Nucleic Acids, Protein Synthesis, and Molecular Genetics
Identification, Cloning, and Characterization of Cystatin M, a Novel Cysteine Proteinase Inhibitor, Down-regulated in Breast Cancer*

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A novel human cystatin gene was identified in a differential display comparison aimed at the isolation of transcriptionally regulated genes involved in invasion and metastasis of breast cancer. Messenger RNAs from primary and metastatic tumor cells isolated from the same patient were compared. A partial cDNA was isolated that was expressed in the primary tumor cell line but not in the metastatic line. The full-length cDNA was cloned and sequenced, and the inferred amino acid sequence was 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 coli as a glutathione S-transferase fusion protein using the pGEX-2T expression system and purified by affinity chromatography. The cystatin M fusion protein displayed inhibitory activity against papain. Native cystatin M protein of approximately 14.5 kDa is secreted and was immunoprecipitated from supernatants of mammary cell cultures using affinity-purified antisera raised against recombinant cystatin M. An N-glycosylated form of cystatin M of 20-22 kDa was co-immunoprecipitated and accounted for about 30-40% of total cystatin M protein. Both forms of native cystatin M also occurred intracellularly. Consistent with the mRNA differential expression, no cystatin M protein was detected in metastatic mammary epithelial tumor cells. Loss of expression of cystatin M is likely associated with the progression of a primary tumor to a metastatic phenotype.

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This work was supported in part by National Institutes of Health Grant CA61253 (to R. S) and an award from the Fulbright Program (CIES, Council for International Exchange of Scholars) (to G. S.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) U62800.

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Present address: Dept. of Pharmacy, School of Health Sciences, University of Patras, Rion 261 10, Patras, Greece. Fax: 30-61-994941;