Carboxypeptidases cathepsins X and B display distinct protein profile in human cells and tissues
Introduction
Cathepsins X and B are lysosomal cysteine proteases, an important group of papain-like enzymes (clan CA) which are involved in physiological processes such as intracellular protein turnover, remodeling of extracellular matrix (ECM), hormone processing, bone resorption, and antigen presentation [1]. Cathepsin B is the best characterized cysteine protease. It is present in all mammalian cells and exhibits a relatively broad substrate specificity. The presence of a signal sequence and N-glycosylation sites shows that cathepsin B is targeted to the endosomal/lysosomal compartment via the mannose-6-phosphate receptor pathway [2]. Under certain pathological conditions, cathepsin B is translocated to the peripheral cytoplasmic and plasma membrane region or secreted from cells [3]. Access of substrate into the active site of cathepsin B is controlled by an 18-residue-long insertion (Pro 107–Asp 124), termed the occluding loop [4] which provides two His residues to bind the carboxylic group of the C-terminus of the substrate [5]. This explains the preferred carboxypeptidase activity of the enzyme. However, cathepsin B can also act as an endopeptidase since the occluding loop is flexible and can move from the active site cleft when endopeptidase substrate binds to the enzyme [6], [7]. The endopeptidase activity of cathepsin B should be important in pathological processes associated with the remodeling of ECM, such as cancer, since it is capable of degrading ECM proteins laminin, fibronectin, and collagen IV, facilitating tumor cell invasion and metastasis [8]. In addition, cathepsin B can activate other enzymes, such as urokinase-type plasminogen activator, which act downstream in the proteolytic cascade and cause even more extensive degradation of ECM. The active role in processes of tumor progression makes cathepsin B a promising marker for prognosis and diagnosis and a potential target for therapeutic intervention [9].
In contrast to cathepsin B, cathepsin X was discovered only recently [10]. Like cathepsin B, it acts as a carboxypeptidase, although with a different profile against substrates and inhibitors [11]. Its structure shares common features with other papain-like enzymes, but its unique mini-loop, formed of a three-residue insertion, protrudes into the active site of the protease and modulates its carboxypeptidase activity [12]. Cathepsin X exhibits mono- and di-carboxypeptidase activity [12], but, in contrast to cathepsin B, does not act as endopeptidase [11], [12]. The biological function of cathepsin X has not been defined. Northern blot analysis provided evidence of high mRNA levels in various tumor cell lines and primary tumors, suggesting a similar role in tumor progression as that for cathepsin B and other cathepsins [13]. High mRNA levels were observed also in human placenta, lung, liver, kidney, pancreas, colon, ovary, peripheral blood leucocytes, prostate, small intestine, spleen [10] and inflamed gastric mucosa [14]. By using active site directed probe, cathepsin X has been determined in antigen presenting cells such as macrophages and dendritic cells [15]. However, the protein levels of cathepsin X, needed to confirm the widespread presence of this enzyme in tissues and cells, have not yet been determined.
Since cathepsins X and B display overlapping enzymatic specificities and apparently similar distribution profiles, the question arises as to which of the two enzymes has actually been monitored in experiments on biological or pathological samples, using substrates, inhibitors and antibodies, presumed to be specific for cathepsin B. In order to distinguish between these enzymes and to detect protein levels of cathepsin X in biological samples, monoclonal antibodies specific for cathepsin X, isolated from human liver, have been raised. We were thus able to localize and to quantitate the antigen in a variety of human tissues and cell lines. The results showed a distribution profile, different from that for cathepsin B, with the highest levels in lung bronchoepithelial cells, alveolar and tingible body macrophages, pro-monocytic cell line U-937, and monocytes and dendritic cells, suggesting that the function of cathepsin X is linked to the processes of inflammatory and immune response rather than the progression of cancer, dependent on degradation of ECM.
Section snippets
Cells lines and media
Breast tumor epithelial cell line MDA-MB-231 (ATCC, Manassas, VA) was maintained in Leibovitz's L-15 medium, supplemented with 10% fetal bovine serum. U-937 pro-monocytic cell line (ATCC, Manassas, VA) was grown in RPMI 1640, supplemented with 2 mM glutamine, 1.5 g/l sodium bicarbonate, 4.5 g/l glucose, 10 mM HEPES, 1.0 mM sodium pyruvate and 10% fetal bovine serum. MCF-10A neoT cell line was provided by Prof. Bonnie F. Sloane (Wayne State University, Detroit, MI). Its origin is MCF-10 human
Antibody specificity
2B2, 3B10 and 2F12 MAbs, selected for analyses of cathepsin X, were tested for specificity by Western blots and indirect ELISA on immobilized antigen. All three monoclonal antibodies reacted with the mature form of cathepsin X but not with cathepsin B (Fig. 1) or other related human cathepsins (L, H, S, C, results not shown). Similarly, the antibodies bound cathepsin X specifically in antigen-immobilized ELISA (Fig. 2) and in direct quantitative ELISA. For the latter, the working range of 4–125
Discussion
Eleven human cathepsins, B, H, L, X, S, C, K, V, W, F, and O, belong to the papain family of cysteine proteases and share several genetic, structural, and functional similarities [29]. For many years, their function has been known as nonspecific protein degradation within the endosomal/lysosomal system. Although cathepsins exhibit diversity in cell and tissue localization, substrate specificity and pH stability, there is a strong belief that functional redundancy is typical of this group of
Acknowledgments
The authors thank Prof. Roger Pain for critical reading of the manuscript. This work was supported by Ministry of Education, Science and Sport of the Republic of Slovenia.
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