IDENTIFICATION, CLONING, AND CHARACTERIZATION OF CYSTATIN-M, A NOVEL CYSTEINE PROTEINASE-INHIBITOR, DOWN-REGULATED IN BREAST-CANCER

A novel human cystatin gene was identified in a differential display c omparison aimed at the isolation of transcriptionally regulated genes involved in invasion and metastasis of breast cancer. Messenger RNAs f rom primary and metastatic tumor cells isolated from the same patient were compared. A partial cDNA was isolated that was expressed in the p rimary tumor cell line but not in the metastatic line. The full-length cDNA was cloned and sequenced, and the inferred amino acid sequence w as found to encode a novel protein, which we named cystatin M, with 40 % homology to human family 2 cystatins and similar overall structure, Cystatin M is expressed by normal mammary cells and a variety of human tissues. The mature cystatin M protein was produced in Escherichia co li as a glutathione S-transferase fusion protein using the pGEX-2T exp ression system and purified by affinity chromatography. The cystatin M fusion protein displayed inhibitory activity against papain. Native c ystatin M protein of approximately 14.5 kDa is secreted and was immuno precipitated from supernatants of mammary cell cultures using affinity -purified antisera raised against recombinant cystatin M. An N-glycosy lated form of cystatin M of 20-22 kDa was co-immunoprecipitated and ac counted for about 30-40% of total cystatin M protein. Both forms of na tive cystatin M also occurred intracellularly. Consistent with the mRN A differential expression, no cystatin M protein was detected in metas tatic mammary epithelial tumor cells. Loss of expression of cystatin M is likely associated with the progression of a primary tumor to a met astatic phenotype.