ISOLATION AND MOLECULAR-CLONING OF HUMAN SORCIN A CALCIUM-BINDING PROTEIN IN VINCRISTINE-RESISTANT HOB1 LYMPHOMA-CELLS

A vincristine-resistant lymphoma cell line (HOB1/VCR(1.0)) that is res istant to 1.0 mu M Of vincristine has been established from a human im munoblastic B lymphoma cell line, HOB1. HOB1/VCR(1.0) cells demonstrat ed the typical multidrug resistant phenotypes. Using two-dimensional g el electrophoresis, we discovered one protein with a molecular mass of 22 kDa and pi 5.7 that was overexpressed in HOB1/VCR(1.0) cells. This protein was purified to the degree of apparent homogeneity by prepara tive isoelectric focusing and sodium dodecylsulfate-polyacrylamide gel electrophoresis. The identification of this protein with sorcin was r evealed by comparing the internal amino acid sequence of three Lys-C d igested peptides from the purified protein with the sequence previousl y determined for hamster sorcin. The complete primary structure of the human sorcin was deduced from nucleotide sequence analysis of its cDN A clones. It is composed of 198 amino acid residues with a calculated molecular weight of 21 676, and its sequence is highly similar to that of hamster sorcin (95%). Direct-binding assay with calcium showed tha t human sorcin is a calcium-binding protein with four 'E-F hand' struc tures typical of calcium-binding sites. Like the sorcin of hamster, tw o of the calcium-binding sites of human sorcin contain putative recogn ition sites for cAMP-dependent protein kinase. Southern and Northern b lot analyses showed that the human sorcin gene was greatly amplified a nd overexpressed in resistant HOB1/VCR(1.0) cells but not detected in the parental HOB1 cells. The overproduction of this protein in resista nt cells implies that sorcin plays a role in expression of the resista nt phenotype.