title: Identification, cloning, and characterization of cystatin M, a novel cysteine proteinase inhibitor, down-regulated in breast cancer.
abstract: 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.
authors.affiliation: Division of Cancer Genetics, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA. G.Sotiropoulou@upatras.gr
authors.forename: G
authors.initials: G
authors.lastname: Sotiropoulou
authors.forename: A
authors.initials: A
authors.lastname: Anisowicz
authors.forename: R
authors.initials: R
authors.lastname: Sager
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D001943:Breast Neoplasms / Q000235:genetics*; D055332:Cystatin M; D015891:Cystatins / Q000737:chemistry / Q000235:genetics*; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry / Q000235:genetics*; D015536:Down-Regulation; D005260:Female; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016415:Sequence Alignment
chemical_list: C525104:CST6 protein, human; D055332:Cystatin M; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors