Molecular characterization and spatiotemporal expression of prohormone convertase 2 in the Pacific abalone, Haliotis discus hannai

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.

Carbonic Anhydrase in Pacific Abalone Haliotis discus hannai: Characterization, Expression, and Role in Biomineralization

“…Cerebral ganglion, mantle, gill, heart, shell muscle, hemocyte, and gonadal tissues (testis and ovary) were dissected, immediately frozen in liquid N 2 , and stored at −80°C for subsequent molecular analyses. Cryosection preparation from shell-forming mantle tissue was performed as described previously ( Sharker et al, 2020a , b ). All animal experiments were performed in accordance with guidelines of the Institutional Animal Care and Use Committee of Chonnam National University (approval number: CNU IACUC-YS-2020-5).…”

Molecular Identification, Characterization, and Expression Analysis of a Gonadotropin-Releasing Hormone Receptor (GnRH-R) in Pacific Abalone, Haliotis discus hannai

“…ISH was carried out according to a method described previously [ 44 , 45 , 46 ]. Briefly, tissue sections were pre-hybridized with hybridization buffer and yeast total RNA (50 μL) for 2 h and then hybridized with the RNA probe at 65 °C overnight.…”

Characterization of Insulin-Like Growth Factor Binding Protein 7 (Igfbp7) and Its Potential Involvement in Shell Formation and Metamorphosis of Pacific Abalone, Haliotis discus hannai

“…All samples were immediately frozen in liquid nitrogen and stored at −80 °C until total RNA extraction. For in situ hybridization, cryosections were prepared from mantle and gill tissues following a previously reported method by Sharker et al [ 49 , 50 , 51 ].…”