Molecular cloning of a histidine-rich Ca2+-binding protein of sarcoplasmic reticulum that contains highly conserved repeated elements

We reported previously the purification of a 165-kDa muscle-specific protein identified by virtue of its ability to bind 125I-labeled low density lipoprotein with high affinity after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Hoffmann, S. L., Brown, M. S., Lee, E., Pathak, R. K., Anderson, R. G. W., and Goldstein, J. J. (1989) J. Biol. Chem. 264, 8260-8270). The protein is located in the lumen of the sarcoplasmic reticulum, where it has no access to plasma lipoproteins. It binds to 45Ca2+ on nitrocellulose blots and stains metachromatically blue with Stains-all, a cationic dye that stains Ca2+-binding proteins. In the current paper, we have isolated a full-length rabbit cDNA clone for the 165-kDa protein. The deduced amino acid sequence reveals a 852-amino acid protein with the following structural features: 1) an NH2-terminal 27-residue putative signal sequence; 2) a highly repetitive region containing nine nearly identical tandem repeats of 29 residues, each consisting of a histidine-rich sequence HRHRGH, a stretch of 10-11 acidic amino acids, and a sequence containing 2 serines and a threonine in a negatively charged context; 3) a 13-residue stretch of polyglutamic acid; and 4) a COOH-terminal cluster of 14 closely spaced cysteine residues with the repeating pattern of Cys-X-X-Cys suggestive of a heavy metal binding domain. Histidine, aspartic acid, and glutamic acid accounted, respectively, for 13, 12, and 19% of the amino acids. The protein does not share any significant sequence homology with the cell surface low density lipoprotein receptor. Stretches of acidic amino acids are a feature of two other luminal sarcoplasmic reticulum proteins, suggesting that these may be a general feature of luminal sarcoplasmic reticulum proteins. We suggest that the histidine-rich Ca2+-binding protein described in the current study be designated HCP. The role of HCP in Ca2+ homeostasis in the sarcoplasmic reticulum of skeletal and cardiac muscle remains to be determined.