MOLECULAR-CLONING, FUNCTIONAL EXPRESSION AND TISSUE DISTRIBUTION OF THE CDNA-ENCODING FROG SKELETAL-MUSCLE CALSEQUESTRIN

We have cloned, sequenced and expressed the cDNA encoding frog skeletal muscle calsequestrin. The processed frog calsequestrin is 398 residues long, with an M(r) of 45941 (unglycosylated form), and exhibits 77% sequence similarity with its rabbit counterpart. Consensus sequences for glycosylation and phosphorylation of the protein were conserved. Compared with rabbit calsequestrin, the mature amphibian protein has peculiar structural properties, which include (i) a higher content of negatively charged residues (142 versus 109), and (ii) a striking repeat sequence at the C-terminal region of 44 aspartic acid residues. Furthermore, this is the first report on the expression of calsequestrin cDNA in COS-1 cells: the expressed protein exhibited an M(r) and antigenic properties which were indistinguishable from those of the native protein. In addition, it was capable of binding Ca-45 in a ligand overlay. Northern blot analysis of frog skeletal muscle, liver, heart and brain RNA showed that the protein is mainly expressed in skeletal muscle. The high density of negative charges at the C-terminus might constitute high-capacity low-affinity Ca2+-binding sites, which may account for the higher Ca2+-binding capacity of frog calsequestrin compared with other members of the calsequestrin family (56 mol/mol versus 40-44 mol/mol of protein).