Abstract
Prohormone convertases (PCs) are subtilisin-like proteases responsible for the intracellular processing of prohormones and proneuropeptides in vertebrates and invertebrates. The full-length PC2 cDNA sequence was cloned from pleuropedal ganglion of Haliotis discus hannai, consisted of 2254-bp with an open reading frame of 1989-bp and encoded a protein of 662 amino acid residues. The architecture of Hdh PC2 displayed key features of PCs, including a signal peptide, a pro-segment domain with sites for autocatalytic activation, a catalytic domain, and a pro-protein domain (P-domain). It shares the highest homology of its amino acid sequence with the PC2 from H. asinina and to lesser extent with that of Homo sapiens and Rana catesbeiana PC2. Sequence alignment analysis indicated that Hdh PC2 was highly conserved in the catalytic domain, including a catalytic triad of serine proteinases of the subtilisin family at positions Asp-195, His-236, and Ser-412. The cloned sequence contained a canonical integrin binding sequence, and four cysteine residues involved in the formation of an intramolecular disulfide link. Phylogenetic analysis revealed that the Hdh PC2 is robustly clustered with the Has PC2. Quantitative PCR assay demonstrated that the Hdh PC2 was predominantly expressed in the pleuropedal ganglion rather than in other examined tissues. Although PC2 mRNA was expressed throughout the gametogenetic cycle of male and female abalone, the expression level was significantly higher in the ripening stage of female abalone. Also, a significantly higher expression was observed in the pleuropedal ganglion and gonadal tissues at a higher effective accumulative temperature (1000°C). In situ hybridization revealed that the PC2 mRNA expressing neurosecretory cells were distributed in the cortex region of the pleuropedal ganglion. According to the results, it can be concluded that pleuropedal ganglion is the highest site of PC2 activity, and this enzyme might be involved in the abalone reproduction process.
Highlights
Prohormone convertases (PCs) are Ca2+ dependent subtilisin-like endoproteases and are thought to be involved in the post-translational process of hormones, neuropeptides, and other regulatory proteins [1,2]
The full-length PC2 cDNA sequence was obtained from the pleuropedal ganglion and referred to as Hdh PC2
A BLASTP search indicated that this translated protein sequence showed the highest homology (95%) with the H. asinina PC2 (Has PC2)
Summary
Prohormone convertases (PCs) are Ca2+ dependent subtilisin-like endoproteases and are thought to be involved in the post-translational process of hormones, neuropeptides, and other regulatory proteins [1,2]. Several subtypes of PCs have been identified by molecular cloning and categorized as members of the subtilisin-like endoproteases family These enzymes include furin, PC1/3, PC2, PC4, PACE4, PC5/6, PC7, SKI-1 (Mbtps1), and PC9 [6]. Some PCs (furin and PACE4) exhibited a ubiquitous tissue distribution, whereas the expression of others, including PC1 and PC2, is restricted to neural and endocrine cells [9] Each of these subtypes has distinct characteristics and specificities, similar biochemical properties are found among the members of PCs in both vertebrates and invertebrates [6]. In Xenopus, PC2 plays a crucial role in the processing of proopiomelanocoretin (POMC) to α-MSH [13] It is involved in the processing of egg-laying hormone-related precursors in atrial-gland secretory cells of Aplysia [14]. Homologues of PC2 have been characterized in only few invertebrates, including the nematode Caenorhabditis elegans [16], gastropod mollusk species Lymnaea stagnalis [17], Aplysia californica [14], H. asinina [18], arthropod species Lucilia cuprina [19], Drosophila melanogaster [20], Orconectes limosus [15], and Penaeus monodon [21]
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