New insights into evolution of crustacean hyperglycaemic hormone in decapods--first characterization in Anomura.

The neuropeptides of the crustacean hyperglycaemic hormone (CHH) family are encoded by a multigene family and are involved in a wide spectrum of essential functions. In order to characterize CHH family peptides in one of the last groups of decapods not yet investigated, CHH was studied in two anomurans: the hermit crab Pagurus bernhardus and the squat lobster Galathea strigosa. Using RT-PCR and 3' and 5' RACE methods, a preproCHH cDNA was cloned from the major neuroendocrine organs (X-organs) of these two species. Hormone precursors deduced from these cDNAs in P. bernhardus and G. strigosa are composed of signal peptides of 29 and 31 amino acids, respectively, and CHH precursor-related peptides (CPRPs) of 50 and 40 amino acids, respectively, followed by a mature hormone of 72 amino acids. The presence of these predicted CHHs and their related CPRPs was confirmed by performing MALDI-TOF mass spectrometry on sinus glands, the main neurohaemal organs of decapods. These analyses also suggest the presence, in sinus glands of both species, of a peptide related to the moult-inhibiting hormone (MIH), another member of the CHH family. Accordingly, immunostaining of the X-organ/sinus gland complex of P. bernhardus with heterologous anti-CHH and anti-MIH sera showed the presence of distinct cells producing CHH and MIH-like proteins. A phylogenetic analysis of CHHs, including anomuran sequences, based on maximum-likelihood methods, was performed. The phylogenetic position of this taxon, as a sister group to Brachyura, is in agreement with previously reported results, and confirms the utility of CHH as a molecular model for understanding inter-taxa relationships. Finally, the paraphyly of penaeid CHHs and the structural diversity of CPRPs are discussed.

Posttranslational isomerization of a neuropeptide in crustacean neurosecretory cells studied by ultrastructural immunocytochemistry.

Isomerization of the third amino acid residue (a phenylalanine) of crustacean hyperglycemic hormone (CHH) has been previously reported to occur as a late step of hormone precursor maturation in a few neurosecretory cells in the X-organ-sinus gland complex of the crayfish Orconectes limosus. In the present report, using conformation-specific antisera combined with immunogold labeling, we have studied, at the ultrastructural level, the distribution of L- and D-CHH immunoreactivity in CHH-secreting cells of the crayfish Astacus leptodactylus. Two CHH-secreting cell populations were observed, the first one (L-cells), the most numerous, exhibited only labeling for L-CHH. In the second one (D-cells), four secretory granule populations were distinguished according to their labeling: unlabeled, either L- or D- exclusively or both L- and D-granules. Labeling quantification by image analysis in D-cells showed a marked increase in D-labeling from the cell body to the axon terminal. However some L- and mixed granules remain in axon terminals. Our results demonstrate that Phe3 isomerization of CHH occurs within the secretory granules of specialized neurosecretory cells and progresses as the granules migrate along the axonal tract. The observation that not all the CHH synthesized is isomerized, and the great variability in the proportion of L- and D-immunoreactivity in granules in every cell region may suggest an heterogeneous distribution of the putative enzyme involved in Phe3 isomerization, a peptide isomerase, within the secretory pathway.

Molecular and cellular specificity of post-translational aminoacyl isomerization in the crustacean hyperglycaemic hormone family.

D-aminoacyl residues have been detected in various animal peptides from several taxa, especially vertebrates and arthropods. This unusual polymorphism was shown to occur in isoforms of the crustacean hyperglycaemic hormone (CHH) of the American lobster because a D-phenylalanyl residue was found in position 3 of the sequence (CHH and D-Phe3 CHH). In the present study, we report the detailed strategy used to characterize, in the lobster neuroendocrine system, isomers of another member of the CHH family, vitellogenesis inhibiting hormone (VIH). We have demonstrated that the fourth residue is either an L- or a D- tryptophanyl residue (VIH and D-Trp4 VIH). Furthermore, use of antisera specifically recognizing the epimers of CHH and VIH reveals that aminoacyl isomerization occurs in specialized cells of the X organ-sinus gland neurosecretory system and that the D-forms of the two neuropeptides are not only present in the same cells, but, importantly, also are co-packaged within the same secretory vesicles.