[CANCERRESEARCH58,1624-1630, April 15. 19981 Advances in Brief Cathepsin L2, a Novel Human Cysteine Proteinase Produced by Breast and Colorectal Carcinomas1 Ifligo Santamarla, Gloria Velasco, Maite Cazorla, Antonio Fueyo, Elias Campo, and Carlos López-Otmn2 Departamento de Bioquimica y Biolog(a Molecular (I .S., G. V., C. L-O.J and Biolog(a Funcional (A F], Facultad de Medicina. Universidad de Oviedo, 33006 Oviedo. and Departamento de Anatom(a Patoldgica, Hospital Clmnico—Barcelona,08036Barcelona, [M. C., E. C.!, Spain Abstract (3); (b) cathepsin L (4); (c) cathepsin H (5); (d) cathepsin S (6); (e) cathepsin C (7); (/) cathepsin 0 (8); (g) cathepsin K (9); and (h) We have identified and cloned a new member of the papain family of cathepsin W (10). Furthermore, several groups have described the cysteine proteinases from a human brain eDNA library. The isolated existence of additional cysteine proteinases including cathepsins M, cDNA codes for a polypeptide of 334 amino acids that exhibits all of the structural features characteristic of cysteine proteinases, including the N, P. and T, which were originally identified because of their degrad active site cysteine residue essential for peptide hydrolysis. PairWise corn ing activity on specific substrates such as aldolase, collagen, prom parisons of this amino acid sequence with the remaining human cysteine sulin, or tyrosine aminotransferase, but whose characterization at the proteinases identified to date showed a high percentage of identity (78%) molecular level has not yet been reported (11—14). with cathepsin L; the percentage of identity with all other members of the Structural comparisons between the different members of the cys family was much lower (<40%). On the basis of these structural charac teine proteinase family have shown that they are synthesized as teristics, we have tentatively called this novel protein cathepsin L2. The preproenzymes, which are processed to the corresponding proen eDNA encoding the mature cathepsin L2 was expressed In Escherichia zymes and targeted to the lysosomes by the mannose 6-phosphate coli, and after purification, the recombinant protein was able to degrade signal attached to them. However, in some cases, the precursors of the synthetic peptide benzyloxycarbonyl-L-phenylalanyl-L-arginine-7- these lysosomal enzymes escape from this processing pathway and amido-4-rnethylcoumarln, which is commonly used as a substrate for cysteine proteinases. Cathepsin L2 proteolytic activity on this substrate continue along the secretory route, entering storage granules and wasabolishedby trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane,finally being released into the extracellular space (15). Amino acid an inhibitor of cysteine proteinases, thus providing additional evidence sequence comparisons between all members of the family have re that the isolated eDNA encodes a functional cysteine proteinase. Northern vealed that they are not closely related; the percentage of identity blot analysis of polyadenylated RNAs isolated from a variety of human between them is less than 50%. Nevertheless, in their amino acid tissues demonstrated that cathepsin L2 is predominantly expressed in the sequences, all of them contain a series of amino acids that is abso thymus and testis. This finding is in marked contrast with the wide tissue lutely conserved and essential for their catalytic activity (1). distribution of most cysteine protelnases characterized to date, including Because it seems clear that cysteine proteinases play essential roles cathepsin L, and suggests that cathepsin L2 may play a specialized role in in both normal and pathological conditions, including tumor pro the thymus and testis. Expression analysis of cathepsin L2 in human cesses, over the last few years, we have been interested in examining tumors revealed a widespread expression in colorectal and breast carci nomas but not in normal colon or mammary gland or in peritumoral the possibility that additional, uncharactenzed members of this family tissues. Cathepsin L2 was also expressed by colorectal and breast cancer of proteolytic enzymes could be produced by human tumors. This cell lines as well as by some tumors of diverse origin, including ovarian search for new human cysteine proteinases led us to identify cathepsin and renal carcinomas. These results open the possibility that this novel 0, which was originallycloned from a breastcarcinomabut is widely enzyme may be involved in tumor processes, as already reported for other distributed in human tissues (8). Furthermore, we have recently re cysteine proteinases, including cathepsin L. ported the cloning and characterization of human bleomycin hydro lase, a cytosolic cysteine proteinase that is distantly related to other Introduction members of the papain family and is involved in chemotherapy The cysteine proteinases are a family of enzymes involved in many resistance (16). In this study, we describe the molecular cloning and normal cellular processes, including the turnover of intracellular pro complete nucleotide sequence3 of a cDNA encoding a novel member teins, prohormone activation, and bone remodeling (1). In addition, it of this family of enzymes, which has tentatively been called cathepsin has been suggested that these proteolytic enzymes play important L2. We also report the expression of the cDNA in Escherichia coli roles in a number of pathological conditions such as Alzheimer's and demonstrate that recombinant cathepsin L2 is a functional cys disease, pulmonary emphysema, rheumatoid arthritis, muscular dys teine proteinase. Furthermore, we analyze its expression in human trophy, and cancer invasion and metastasis (1, 2). At present, eight tissues, showing that it is mainly expressed in the thymus and testis. human cysteine proteinases of the papain family have been isolated Finally, we present evidence that cathepsin L2 is widely expressed in and characterized at the amino acid sequence level: (a) cathepsin B colorectal and breast carcinomas. Received I 1/21/97; accepted 2/25/98. Materials and Methods The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with Materials. cDNA libraries from human brain, mouse embryo, and mouse 18 U.S.C. Section 1734 solely to indicate this fact. brain constructed in Agtll and Northern blots containing poly(A)@ RNAs t Supported by Grant 5AF97—0258 from the Comisión Interministerial de Ciencia y Tecnologla, Grant BMH4-CT96-00l7 from European Union-BIOMED II, Maraton TV3 prepared from different human tissues and cancer cell lines were purchased Cancer (to E. C.), and Glaxo-Wellcome, Spain. I. S. is a recipient of a fellowship from from Clontech (Palo Alto, CA). Restriction endonucleases and other reagents Ministerio de Educacióny Ciencia (Spain). used for molecular cloning were from Boehringer Mannheim (Mannheim, 2 To whom requests for reprints should be addressed, at Departamento de Bio quimica y BiologIa Molecular, Facultad de Medicina, Universidad de Oviedo, 33006 Oviedo, Spain. Phone: 34-85-104201; Fax: 34-85-103564/34-85-232255; E-mail: 3 The nucleotide sequence reported in this paper has been submitted to the GenBank/ [email protected]. European Molecular Biology Laboratory Data Bank with accession number Y14734. 1624 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1998 American Association for Cancer Research. CATHEPSIN L2 IN HUMAN TUMORS Germany). Oligonucleotides were synthesized by the phosphoramidite method volume of PBS, lysed by using a French press, and centrifuged at 20,000 X g in an Applied Biosystems DNA synthesizer (model 392 A) and used directly for 20 mm at 4°C.The soluble extract was treated with glutathione-Sepharose after synthesis. Double-stranded DNA probes were radiolabeled with 4B and eluted with glutathione elution buffer [10 mMreduced glutathione in 50 [cs-32P]dCTP (3000 Ci/mmol) from Amersham (Amersham, Buckinghamshire, mM Tris-HC1 (pH 8.0)]. United Kingdom) using a commercial random-priming kit purchased from Enzyme Assays. The enzymatic activity of purified cathepsin L2 produced Pharmacia LKB Biotechnology AB (Uppsala, Sweden). Synthetic peptides in E. coli was measured by using 20 @.tMZ-Phe-Arg-AMC or Z-Arg-Arg-AMC Z-Phe-Arg-AMC4 and Z-Arg-Arg-AMC were from BACHEM, Inc. (Buben as the substrate and following the procedure described by Barrett and Kirschke dorf, Switzerland), and proteinase inhibitor E-64 was from Sigma (St. Louis, (17) with minor modifications. Assays were performed at 30°C in 100 mM MO). sodium acetate buffer (pH 4.5) containing 10 mM DTT and 2 mM EDTA. Screening of a Human Brain cDNA Library. After searching the Gen Substrate hydrolysis was monitored in a Cytofluor 2350 fluorometer (Milli Bank database of human ESTs for sequences with homology to human cys pore, Bedford, MA) at excitation and emission wavelengths of 360 and 460 teine proteinases, we identified a mouse sequence (AA013726; deposited by nm, respectively. For inhibition assays, the reaction mixture was preincubated M. Marra et aL, WashingtonUniversity-HowardHughesMedicalInstitute with 20 @.LME-64at 30°Cfor 15 mmn, and the remaining activity was deter Mouse EST Project) that, when translated, showed a significant similarity to mined using the fluorogenic substrate Z-Phe-Arg-AMC as described previ amino acid sequences previously determined for other cathepsins. This DNA ously. As a positive control of enzyme assays, we used recombinant