Biochemical characterization of human cathepsin X revealed that the enzyme is an exopeptidase, acting as carboxymonopeptidase or carboxydipeptidase

Abstract

Cathepsin X, purified to homogeneity from human liver, is a single chain glycoprotein with a molecular mass of < 33 kDa and pI 5.1±5.3. Cathepsin X was inhibited by stefin A, cystatin C and chicken cystatin (K i 1.7±15.0 nm), but poorly or not at all by stefin B (K i . 250 nm) and l-kininogen, respectively. The enzyme was also inhibited by two specific synthetic cathepsin B inhibitors, CA-074 and GFG-semicarbazone. Cathepsin X was similar to cathepsin B and found to be a carboxypeptidase with preference for a positively charged Arg in P1 position. Contrary to the preference of cathepsin B, cathepsin X normally acts as a carboxymonopeptidase. However, the preference for Arg in the P1 position is so strong that cathepsin X cleaves substrates with Arg in antepenultimate position, acting also as a carboxydipeptidase. A large hydrophobic residue such as Trp is preferred in the P1H position, although the enzyme cleaved all P1 H residues investigated (Trp, Phe, Ala, Arg, Pro). Cathepsin X also cleaved substrates with amide-blocked C-terminal carboxyl group with rates similar to those of the unblocked substrates. In contrast, no endopeptidase activity of cathepsin X could be detected on a series of o-aminobenzoic acid-peptidyl-N-[2,-dinitrophenyl]ethylenediamine substrates. Furthermore, the standard cysteine protease methylcoumarine amide substrates (k cat /K m < 5.0 Â 10 3 m 21´s21 ) were degraded < 25-fold less efficiently than the carboxypeptidase substrates (k cat /K m < 120.0 Â 10 3 m 21´s21 ).

Synergistic antitumor effects of combined cathepsin B and cathepsin Z deficiencies on breast cancer progression and metastasis in mice

“…Most importantly, Ctsb and Ctsz double-deficient tumor cells that expressed no active cysteine cathepsins on their cell surfaces exhibited strongly reduced formation of invasive strands. Because Ctsb and Ctsz are the only enzymes with carboxypeptidase activity among the cysteine cathepsins (27), these data suggest that cathepsin carboxypeptidase activity significantly contributes to cancer cell invasion and thus to metastasis. The spontaneously occurring metastatic process in PymT mice depends largely on the rate of primary breast cancer development.…”

“…Ctsz shows unique features among these proteases, that is, the presence of a RGD motif for integrin binding in its propeptide and strict exopeptidase activity (25,26). Notably, Ctsb and Ctsz are the only carboxypeptidases in the cysteine cathepsin family, although Ctsb also exhibits endopeptidase activity (27,28). To elucidate specific functions of Ctsz in cancer progression, we generated Ctszdeficient mice and bred them with transgenic mice expressing PymT under control of the mammary epithelium-specific mouse mammary tumor virus (MMTV) LTR promoter (21).…”

Cysteine protease cathepsin X modulates immune response via activation of β2 integrins

“…The proliferation of both, CD4 + and CD8 + T lymphocytes was increased by these inhibitors. Proliferation increased by 24Á1% with CA-074 (inhibitor of cathepsins B and X) 22 and by 11Á7% with 2A2 mAb (specific inhibitor of cathepsin B) (Fig. 3b).…”

Inhibition of papain-like cysteine proteases and legumain by caspase-specific inhibitors: when reaction mechanism is more important than specificity

“…Cathepsin inhibitors E-64, E-64d and CA-074 were from Peptide Research Institute (Osaka, Japan). The specific cathepsin X substrate Dnp-GFFW-OH 37 was a kind gift of Dr. Luiz Juliano (University of Sao Paulo, Brazil)(Dnp¼2,4-dinitrophenyl).…”