Biosynthesis of Cathepsin B in Cultured Normal and I-Cell Fibroblasts*

Biosynthesis and processing of cathepsin B in cul- tured human skin fibroblasts were investigated using immunological procedures. Upon metabolic labeling with [3SS]methionine for 10 min, a precursor form with M, 44,500 was identified. During an 80-min chase, about 50% of it was converted to an M, 46,000 form. Further processing yielded mature forms with M, 33,000 and 27,000, in a final quantitative ratio of about 3:l. Processing of cathepsin B was inhibited by leupeptin, which led to an accumulation of the M, 33,000 polypeptide. The M, 33,000 form appeared to be the most active form and showed a half-time of about 12 h. About 5% of newly synthesized enzyme was secreted as precursor, being detectable extracellularly already after 40 min. NH4Cl enhanced the secretion of the precursor about 20-fold. The precursor and the 33- kDa form contained phosphorylated N-linked oligosaccharides. Cleavage by peptide N-glycosidase F or biosynthesis in the presence of tunicamycin yielded a pre- cursor with M, 39,000. Evidence of a mannose 6- phosphate-dependent transport of cathepsin B in fibroblasts was obtained on the basis of the following re- sults: (i)


From the Slnstitut fur Physwlogische Chernie und Pathobiochemie der Westfdischen Wilhelms Universitat, 0-4400 Miinster, Federal Republic of Germany and SZentrurn fur Angewandte Genetik, Universitat fur Bodenkultur, A-1180 Wien, Austria
Biosynthesis and processing of cathepsin B in cultured human skin fibroblasts were investigated using immunological procedures. Upon metabolic labeling with [3SS]methionine for 10 min, a precursor form with M, 44,500 was identified. During an 80-min chase, about 50% of it was converted to an M, 46,000 form. Further processing yielded mature forms with M, 33,000 and 27,000, in a final quantitative ratio of about 3:l. Processing of cathepsin B was inhibited by leupeptin, which led to an accumulation of the M, 33,000 polypeptide. The M, 33,000 form appeared to be the most active form and showed a half-time of about 12 h. About 5% of newly synthesized enzyme was secreted as precursor, being detectable extracellularly already after 40 min. NH4Cl enhanced the secretion of the precursor about 20-fold. The precursor and the 33-kDa form contained phosphorylated N-linked oligosaccharides. Cleavage by peptide N-glycosidase F or biosynthesis in the presence of tunicamycin yielded a precursor with M, 39,000. Evidence of a mannose 6phosphate-dependent transport of cathepsin B in fibroblasts was obtained on the basis of the following results: (i) cathepsin B precursor from NH4C1-stimulated secretions was internalized in a mannose 6-phosphate inhibitable manner, and (ii) I-cell fibroblasts secreted more than 95% of newly synthesized cathepsin B precursor. In conclusion, cathepsin B from human skin fibroblasts shows an analogous biosynthetic behavior as other lysosomal enzymes.
Cathepsin B (EC 3.4.22.1) is a well-characterized lysosomal thiol proteinase (for review see Barrett and Kirschke, 1981;Katunuma and Kominami, 1983). Besides its lysosomal role cathepsin B or cathepsin B-like enzymes have been suggested to be involved in a variety of physiological and pathological processes, for example in the proteolytic maturation of proinsulin Steiner et al., 1984), or proalbumin (Judah and Quinn, 1978;Matsuda et al., 1986) and of proapolipoprotein A-I1 (Gordon et al., 1985) to their respective mature forms. They are also thought to play a role in inflammatory and neoplastic disease states (e.g. Mort et al., 1983;Koppel et al., 1984;Sloane et al., 1986aSloane et al., , 1986b. Wide variations in tissue levels of cathepsin B are compatible with * This work was supported in part by Deutsche Forschungsgemeinschaft SFB 310. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ll To whom correspondence should be addressed Zentrum fiir Angewandte Genetik, Universitiit fiir Bodenkultur, Gregor-Mendel-Str. 33, A-1180 Wien, Austria. specialized functions of this enzyme in distinct tissues (Kominami et al., 1985;San Segundo et al., 1986). Recent studies revealed a growth-regulated expression of thiol-dependent proteinases in different organisms (Williams et al., 1985;Portnoy et al., 1986;Denhardt et al., 1986).
The mature form of cathepsin B is well characterized by amino acid sequencing of the enzyme from rat (Takio et al., 1983) and human liver (Ritonja et al., 1985). Analysis of cDNA clones confirmed that cathepsin B of human, rat, and mouse origin is highly conserved at both the protein and nucleotide level and that it is closely related to other cysteine proteinases of animal and plant origin (San Segundo et at., 1985;Fong et al., 1986;Chan et al., 1986).
The lysosomal localization of cathepsin B is well established (Mort et al., 1981). In most instances, lysosomal enzymes are synthesized as proenzyme forms of higher molecular size and they are targeted to lysosomes by the mannose 6-phosphate recognition marker (Hasilik and von Figura, 1984). Mannose 6-phosphate residues are components of high mannose and hybrid oligosaccharides (Goldberg et al., 1984). However, cathepsin B showed only negligible affinity to concanavalin A-Sepharose ( Barrett and Kirschke, 1981), and the structure of the carbohydrate moiety of cathepsin B from porcine spleen (Takahashi et al., 1984) and rat liver (Taniguchi et al., 1985) differed drastically from that of other lysosomal enzymes (Goldberg et al., 1984).
In view of the differences in the carbohydrate structure and of the proposed extra-and intralysosomal roles of cathepsin B, the question of its biosynthetic pathway is of high relevance. Nevertheless, detailed information on biosynthesis and intracellular targeting of cathepsin B is not yet available. As cultured human skin fibroblasts are a well-established model for studying lysosomal enzymes (Hasilik and von Figura, 1984), we chose this system to elucidate the biosynthesis and maturation of cathepsin B using immunological procedures. In addition, the behavior of cathepsin B in fibroblasts from patients with I-cell disease was investigated. In I-cell disease, the synthesis of the mannose 6-phosphate recognition signal on lysosomal enzymes is hindered due to a deficient phosphotransferase. This defect leads to a secretion of biosynthetic precursors of most soluble lysosomal enzymes and to an intracellular deficiency of enzyme activity in cultured fibroblasts (for review see Creek and Sly, 1984).
In this report we will show that cathepsin B in cultured fibroblasts is synthesized analogously as other lysosomal enzymes. It carries the mannose 6-phosphate recognition marker, and consequently it is localized inappropriately in Icell fibroblasts.

DISCUSSION
The goal of the present study was to get some insight into post-translational modifications of cathepsin B. The antiserum used was directed against an electrophoretically purified M, 24,000 protein band which is the main cathepsin B form in human liver (Barrett and Kirschke, 1981;Mort et al., 1983). The specificity of the antiserum and of the immune precipitation was assured using three independent methods. Protein bands with corresponding M, values were recognized by Western blotting and by two-dimensional immunoelectrophoresis using unlabeled cell and medium extracts as antigen sources. The same protein bands were shown to be cathepsin B related by a quenching experiment with purified human liver cathepsin B.
For immune precipitation of cathepsin B from labeled cell and medium extracts, it was necessary to denature the extracts prior to immune precipitation. The order of treatment was crucial since a specific immunoprecipitation was obtained only if SDS was added after reduction and alkylation of the extracts. The fact that the antiserum had been prepared against a cathepsin B preparation purified on a preparative SDS-polyacrylamide gel electrophoresis should account for that phenomenon. However, the recognition of only denatured cathepsin B is a common phenomenon since other antisera against human cathepsin B had similar properties (e.g. Barrett, 1973;Mort et d., 1981).
Human fibroblast cathepsin B is synthesized as an Nglycan-containing precursor with an M, of about 44,500, a part of which becomes converted to an M, 46 This M, value is close to the calculated molecular mass of 35,900 for human hepatoma procathepsin B, deduced from the cDNA sequence . In rat pancreatic islets, a cathepsin B precursor of approximately 44 kDa was found. , which is in good agreement with the size of the glycosylated precursor from human skin fibroblasts.
In skin fibroblasts, the precursor is processed first to an M, 33,000 form and then in part to an M, 27,000 polypeptide.
The latter proteolytic step was sensitive to leupeptin. Leupeptin treatment of fibroblast cultures led to an accumulation of the M, 33,000 form as well as to an increase of specific enzymatic activity of cathepsin B, both to a similar extent. An increased activity of cathepsin B after leupeptin loading is in agreement with observations in galactosialidosis fibroblasts (Kato et al., 1983;Takeda et ul., 1986) and in mouse 'Portions of this paper (including "Experimental Procedures," "Results," Figs. 1-9, and Table I)  organs (Sutherland and Greenbaum, 1983). Leupeptin is a reversibly acting competitive inhibitor of thiol-and some serine proteases (Knight, 1980;Umezawa, 1976). Therefore, cathepsin B from leupeptin-treated cells might be of normal activity after harvesting the cells in a leupeptin-free buffer in spite of a possible complete inhibition in uiuo. Taken together with the results obtained by immunological detection of nonlabeled cathepsin B in fibroblast extracts, it is suggested that the M, 33,000 polypeptide is the main form as well as the most active form of cathepsin B in cultivated skin fibroblasts. Similar molecular weights were observed for cathepsin B forms from rat islets of Langerhans (Docherty et ul., 1984) and for a cathepsin B-like mouse tumor thiol proteinase (Olstein and Liener, 1983). Additionally, an extracellular form of cathepsin B with an M, of 40,000, representing tentatively a precursor of cathepsin B, was detected in ascitic fluid of cancer patients (Mort et al., 1983;Mort and Recklies, 1986).
This latent form could be rendered active by pepsin treatment, yielding an M, of 33,000. A 5-kDa light chain of cathepsin B, representing most likely a product of limited proteolysis in the N-terminal part of the enzyme (Takio et al., 1983;Ritonja et al., 1985) was not detected in skin fibroblasts.
The proposed extralysosomal functions of cathepsin B (for review see Katunuma and Kominami, 1983;Docherty et al., 1984, Gordon et al., 1986Sloane et al., 1986a,b) raises the question of its intracellular targeting. Our results demonstrate that at least in skin fibroblasts cathepsin B, as most other lysosomal enzymes, is targeted to the lysosomes via the mannose 6-phosphate recognition marker. This is shown by several lines of evidence. (i) Phosphate label was incorporated into the N-glycan containing cathepsin B precursor, which could be removed by peptide N-glycosidase F. (ii) NH4C1, which inhibits targeting of lysosomal enzymes due to a disturbance of pH-gradients in acidic cell organelles (Hasilik and von Figura, 1984;Creek and Sly, 1984), enhanced secretion of newly synthesized cathepsin B precursor and inhibited proteolytic processing of the precursor. (iii) Cathepsin B precursor from ammonium chloride secretions was internalized by fibroblasts in a mannose 6-phosphate inhibitable manner and further processed intracellularly. (iv) I-Cell fibroblasts secreted most of their newly formed cathepsin B precursor. Accordingly, no incorporation of phosphate was detectable in cathepsin B from I-cell fibroblasts. However, we estimated with almost 15% a relatively high residual enzyme activity of cathepsin B in I-cell fibroblasts. This is not consistent with the observed secretion of about 95% of newly synthesized enzyme. A similar but more extreme situation was observed for acid phosphatase (Lemansky et al., 1985). In I-cell fibroblasts about one-third of the synthesized acid phosphatase polypeptides remains intracellularly, whereas the intracellular activity of that enzyme is normal.
In conclusion, cathepsin B shows a biosynthetic behavior principally similar to that of most other lysosomal enzymes. At least in, skin fibroblasts, it is targeted to the lysosomes via the mannose 6-phosphate recognition system and its secretion in I-cell fibroblasts is increased. However, it is likely that in other tissues, due to postulated specialized functions of this enzyme, cathepsin B is sorted and processed in a different way than in fibroblasts. After endocytosis, a t least 5 polypeptides were detectable intracellularly, then followed in the absence or presence of 5 mJ4 mannose 6-phosphate (Piq. 91.
wlth Ur values of 46,000, 3 3 , 0 0 0 , 27,000 and two bands with l w a r n o l e c~l a r weiqht. HO endocytosis followed by degradation to mailer polypeptides occurred in the presence of mannose 6-phosphate. Hwevel'. some mannose 6-phoaphste independent asIOCiatiOn of the cathepsin 8 precursor with the cell layer was observed. Thls phenomenon was not further investiqared 80 f a r .