immunohistochemistry; D007447:Invertebrate Hormones; D008297:Male; D008969:Molecular Sequence Data; D008121:Nephropidae / Q000235:genetics*; D009479:Neuropeptides / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D002352:Carrier Proteins; D018076:DNA, Complementary; D007447:Invertebrate Hormones; D009479:Neuropeptides; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C073189:vitellogenesis inhibiting hormone