RESUMEN
The neuro-endocrine X-organ sinus-gland complex of crustaceans produces and releases the neuropeptides of the crustacean hyperglycemic hormone (cHH)/molt-inhibiting hormone (MIH)/gonad-inhibiting hormone (GIH) family that regulate important physiological processes, such as growth, reproduction and molting. We cloned two full-length cDNAs encoding the preprocHH-A and preprocHH-B of the Norway lobster Nephrops norvegicus of 132 and 131 amino acid residues. The two cHHs differ in the preprohormone but not in the mature peptide sequence. The mature cHH was expressed in bacteria as GST fusion protein that, in bioassay, shows a hyperglycemic activity similar to that of native cHH present in an eyestalk extract.
Asunto(s)
Hormonas de Invertebrados/química , Nephropidae/genética , Proteínas del Tejido Nervioso/química , Precursores de Proteínas/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Proteínas de Artrópodos , Secuencia de Bases , Bioensayo , Western Blotting , Clonación Molecular , Secuencia Conservada , ADN Complementario/química , ADN Complementario/genética , Expresión Génica , Glutatión Transferasa/metabolismo , Hormonas de Invertebrados/metabolismo , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/metabolismo , Neuropéptidos/genética , Neuropéptidos/metabolismo , Reacción en Cadena de la Polimerasa , Precursores de Proteínas/metabolismo , ARN Mensajero/análisis , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de AminoácidoRESUMEN
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.