Cystatin E is a Novel Human Cysteine Proteinase Inhibitor with Structural Resemblance to Family 2 Cystatins

Abstract

A new member of the human cystatin superfamily, called cystatin E, has been found by expressed sequence tag (EST) sequencing in amniotic cell and fetal skin epithelial cell cDNA libraries. The sequence of a fulllength amniotic cell cDNA clone contained an open reading frame encoding a putative 28-residue signal peptide and a mature protein of 121 amino acids, including four cysteine residues and motifs of importance for the inhibitory activity of Family 2 cystatins like cystatin C. Recombinant cystatin E was produced in a baculovirus expression system and isolated. An antiserum against the recombinant protein could be used for affinity purification of cystatin E from human urine, as confirmed by N-terminal sequencing. The mature recombinant protein processed by insect cells started at amino acid 4 (cystatin C numbering), and displayed reversible inhibition of papain and cathepsin B (K i values of 0.39 and 32 nM, respectively), in competition with substrate. Cystatin E is thus a functional cysteine proteinase inhibitor despite relatively low amino acid sequence similarities with human cystatins (26 -34% identity with sequences for the Family 2 cystatins C, D, S, SN, and SA; <30% with the Family 1 cystatins, A and B, and domains 2 and 3 of the Family 3 cystatin, kininogen). Unlike other human low M r cystatins, cystatin E is a glycoprotein, carrying an N-linked carbohydrate chain at position 108. Northern blot analysis revealed that the cystatin E gene is expressed in most human tissues, with the highest mRNA amounts found in uterus and liver. A strikingly high incidence of cystatin E clones in cDNA libraries from fetal skin epithelium and amniotic membrane cells (>0.5% of clones sequenced) indicates a protective role of cystatin E during fetal development.

Frequent epigenetic inactivation of cystatin M in breast carcinoma

“…Cystatin M, a potent inhibitor of human lysosomal proteases, was identified as a gene frequently transcriptionally downregulated during progression of breast cancer (Ni et al, 1997;Sotiropoulou et al, 1997;Zhang et al, 2004). In our study, we demonstrated that hypermethylation of the cystatin M promoter occurs frequently in primary breast carcinomas and breast cancer cell lines.…”

“…Thus, it has been proposed that cystatin M may function as candidate tumour suppressor for breast carcinogenesis . Cystatin M is a potent inhibitor of cysteine peptidases, including papain, cathepsin B, cathepsin V and cathepsin L (Ni et al, 1997;Cheng et al, 2006). These peptidases play key roles in intracellular protein degradation.…”

“…Disturbance of the balance between proteases and their cystatins can lead to irreversible damage and tumour development (Calkins and Sloane, 1995;Henskens et al, 1996). Cystatin M is a new member of the human cystatin gene family and demonstrates more diverse tissue distribution and target specificity than other cystatins (Ni et al, 1997;Sotiropoulou et al, 1997). Cystatin M was first identified and cloned by differential RNA display as a transcript that is downregulated in metastatic breast cancer cells when compared to primary breast cancer cells (Ni et al, 1997;Sotiropoulou et al, 1997).…”

“…Cystatin M is a low molecular mass protein sharing 27-32% homology to other cystatins. The protein can be secreted from cells in a glycosylated (17 kDa) and unglycosylated (14.4 kDa) form (Ni et al, 1997). It has been reported that constitutive expression of cystatin M in human breast cancer cells MDA-MB-435S significantly reduces in vitro cell proliferation, migration, Matrigel invasion and tumour-endothelial cell adhesion .…”

Methylation-dependent silencing of CST6 in primary human breast tumors and metastatic lesions

“…Cystatin M (CST6) functions as an endogenous inhibitor of lysosomal cysteine proteases (Ni et al, 1997;Sotiropoulou et al, 1997), which are known to contribute to tumor cell invasion by degrading extracellular matrix components (Buck et al, 1992;Maciewicz et al, 1990;Mai et al, 2002). Cathepsin B and cathepsin L are two important cysteine proteases that have been implicated in tumor cell invasion and metastasis (Kane and Gottesman, 1990;Roshy et al, 2003;Slone et al, 1981).…”