Characterization of cDNAs for calmodulin and calmodulin-like protein in the freshwater mussel Hyriopsis cumingii: Differential expression in response to environmental Ca2 + and calcium binding of recombinant proteins

Calmodulin and calmodulin-like protein are two crucial calcium regulators in bivalves. However, molecular characteristics and expression patterns of these genes in the freshwater mussel are poorly understood. In this study, two cDNAs encoding novel calmodulin and calmodulin-like protein (HcCaM and HcCaLP) were cloned and characterized from the freshwater pearl mussel Hyriopsis cumingii. The full-length cDNA of HcCaM was 726 bp, including a 118-bp 5′-untranslated region (UTR), a 447-bp open reading frame (ORF), and a 161-bp 3′-UTR. The 1217-bp HcCaLP cDNA comprised of a 51-bp 5′-UTR, a 447-bp ORF, and a 716-bp 3′-UTR. The potential phosphorylation sites of, Arg80 and Phe100 in deduced HcCaM were mutated to Thr80 and Tyr100 in HcCaLP. Tissue-specific expression analysis revealed that HcCaM mRNA was prominently expressed in the gill, mantle center, and foot. In contrast, HcCaLP mRNA was mainly expressed in the mantle edge. The recombinant HcCaM and HcCaLP proteins expressed in Escherichia coli showed the typical Ca2 + dependent electrophoretic shift characterization as CaM and differed in the calcium binding affinity. The calcium stimulation test that lasted 5 weeks implied that HcCaM and HcCaLP had differential expression patterns in response to various environmental Ca2 + concentrations (0.25–1.25 mM). The expression of HcCaM mRNA was up-regulated by low Ca2 + concentration (0.25 mM), and the highest expression of HcCaLP mRNA occurred under Ca2 + concentration of 1 mM.