Molecular Cloning And Characterization Analysis Of A Gene Encoding Voltage-dependent Calcium Channel From Sea Cucumber Stichopus Japonicus

Voltage-dependent calcium channels (VDCC) are special proteins that are located in theplasma membrane of virtually all excitable cell types and serve to control the influx of Ca2+ions into the cell. VDCC β subunits can modulate channel activities of VDCC. In this study,the degenerate primers were designed on the basis of conserved sequences of VDCC βsubunit genes in echinoderms (including starfish and sea urchin) and other invertebrates. A443bp fragment of VDCC β subunit cDNA from Stichopus japonicus(SjCaβ) was obtainedusing RT-PCR technique. Based on the sequence of obtained VDCC β subunit, primers weredesigned for the5′and3′extensions by RACE techniques. As a result, two fragments879bpand1019bp from the5′and3′ends were amplified and sequenced. After assembling theabove three cDNA together, the complete gene encoding voltage-dependent calcium channel βsubunit from the intestine of the sea cucumber Stichopus japonicus(SjCaβ)was successfullycloned and analysed. The full length cDNA of SjCaβ was1867bp (GenBank: EU853658)with a complete open reading frame of1614bp, encoding537amino acid residues. Themolecular weight of this protein was estimated to be59.8kD, the pI was8.77and thechemical formula was C2583H4151N771O834S13. There were three cysteine residues were in theamino acid sequences which two of them (Cys410and Cys428) can form the S-S bondspredicted by Dipro. This putative VDCC β subunit protein was hydrophilicity analysed by theprogram ProtScale.Based on sequence analysis and comparison, SjCaβ showed significant homology withcalcium channel β subunit genes from other known invertebrate species. The BID domain (βinteraction domain), PYDVVP--RP---VGPSLKGYEVTDMMQKALFDF, which highlyconserved in voltage-dependent calcium channel β subunits, were detected in the amino acidsequence of SjCaβ. Other two conserved functional domains (src-homology3domain andguanylate kinase domain) of calcium channel β subunit were also found in SjCaβ. In the study,the secondary structure of SjCaβ was predicted, the phylogenetic tree was constructed, andthe three-dimensional molecular structure was generated by homologous modeling to furthercharacterize the gene product. Based on three-dimensional molecular structure modeling, the residues Pro262，Pro266，Pro272and Tyr277were postulated to be the important residues forbinding the β subunit to the α1subunit. These results are important to understanding of thestructure of this channel protein in Stichopus japonicus and its role in signal transduction, andare essential to elucidate some autolysis-related issues in sea cucumber production.