G. Sotiropoulou et al., IDENTIFICATION, CLONING, AND CHARACTERIZATION OF CYSTATIN-M, A NOVEL CYSTEINE PROTEINASE-INHIBITOR, DOWN-REGULATED IN BREAST-CANCER, The Journal of biological chemistry, 272(2), 1997, pp. 903-910
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.