Carboxypeptidases cathepsins X and B display distinct protein profile in human cells and tissues

https://doi.org/10.1016/j.yexcr.2004.12.006Get rights and content

Abstract

Cathepsin X, a recently discovered lysosomal cysteine protease, shares common structural features and activity properties with cysteine protease cathepsin B. Based on its widespread mRNA distribution in primary tumors and tumor cell lines, a redundant function in tumor progression has been proposed. In this study, we have shown that these two related proteases exhibit different profiles with respect to their protein distribution in cells and tissues and to their possible roles in malignancy. Protein level of cathepsin X did not differ significantly between matched pairs of lung tumor and adjacent lung tissue obtained from patients with lung cancer whereas that of cathepsin B was 9.6-fold higher in tumor compared to adjacent lung tissue. Immunohistochemical analysis of lung tumor cathepsin X revealed very faint staining in tumor cells but positive staining in infiltrated histiocytes, alveolar macrophages, bronchial epithelial cells, and alveolar type II cells. Cathepsin X stained positive also in CD68+ cells in germinal centers of secondary follicles in lymph nodes, corresponding to tingible body macrophages. Two cell lines with proven invasive behavior, MCF-10A neoT and MDA-MB 231, showed positive staining for cathepsin B, but negative for cathepsin X. We showed that the invasive potential of MCF-10A neoT cells can be impaired by specific inhibitor of cathepsin B but not by that of cathepsin X. Cathepsin X was found in large amounts in the pro-monocytic U-937 cell line, in monocytes and in dendritic cells, generated from monocytes in vitro. Our results show that cathepsin X is not involved in degradation of extracellular matrix, a proteolytic event leading to tumor cell invasion and metastasis. Its expression, restricted to immune cells suggests a role in phagocytosis and the regulation of immune response.

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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