Báo cáo khoa học: Human proteoglycan testican-1 inhibits the lysosomal cysteine protease cathepsin L pdf

Human proteoglycan testican-1 inhibits the lysosomal cysteine protease cathepsin L Jeffrey P. Bocock 1 , Cora-Jean S. Edgell 2 , Henry S. Marr 2 and Ann H. Erickson 1 1 Department of Biochemistry and Biophysics and 2 Department of Pathology and Laboratory Medicine, The University of North Carolina, Chapel Hill, NC, USA Testican-1, a secreted proteoglycan enriched in brain, has a single thyropin domain that is highly homologous to domains previously shown to inhibit cysteine proteases. We demonstrate that purified recombinant human testican-1 is a strong competitive inhibitor of the lysosomal cysteine pro- tease, cathepsin L, with a K i of 0.7 n M , but it does not inhibit the structurally related lysosomal cysteine protease cathep- sin B. Testican-1 inhibition of cathepsin L is independent of its chondroitin sulfate chains and is effective at both pH 5.5 and 7.2. At neutral pH, testican-1 also stabilizes cathepsin L, slowing pH-induced denaturation and allowing the protease to remain active longer, although the rate of proteolysis is reduced. These data indicate that testican-1 is capable of modulating cathepsin L activity both in intracellular vesicles and in the extracellular milieu. Keywords: cathepsin L; proteoglycan; protease; testican; thyropin. Testican is a proteoglycan first identified in human seminal plasma [1]. The cDNA was subsequently cloned from the human testis [2], hence the name testican, and from human vascular endothelial cells [3,4] and mouse brain [5]. In both human and mouse, testican mRNA is prominent in brain and absent in certain other tissues. Two additional human homologues have been identified, testican-2 [6] and testican- 3 [7]. The amino acid sequences of human and mouse testican-1 are 94% identical, which argues for a significant function for this proteoglycan [5]. Testican is a multidomain protein (Fig. 1), including three domains that have homology to inhibitors of three different classes of proteases. An N-terminal region of testican-1 has been shown to inhibit membrane-type 1 matrix metalloproteinase activation of matrix metallopro- teinase-2 [7]. Adjacent to this domain is a follistatin-like domain that includes a six-cysteine pattern with similarity to Kazal domains found in serine protease inhibitors such as pancreatic secretory trypsin inhibitor [8,9]. The next domain has homology to EF-hands and has been shown to bind calcium when expressed as an independent domain [10]. Finally, near the C-terminus is a 64-amino acid domain highly homologous to protein sequences shown to inhibit cysteine proteases. Such protease inhibition domains have collectively been called thyropins [11] due to their homology with a domain repeated 11 times in thyroglobulin, a precursor of thyroid hormones [12]. The cysteine protease inhibitory function of thyropin domains was established when a fragment of the class II invariant chain, that is normally part of the major histocompatibility complex (MHC), was isolated from human kidney bound to cathepsin L [13]. The class II invariant chain exists in two alternatively spliced forms, p31 and p41. The latter form has a region which shares significant homology with the thyropin domain of thyro- globulin. This domain of the p41 invariant chain was shown to inhibit cathepsin L and to stabilize the active protease at a pH which would normally denature the enzyme [13]. Crystallography of this p41 domain complexed with cath- epsin L revealed that the domain assumes a wedge-shape conformation comprised of three loops stabilized by three disulfide bonds and is lodged in the active site of cathepsin L [14]. Saxiphilin, a bullfrog serum protein that binds a neurotoxin [15], and equistatin, from a sea anemone [16], also have one or more thyropin domains. Like p41, these proteins inhibit cathepsin L proteolytic activity [15,16], but a mammalian proteoglycan has not been demonstrated to serve this role. Cathepsin L is a ubiquitously expressed protease that is normally efficiently segregated into lysosomes, where low pH allows for optimal activity [17]. When expression levels are increased, however, either during specific developmental stages, by cell transformation, or by ectopic expression from a transfected plasmid, the proenzyme is secreted in signifi- cant amounts [18,19]. In response to signaling events, active enzyme can also be released [20,21]. In addition to mediating housekeeping proteolysis in the lysosome, the protease participates in developmental processes and anti- gen processing [22–24]. Many studies also implicate extra- cellular cathepsin L in tumor biology [23,25], where the major role ascribed to secreted lysosomal proteases is degradation of extracellular matrix [26–30]. Correspondence to A. H. Erickson, Department of Biochemistry and Biophysics, CB 7260, Mary Ellen Jones Building, The University of North Carolina, Chapel Hill, NC 27599–7260, USA. Fax: + 1 919 966 2852, Tel.: +1 919 966 4694, E-mail: ann_erickson@med.unc.edu Abbreviations: BCIP, 5-bromo-4-chloro-3-indolylphosphate; MHC, major histocompatibility complex; HEK 293, human embryonic kidney (cells). (Received 18 June 2003, revised 31 July 2003, accepted 12 August 2003) Eur. J. Biochem. 270, 4008–4015 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03789.x Little is known about the function of testicans. We have determined that testican-1, which includes a thyropin domain, is a competitive inhibitor of cathepsin L but not of the related cysteine protease cathepsin B. Inhibition is independent of the chondroitinase ABC-sensitive glycos- aminoglycan chains associated with this proteoglycan. This establishes a new role for testican-1 and provides the first evidence that the protein backbone of a proteoglycan can regulate lysosomal protease activity, thus expanding our understanding of the role proteoglycans play in modulating extracellular events. Experimental procedures Materials Human embryonic kidney 293 (HEK 293) cells were obtained from ATCC (Manassas, VA, USA). Alkaline phosphatase-conjugated goat antibodies to mouse immu- noglobulins were purchased from Jackson Immuno- Research, and mouse monoclonal antibodies specific for the Myc epitope tag, Lipofectamine and Geneticin were obtained from Invitrogen. Centriprep concentrators were from Millipore and Ni-nitrilotriacetic acid agarose was from Qiagen. Rainbow molecular mass markers were from Amersham and Gelcode Blue Staining Reagent was purchased from Pierce (Rockford, IL, USA). 5-Bromo-4- chloro-3-indolylphosphate (BCIP)/nitro blue tetrazolium Color Development Substrate was obtained from Promega. Z-Phe-Arg-4-methyl-7-coumarin (Z-Phe-Arg-NHMec), E64 and Chondroitinase ABC were from Sigma-Aldrich. Fluo- trac 96-well microtiter plates were from Greiner Bio-One (Longwood, FL, USA). Human cathepsin L, purified from liver, was obtained from Athens Research, (Athens, GA, USA) and human cathepsin B, purified from liver, was from Calbiochem. Recombinant testican-1 A complete open reading frame cDNA for human testican- 1 less its last amino acid was assembled from several cDNA clones and inserted between EcoRV and XhoI sites in the Invitrogen expression plasmid, pcDNA3.1/MycHis, keep- ing the Myc epitope tag and the His 6 encoding DNA from the vector in frame at the 3¢-end of the testican-1 open reading frame. The plasmid construct was cloned in Escherichia coli DH5a, and the intended cDNA insert was verified by sequencing. This plasmid was transfected into HEK 293 cells using Lipofectamine according to the manufacturer’s recommendations. Cells that had incorpor- ated plasmid DNA were selected in the presence of Geneticin at 250 lgÆmL )1 . Expression of the recombinant gene was indicated by detecting the Myc epitope in culture fluid from the Geneticin-resistant cells by ELISA. Chondroitinase ABC treatment and purification of recombinant testican-1 Conditioned Opti-MEM culture fluid was collected after 24 h from 810 cm 2 of confluent HEK 293 cells expressing recombinant testican-1. After pelleting cellular debris, the conditioned culture fluid was concentrated to 1 mL using a Centriprep concentrator designed to retain molecules larger than 10 kDa. Half of the concentrated culture fluid was adjusted to basic pH by addition of pH 8 Tris/HCl to 40 m M and sodium acetate to 40 m M and treated with Chondroitinase ABC at 2 UÆmL )1 for 40 min at 37 °C. Recombinant testican-1 was then purified by His 6 binding and elution from Ni-nitrilotriacetic acid agarose, as recom- mended by the manufacturer. For molecular mass analyses, samples were reduced and denatured in the presence of 1m M dithiothreitol and 2% SDS at 100 °Cfor5minand then resolved by standard PAGE using 12% acrylamide with 0.1% SDS. Most of the full-length recombinant testican-1 expressed by HEK 293 cells possessed significant amounts of chondroitin sulfate that prevented the majority of the protein from entering a 12% polyacrylamide gel. Treatment with chondroitinase ABC reduced the effective mass, enabling the use of polyacrylamide gels stained with Gelcode Blue Staining Reagent to assess the purity of the recombinant protein isolated by Ni-nitrilotriacetic acid- affinity chromatography. The size of the testican-Myc- His 6 product was determined by probing gel blots with monoclonal antibodies specific for the recombinant, using Fig. 1. Alignment of the cathepsin-inhibitory domain of mouse p41 invariant chain with homologous domains of mouse and human testican-1. Identical residues are shown on a shaded background. The location of the thyropin domain within testican-1 is illustrated relative to the other known domains of testican-1 (not drawn to scale). Residues 1–21 comprise the signal peptide [45]. The following domain (residues 25–84) is unique to the three testicans. This region of testican-1 is responsible for the inhibition of a membrane-type metalloproteinase [7]. Residues 86–183 have similarity to follistatin domains [55], with a six cysteine Kazal-like sequence. Residues 197–312 comprise an extracellular calcium-binding (EC) module [10]. Thyropin domain homology occurs between residues 310 and 379 [11], comprised of exons 9 and 10. Following the thyropin domain is a region enriched for acidic residues. Twelve of the 13 amino acids within five amino acids of the C-terminus are negatively charged. The serines at 383 and 388 in this domain may have chondroitin or heparan sulfate attached [1], which is designated here as GAG for glycosaminoglycans. Ó FEBS 2003 Testican-1 inhibits cathepsin L (Eur. J. Biochem. 270) 4009 alkaline phosphatase-conjugated goat antibodies to mouse immunoglobulins as the secondary antibody, and localizing the bound alkaline phosphatase activity as a blue precipi- tate using BCIP/nitro blue tetrazolium Color Development Substrate as recommended by the manufacturer. The protein concentrations were determined using Bio-Rad Protein Assay reagent 500–006 in a microtiter plate assay using bovine serum albumin for the standard curve. Cathepsin L active site titration Cathepsin L was diluted in buffer consisting of 340 m M sodium acetate pH 5.5 and 1 m M EDTA, and incubated on ice for 5 min with 5 m M dithiothreitol to activate the enzyme [15]. The concentration of active cathepsin L in the preparation used for these studies was determined by titration with increasing amounts of the stoichiometric inhibitor, E64, at a constant Z-Phe-Arg-NHMec substrate concentration of 6 l M [31]. Liberated fluorophore was detected by excitation at 355 nm and emission at 460 nm using a fluorescence microplate reader and FLUOSTAR 2000 analysis software from BMG Labtechnologies (Durham, NC, USA). Testican-1 inhibition of cathepsin L Cathepsin L was preactivated in the same buffer utilized for active site titration, as described above. Active cathepsin L (0.2 n M ) and varying concentrations (423 p M )100 n M )of recombinant testican-1 were incubated at room temperature for 20 min to allow for complex formation. The tempera- ture was reduced to 0 °C to synchronize the reactions and substrate was added to 6 l M . Reaction mixtures were then incubated at 30 °C for 10 min. The substrate conversion was monitored as described above. The effect of testican-1 on cathepsin B was similarly assayed at a final enzyme concentration of 2 n M in a reaction buffer consisting of 50 m M sodium acetate, pH 5.0, 100 m M NaCl, 1 m M EDTA, 5 m M dithiothreitol, and 6 l M Z-Phe-Arg-NHMec as substrate [15]. Determination of inhibition constant Two approaches were used to determine inhibition con- stants. In the first approach, the enzyme and inhibitor were preincubated in the reaction buffer to allow complex formation, as above. Nonlinear regression analysis of testican-1 titration data obtained on assay of residual enzyme activity was used to determine the inhibition constant K i due to the tight binding of the protease by the inhibitor and the possibility of modification of the inhibitor bytheenzyme[32].Thesedatawerefittedtothetheoretical equation for competitive inhibition: a ¼1À ðE 0 ÞþðI 0 ÞþK i Àf½ðE 0 ÞþðI 0 ÞþK i  2 À4ðE 0 ÞðI 0 Þg 1=2 2ðE 0 Þ where a is the experimentally determined residual enzyme activity in the presence of inhibitor, E 0 is the initial concentration of enzyme, and I 0 is the initial concentration of inhibitor [33]. For these studies, chondroitinase ABC-treated testican-1 was utilized because the preparation purity could be assayed readily by gel electrophoresis. To compare the ability of testican-1 to inhibit cathepsin L at pH 5.5 and 7.2, an alternative method for determination of inhibition constants was necessary to avoid cathepsin L inactivation that would occur during a preincubation at neutral pH. The reactions were initiated by addition of cathepsin L to 0.2 n M into buffer containing a final concentration of 5 m M dithiothreitol, varying concentra- tions of Z-Phe-Arg-NHMec, and varying concentrations of testican-1. To make it possible to detect any change in affinity should the enzyme be allosteric, we chose to emphasize substrate concentrations below the K m [34]. The pH 7.2 buffer was 50 m M sodium phosphate pH 7.2, 100 m M NaCl and 1 m M EDTA [15]. Reactions were monitored fluorometrically every 20 s for up to 20 min. A lag phase up to 100 s was observed to be required for the enzyme to react completely with dithiothreitol and the reaction mixture to warm to assay temperature. The subsequent linear region of each curve was utilized to create the Lineweaver–Burk plots. K i was determined as the x-intercept of a plot of the slopes of these lines vs. inhibitor concentration [34]. Results Testican-1 purity Recombinant testican-1 purified by His 6 affinity chromato- graphy from the conditioned culture fluid of transfected HEK 293 cells before and after treatment with chondroi- tinase ABC was resolved by SDS/PAGE and visualized by Coomassie staining and immunoblotting (Fig. 2 insert). The most abundant proteins in the conditioned culture fluid (lane 1) were absent after Ni-nitrilotriacetic acid affinity chromatography (lane 2). Testican-1 purified after chond- roitinase treatment migrated with a relative molecular mass of 50–60 kDa and was shown to contain the Myc epitope by the Western blot. The mass is consistent with that expected for the recombinant polypeptide less its signal sequence (51 kDa), plus varying amounts of O-linked oligosaccharide that has been reported to be attached in the calcium binding domain [10]. Testican-1 purified before chond- roitinase ABC treatment had the same protein profile but was less intense (data not shown). Treatment with chond- roitinase increased the amount of protein entering the gel by 2.6-fold, indicating that at least 60% of the testican had chondroitin sulfate chains removed by the chondroitinase treatment. Cathepsin L is inhibited by testican-1 To determine whether purified testican-1 could inhibit cathepsin L proteolytic activity, the enzyme was preincu- bated with various concentrations of recombinant testican-1 to allow complex formation prior to assay for cleavage of a synthetic peptide substrate. Greater than 50% of cathep- sin L activity was lost at an inhibitor to enzyme ratio of 2 : 1, while nearly 80% was lost at a 10 : 1 ratio (Fig. 2). This dramatic decrease in enzyme activity at low concentra- tions of inhibitor indicates that inhibitor binding is tight. To determine whether the inhibition of cathepsin L was 4010 J. P. Bocock et al. (Eur. J. Biochem. 270) Ó FEBS 2003 affected by chondroitin sulfate chains on testican-1, cath- epsin L activity was also assayed in the presence of testican- 1 that had not been treated with chondroitinase ABC prior to purification. There was no change in the efficiency of cathepsin L inhibition (Fig. 2), indicating that chondroitin sulfate associated with testican-1 does not mediate or prevent the inhibition of cathepsin L. The inhibition constant, K i ,atpH5.5wasdeterminedto be 0.7 n M using nonlinear regression analysis of enzyme activity remaining after cathepsin L had been preincubated with testican-1 to allow enzyme-inhibitor complexes to form. The data fit the theoretical equation for competitive inhibition [33] with an R 2 value of greater than 0.9. The K m at pH 5.5 was calculated to be 8.5 l M , which is consistent with the reported value of 7 l M [31] for this substrate, although others have reported a lower K m [35]. Testican-1 does not inhibit cathepsin B Certain thyropin domain-containing proteins have been found to inhibit the endopeptidase activity of cysteine proteases other than cathepsin L [15,16]. Therefore, to determine whether testican-1 could inhibit cathepsin B, the enzyme was assayed at 2 n M in the presence of up to 200 n M testican-1. The mean residual activity for cathepsin B was 91.8 ± 8.9%, n ¼ 34. Thus, no significant inhibition of cathepsin B by testican-1 was observed. Testican-1 inhibition of cathepsin L is competitive The thyropins thus far characterized have been found to act as competitive inhibitors of cathepsin L [15,16,36], consis- tent with detection by X-ray crystallography of the p41 thyropin domain in the active site of cathepsin L [14]. To confirm that testican-1 is a competitive inhibitor of cathep- sin L, kinetic assays were performed to measure the rate of cleavage of varying concentrations of substrate in the presence and absence of testican-1. The intersection of the Lineweaver–Burk plots on the y-axis above the origin indicates that cathepsin L is competitively inhibited by testican-1 at pH 5.5 (Fig. 3A). Testican-1 inhibition of cathepsin L at neutral pH Although lysosomal enzymes are assayed commonly at pH 5.5, where the enzymes are most stable, we also assayed cathepsin L inhibition by testican-1 near neutral pH, as Fig. 3. Testican-1 is a competitive inhibitor of cathepsin L at pH 5.5 and pH 7.2. Testican-1 was added at the indicated concentrations to cathepsin L incubated at pH 5.5 or at pH 7.2 with concentrations of Z-Phe-Arg-NHMec between 154 n M and 7.7 l M . The Lineweaver– Burk plots show the lines representing reactions at different testican-1 concentrations that all intercept at the y-axis, as expected for com- petitive inhibition. The error bars represent the standard deviation of at least three replicates. Obvious outliers were discarded. For each replicate, the reaction velocity was determined from the linear region of the curve as the rate of change of fluorescence over a period of at least 200 s. Linear fits for the data at each testican-1 concentration were generated by linear regression, and all R 2 correlation coefficients were >0.92. Fig. 2. Testican-1 with or without chondroitin sulfate inhibits cathep- sin L proteolytic activity. Cathepsin L was incubated with increasing amounts of testican-1 in either its native form (m) or following treat- ment with chondroitinase ABC (n). The inhibitory activity is expressed as residual activity of the enzyme compared to the control reaction without testican-1 set as 100% activity. Residual activity is graphed as a function of the molar ratio of testican-1 added to active cathepsin L present. Each point represents the mean of three repli- cates; error bars represent the standard deviation of each set of repli- cates and the line represents the theoretical curve fit. Testican-1 purified with chondroitin sulfate chains intact and testican-1 purified after chondroitinase ABC digestion were used at the same protein concentration in the enzymatic assays shown. (Inset) Purification of recombinant testican-1. A Coomassie-stained SDS/polyacrylamide gel and a nitrocellulose blot of a parallel gel immunostained for recom- binant testican-Myc-His 6 show its purification from the culture fluid of transfected 293 cells. The first lanes show the unfractionated culture fluid. The second lanes show testican-1 after treatment with chond- roitinase ABC and purification by Ni-nitrilotriacetic acid affinity. The migration distances of Rainbow protein molecular mass markers in these gels are indicated in kDa. Ó FEBS 2003 Testican-1 inhibits cathepsin L (Eur. J. Biochem. 270) 4011 testican-1 is a secreted proteoglycan. As cathepsin L denatures rapidly at neutral pH and above [37], data collection was initiated immediately after addition of enzyme. Lineweaver–Burk plots of the data established that testican-1 also inhibits cathepsin L competitively at pH 7.2 (Fig. 3B). The K m at pH 7.2 was 1.7 l M .TheV max was 385 and 72 fluorescence units RFU per second at pH 5.5 and 7.2, respectively. K i was determined at both pH values from the Line- weaver–Burk plots, as described in Experimental proce- dures, but as such analysis is thought to produce a K i that is less accurate than nonlinear regression analysis for tight- binding inhibitors [33], these values are only presented to compare testican-1¢s inhibitory activity at pH 5.5 to that at pH 7.2. The K i values derived by this method were 13 n M at pH 5.5 and 8 n M at pH 7.2. The linear regression fits for these data had R 2 values greater than 0.9. Thus, testican-1 is similarly effective at inhibiting cathepsin L at pH 5.5 and at pH 7.2. Testican-1 enhances cathepsin L stability at neutral pH At pH 7.2, cathepsin L proteolytic activity in the absence of testican-1 begins to decline before 10 min (Fig. 4), consis- tent with the measurements of others [38]. This is not merely due to depletion of substrate, as indicated by the progress curve of the control reaction at pH 5.5 in the absence of testican-1 (A). When cathepsin L activity was assayed near neutral pH in the presence of testican-1, the loss of activity was noticeably slower (B). This increase in enzyme stability was observed at testican-1 concentrations as low as 5 n M ,a 25 : 1 inhibitor-to-enzyme ratio. Thus at pH values similar to those outside cells, the presence of testican-1 allows the enzyme to remain active longer, at the cost of a reduced rate of proteolytic activity. Cathepsin L could potentially cleave testican within the thyropin domain that would thus act as a competitive substrate, but no change in enzyme velocity was detected over 20 min, as might be expected were the protease degrading the inhibitor (Fig. 4A). This is unlikely to have affected our K i determination (Fig. 2) as these experiments utilized l M concentrations of substrate and n M concentra- tions of testican. Discussion Testican-1, a secreted proteoglycan with a thyropin domain, was determined to be a potent competitive inhibitor of the lysosomal cysteine protease, cathepsin L. At pH 5.5, the physiological pH for a lysosomal enzyme, the proteoglycan inhibited the enzyme with a K i of 0.7 n M .Usingan alternative method, we also demonstrated that testican-1 was similarly effective as an inhibitor of cathepsin L at pH 5.5 and at pH 7.2. The affinity of testican-1 for cathepsin L is similar to that observed for another physio- logical inhibitor, cystatin B [39], but is significantly lower than the affinity of the isolated thyropin domain of the p41 form of the MHC invariant chain for cathepsin L, which has a K i of 1.7 · 10 )3 n M [36]. Proteins containing thyropin domains have been found to inhibit a variety of papain- related cysteine proteases with K i values in the low picomolar to low nanomolar range [15]. Testican-1 is unusual in having multiple specific protease inhibitor activities within a single polypeptide. We show that testican-1 inhibits the cysteine protease cathepsin L, while the N-terminal domain unique to testicans 1–3 has been shown to inhibit pro-matrix metalloproteinase-2 activation by membrane-type 1 and 3 matrix metalloproteinases [7]. In addition, the protein contains a domain homologous to inhibitors of a third family of proteases, the serine proteases. Another human gene family with recognizable homologies to multiple specific protease inhibitors in a single protein has been recognized recently by data bank homology searches and one of the domains similar to Kunitz-type protease inhibitors has been shown to inhibit trypsin [40–42]. Cathepsin L inhibition is a novel activity for the protein core of proteoglycans and thus expands our appreciation of the regulatory role of these molecules. The multidomain structure characteristic of proteoglycans enables them to interact with various molecules including growth factors, cell adhesion proteins, and other extracellular matrix components. Fig. 4. Testican-1 increases the stability of cathepsin L at neutral pH. Cathepsin L at 0.2 n M was incubated at 30 °Cwith350n M Z-Phe- Arg-NHMec at pH 5.5 (A) and at 7.2 (B) without testican-1 (d)and with testican-1 at the indicated concentrations (h, e). Each progress curve is representative of four replicate curves at the given conditions. As fluorescence was measured immediately after the enzyme was added to the dithiothreitol-containing reaction mixture on ice, the initial lag period represents the time required for the enzyme to react with dithiothreitol and the reaction mixture to reach 30 °C. The arrow indicates the time by which human cathepsin L was previously shown to be inactivated when incubated at pH 7 at 30 °Cwiththesame substrate [38]. 4012 J. P. Bocock et al. (Eur. J. Biochem. 270) Ó FEBS 2003 Testican-1 had no significant inhibitory effect on the endopeptidase activity of a related lysosomal cysteine protease, cathepsin B. This is consistent with the finding that the structurally similar p41 thyropin domain inhibits cathepsin L but does not inhibit cathepsin B [36]. Equistatin [16] and saxiphilin [15] both inhibit cathepsin B, but they bind with lower affinity than to cathepsin L. Cathepsin B differs from cathepsin L in that it has an additional loop of approximately 20 amino acids which partially occludes the active site and thus affects interactions with competitive inhibitors such as stefins [43]. Our experiments establish that addition of the full-length testican-1 polypeptide results in inhibition of cathepsin L activity. Specific fragments of the polypeptide can be found in cerebral spinal fluid [44], blood [45] and human semen [1], indicating that testican-1 undergoes maturation or process- ing which might expose, free, or destabilize the thyropin domain. We observed that a preparation containing primarily proteolytic fragments of recombinant testican-1 also inhibited cathepsin L activity (data not shown). This is consistent with isolation of only the thyropin domain of p41 with cathepsin L purified from kidney [13]. This p41 domain is a competitive inhibitor of cathepsin L after it is cleaved from the invariant chain by endosomal proteases [36]. The identification in seminal plasma of testican-1 fragments cleaved within the thyropin domain [1] suggests that this cathepsin L inhibitor can also eventually be degraded by proteases that may be present in blood [45]. Testican interaction with proteases could be mediated by the polypeptide backbone of a proteoglycan, by its glycos- aminoglycans, or by both. Two glycosaminoglycan attach- ment sites are localized near the C-terminus of testican-1, at Ser residues 383 and 388 [2]. Significantly, the two preparations of testican-1, with and without chondroitin sulfate, were equally efficient inhibitors of the protease, suggesting inhibition was mediated by the protein core and not affected by the large glycosaminoglycan moieties. The high homology of the testican-1 thyropin domain to the cathepsin L-inhibitory domain of the p41 variant of the MHC invariant chain is consistent with the conclusion that cathepsin L inhibition is primarily mediated by protein– protein interactions. While cathepsin L is an intracellular protease localized within lysosomes under normal conditions, the protease is secreted when expression levels are elevated by cell trans- formation [19,46,47], in response to signaling [19–21], or during specific developmental stages [48,49]. The thyropin domain in testican-1 could serve merely to reduce the potentially destructive activity of this secreted cysteine protease. Alternatively, a thyropin domain presented in the context of a proteoglycan could alter cathepsin L-mediated proteolysis. There are ample reports of extracellular pro- teolysis ascribed to cathepsin L, however, it has not been clear how a protease unstable at neutral pH mediates extracellular proteolysis. pH-induced unfolding has been reported to cause rapid inactivation of mature cathepsin L [38]. While the presence of testican-1 reduced the rate of enzymatic cleavage of substrate, it also significantly slowed the expected loss of cathepsin L activity due to denaturation at neutral pH. Thus, our data suggest that testican-1 may actually stabilize the mature cathepsin L protease, so that its half-life is increased, although its velocity is reduced. A role for testican-1 in inhibiting, yet also stabilizing, protease activity is completely consistent with the recent findings that the p41 alternatively spliced variant of the MHC invariant chain is not merely an inhibitor of cathepsin L activity but also serves as a chaperone that helps to maintain a pool of active protease in late-endocytic compartments of antigen presenting cells [50]. Precedent for this role comes from the observation that coexpression of p41 with p31 modifies endosomal proteolysis of p31 [51]. Cathepsin L activity is also stabilized extracellularly when this p41–enzyme complex is secreted by activated macro- phages [52]. Heparin-like glycosaminoglycans have recently been reported to protect human cathepsin B from pH-induced inactivation in vitro [53], while heparan sulfate on ectodomains of cell membrane proteoglycans shed to wound fluids are known to protect serine proteases from interaction with their endogenous inhibitors, thus modify- ing the proteolytic balance of the fluid [54]. This physio- logical modulation of proteolysis primarily depends on protease interactions with the glycosaminoglycans of proteoglycans and does not require specific protein–protein interaction as occurs between testican-1 and cathepsin L. Through regulation of testican-1 expression levels, the more specific protein–protein interaction may spatially and temporally control the activity of secreted cathepsin L, allowing the enzyme to serve multiple, specific roles in different tissues. Acknowledgements We thank Dr Tom Traut for his expert advice on enzyme kinetics, Dr Mike Caplow for helpful suggestions, Susan Jones for assistance with the fluorescence microplate reader, and Dr Mohammad BaSalamah for stimulating the initiation of this study. This work was supported in part by National Institutes of Health RO1 HL55452 to C J. E and by a University of North Carolina Medical Faculty Award to A. E. References 1. Bonnet, F., Perin, J P., Maillet, P., Jolles, P. & Alliel, P.M. (1992) Characterization of a human seminal plasma glycosaminoglycan- bearing polypeptide. Biochem. J. 288, 565–569. 2. Alliel, P.M., Pedrin, J P., Jolles, P. & Bonnet, F.J. (1993) Testi- can, a multidomain testicular proteoglycan resembling modulators of cell social behaviour. Eur. J. Biochem. 214, 347–350. 3. Rieber, A.J., Marr, H.S., Comer, M.B. & Edgell, C.J.S. (1993) Extent of differentiated gene expression in the human endo- thelium-derived EA.hy926 cell line. Thrombo. Haemost. 69, 476–480. 4. 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Human proteoglycan testican-1 inhibits the lysosomal cysteine protease cathepsin L Jeffrey P. Bocock 1 , Cora-Jean S. Edgell 2 , Henry S.