Biochemical and Biophysical Research Communications 268, 904 –908 (2000) doi:10.1006/bbrc.2000.2207, available online at http://www.idealibrary.com on Characterization of Endopeptidase Activity of Tripeptidyl Peptidase-I/CLN2 Protein Which Is Deficient in Classical Late Infantile Neuronal Ceroid Lipofuscinosis Junji Ezaki,* Mitsue Takeda-Ezaki,* Kohei Oda,† and Eiki Kominami* ,1 *Department of Biochemistry, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan; and †Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Sakyo-ku, Kyoto, 606-8585, Japan Received December 22, 1999 Endopeptidase activities of the CLN2 gene product (Cln2p)/tripeptidyl peptidase I (TPP-I), purified from rat spleen, were studied using the synthetic fluorogenic substrates We designed and constructed decapeptides, based on the known sequence cleavage specificities of bacterial pepstatin-insensitive carboxyl proteases (BPICP) MOCAc-Gly-Lys-Pro-Ile-Pro-Phe-Phe-Arg-LeuLys(Dnp)r-NH is readily hydrolyzed by Cln2p/TPP-I (K cat/K m ؍7.8 s ؊1 mM ؊1) The enzyme had a maximal activity at pH 3.0 for an endopeptidase substrate, but at pH 4.5 with respect to tripeptidyl peptidase activity Both endopeptidase and tripeptidyl peptidase activities were strongly inhibited by Ala-Ala-Phe-CH2Cl, but not inhibited by tyrostatin, an inhibitor of bacterial pepstatininsensitive carboxyl proteases, pepstatin, or inhibitors of serine proteases Fibroblasts from classical late infantile neuronal ceroid lipofuscinosis patients have less than 5% of the normal tripeptidyl peptidase activity and pepstatin-insensitive endopeptidase activity Cln2p/ TPP-I is a unique enzyme with both tripeptidyl peptidase and endopeptidase activities for certain substrate specificity © 2000 Academic Press The neuronal ceroid lipofuscinosis (NCLs) represent a diverse group progressive neurodegenerative disorders which involve an autosomal recessive mode of inheritance (1, 2) Late infantile neuronal ceroid lipofuscinosis (LINCL) is characterized by the onset of symptoms at a relatively early age and the ultrastructure of storage bodies In cells with LINCL, subunit c of the mitochondrial ATP synthase complex (F 0F 1ATPase) is stored exclusively in the lysosomal compartment (3–5) Subunit c is a small (Mr 7608 Da), very hydrophobic protein We have been interested in the mechanism of lysosomal accumulation of subunit c in To whom correspondence should be addressed Fax: ϩ81-3-58025889 E-mail: Kominami@med.juntendo.ac.jp 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved fibroblasts with LINCL, and our earlier findings suggest that lysosomal proteolytic dysfunction is responsible for the specific delay in the degradation of subunit c (6 – 8) Sleat et al detected the absence of a single lysosomal protein in cases of LINCL, and identified mutations in the CLN2 gene which encodes for this protein in LINCL patients (9) The CLN2 gene product (Cln2p) showed significant amino acid sequence similarity with BPICP (10, 11) These workers also showed the presence of pepstatin insensitive hemoglobin hydrolase activity in normal brain, but a quite low level of activity in brain tissue from LINCL patients (12) We raised specific antibodies against Cln2p and showed that Cln2p is missing in fibroblasts with LINCL, and that lysosomal CLN2p is essential for the degradation of subunit c of ATP synthase, but not for the degradation of the  subunit of ATP synthase (13) Recently, Vines and Warburton reported that tripeptidyl peptidase I (TPP-I; EC 3.4.14.9) is identical to Cln2p, as evidenced by a comparison of TPP-I amino acid sequences with sequences derived from the EST database, Cln2p (14, 15) TPP-I is a lysosomal enzyme that cleaves tripeptides from the N-terminus of oligopeptides (14, 16) LINCL fibroblasts are devoid of TPP-I activities (15) However, the endopeptidase activity of Cln2p/TPP-I has not been completely characterized The present study was initiated in order to better understand the enzymological natures of Cln2p/ TPP-I and the cellular pathology of LINCL MATERIALS AND METHODS Peptide substrates and enzymes Cln2p/TPP-I was purified to apparent homogeneity from rat spleen by the methods described by Vines and Warburton (14) Ala-Ala-Phe-MCA (7-amino-4-methylcoumaline) and Ala-Ala-Phe-CH2Cl were purchased from Sigma Chemical Co (St Louis, MO) The five fluogenic peptides substrates MOCAc (7-methoxycoumaline-4yl)acetyl)-Gly-Lys-Pro-Ile-Pro-Phe-Phe-ArgLeu-Lys(Dnp)r-NH (substrate 1), MOCAc-Gly-Lys-Pro-Ile-Leu-Phe- 904 Vol 268, No 3, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE Substrate Specificities of Cln2p/TPP-I and Some Kinetic Parameters for the Hydrolysis of Fluorogenic Substrates Specific activity (nmol/min/mg) Substrate Ala-Ala-Phe-MCA P6 P5 P4 P3 P2 P1 P1Ј P2Ј P3Ј P4Ј MOCAc-Gly-Lys-Pro-Ile-Pro-Phe-Phe-Arg-Leu-Lys-(Dnp)r-NH -Leu-Ala-Leu-Leu-Glu-SerMOCAc-Ala-Pro-Ala-Lys- % 250.6 29.85 4.20 1.78 1.24 3.16 100 14.3 6.1 4.2 10.7 Km (mM) K cat (s Ϫ1) K cat/K m (s Ϫ1 mM Ϫ1) 0.25 17.0 68.0 0.40 — — — — 3.1 — — — — 7.8 — — — — Note All reactions were carryed out at pH 4.0 in sodium acetate buffer at 37°C for 10 Kinetic parameters were determined as described under Materials and Methods Phe-Arg-Leu-Lys(Dnp)r-NH (substrate 2), MOCAc-Gly-Lys-Pro-AlaLeu-Phe-Phe-Asp-Ser-Lys(Dnp)r-NH2 (substrate 3), MOCAc-Gly-LysPro-Ile-Leu-Phe-Phe-Glu-Ser-Lys(Dnp)r-NH (substrate 4), MOCAcAla-Pro-Ala-Lys-Phe-Phe-Arg-Leu-Lys(Dnp)r-NH2 (substrate 5) were custom-synthesized at the Peptide Institute (Osaka, Japan) They were dissolved in dimethyl sulfoxide at 10 mM and stored at Ϫ80°C until required for use Tyrostatin (N-isovaleryl-tyrosyl-leucyl-tyrosinal), a specific inhibitor of Pseudomonas carboxyl proteinase (PCP) and Xanthomonas carboxyl proteinase (XCP) was prepared as previously described (17) All chemicals were of reagent grade and were purchased from a variety of chemical sources Assays The tripeptidyl peptidase activity was determined by using Ala-Ala-Phe-MCA The substrate, 100 M Ala-Ala-Phe-MCA, 0.1 M sodium acetate buffer (pH 4.0 for routine assay) and enzyme were incubated in a total volume of 100 l at 37°C The reaction was terminated by the addition of 100 l of 10% SDS The liberated 7-amino-4-methylcoumaline was measured fluorometrically at an alkaline pH by adding ml of 0.1 M Tris–HCl, pH 9.0 (18) The hydrolysis of five fluorogenic substrates at the Phe-Phe bonds was determined as described by Yasuda et al (19) A typical reaction mixture contained 25 M substrate, 0.04 M sodium acetate buffer (pH 4.0 for routine assays) and sample solutions were incubated in a total volume of 100 l at 37°C The reaction was terminated by the addition of ml of 5% trichloroacetic acid Kinetic constants K m and V max were calculated from Lineweaver–Burk plots K cat was derived from V max ϭ k cat[E] 0, where [E] is the enzyme concentration The activity, with respect to hemoglobin hydrolysis, was determined by measuring acid-soluble products as described previously (20) Acidsoluble products from hemoglobin were quantified by the use of fluorescamine Three hundred microliters of acid-soluble products was mixed with ml of 0.2 M borate buffer (pH 8.9) and then added to ml of fluorescamine (0.1 mg/ml in acetone) The resulting fluorescence was measured within 10 at 475 nm (excitation at 390) Fibroblast cultures and preparation of mitochondrial-lysosomal fractions Skin fibroblasts were obtained from the McGill University Repository for Mutant Human Cell Strains, the Kennedy Krieger Institute Repository for Mutant Human Strains, and the Edmonton Genetic Clinic, University of Alberta repository for Mutant Human Cell Strains Fibroblasts from control and patients with JNCL (the juvenile form of NCL) and LINCL were cultured at 37°C under 5% CO in air in Dulbecco’s modified Eagle’s medium, supplemented with 10% (v/v) heat-inactivated fetal calf serum and antibiotic–antimycotic solution (GIBCO) Mitochondrial-lysosomal fractions from fibroblasts were obtained by differential centrifugation as described previously (5) RESULTS AND DISCUSSION Endopeptidase activity of Cln2p/TPP-I The CLN2 gene product (Cln2p) has been identified as a homo- logue of the bacterial pepstatin-insensitive carboxyl proteases (BPICP) from Pseudomonas (PCP) and Xanthomonas (XCP) (10, 11) To examine the endopeptidase activity of Cln2p/TPP-I and its substrate specificity, we synthesized fluorogenic substrates with decapeptides, based on the substrate specificity of BPICP and pepstatin-sensitive carboxyl proteinases, such as cathepsin D and E (Table 1) Of the five substrates tested, Cln2p/TPP-I was found to cleave the synthetic decapeptide MOCAc-Gly-Lys-Pro-Ile-ProPhe-Phe-Arg-Leu-Lys(Dnp)r-NH (substrate-1) most effectively (Table 1) MOCAc-Gly-Lys-Pro-Ile-Leu-PhePhe-Arg-Leu-Lys(Dnp)r-NH (substrate-2) was originally synthesized for the assay of cathepsin D and E (19) In fact, rat cathepsin D cleaved substrate-2 much more effectively than substrate-1 (not shown) A chromogenic substrate with the same amino acid sequence as substrate-2 is the best substrate for PCP and a good substrate for XCP (11, 21) Thus the specificity of endopeptidase activity of Cln2p/TPP-I appears to be different from that of cathepsin D, PCP or even XCP Another BPICP from Bacillus coagulans J-4 (J-4) catalyzes the hydrolysis of chromogenic peptides with the same amino acid sequence as substrate-1 quite effectively (22), suggesting that the endopeptidase activity of Cln2p/TPP-I differs considerably in substrate specificity from PCP and XCP and, of BPICP, that it is rather similar to J-4 Cln2p/TPP-I also cleaved Ala-Ala-Phe-MCA, a substrate for tripeptidyl peptidase at pH 4.0 more effectively than substrate-1 The K m and K cat values for Ala-Ala-Phe-MCA and substrate-1 of Cln2p/TPP-I were 0.25 mM, 17.0 s Ϫ1 and 0.4 mM, 3.1 s Ϫ1 respectively (Table 1) To confirm the cleavage site, substrate-1 was incubated with purified TPP-I at pH for h at 37°C The resulting cleaved peptides were chromatographed by reverse-phase HPLC on a 4.6 ϫ 250 mm Cosmosil 5C18-AR (Nacalai tesque, Kyoto, Japan) using a –52.5% acetonitrile/0.1% trifluoroacetic acid gradient over 80 The substrate was cleaved into two peptides, and the N-terminal sequence of one of these was 905 Vol 268, No 3, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Phe-Arg-Leu (not shown) This finding indicates that TPP-I acts as an endopeptidase and cleaves the substrate at the Phe-Phe bond These results clearly indicate that purified Cln2p/TPP-I shows both tripeptidyl peptidase activity and endopeptidase activity Characterization of Cln2p/TPP-I with both tripeptidyl peptidase and endopeptidase activities When Ala-Ala-Phe-MCA was incubated with purified Cln2p/ TPP-I over a wide range of pH (2.5– 6.0) at 37°C for 10 min, a single peak was observed at around pH 4.5, in agreement with a previous report (14) At pH 3.0, the tripeptidyl peptidase activity of Cln2p/TPP-I was less TABLE Effect of Inhibitors on Activities of Tripeptidy Peptidase and Endopeptidase of Cln2p/TPP-I Inhibition (%) Inhibitor AAFCH 2Cl Pepstatin Tyrostatin DFP PMSF Concentration (M) AAF-MCA Substrate-1 10 0.1 10 0.1 10 0.1 10,000 1,000 1,000 99.5 94.1 67.4 5.2 0 3.2 0 0 95.9 84.1 21.5 0 0 0 0 Note All reactions were carryed out at pH 4.0 in sodium acetate buffer at 37°C for 10 Substrate-1 is MOCAc-Gly-Lys-Pro-IlePro-Phe-Phe-Arg-Leu-Lys(Dnp)r-NH FIG Determination of the pH profile for the activity (A) and pH stability (B) of Cln2p/TPP-I The conditions for the hydrolysis of substrates are described under Materials and Methods Substrates used are Ala-Ala-Phe-MCA (E, F), MOCAc-Gly-Lys-Pro-Ile-Pro-PhePhe-Arg-Leu-Lys(Dnp)r-NH (substrate-1) (‚, Œ), and hemoglobin (ᮀ, ■) (A) Buffers used were sodium formate (E, ‚, ᮀ) or sodium acetate buffer (F, Œ, ■) The point of maximum activity was taken as 100% in each case (B) The enzyme was incubated for 30 at 37°C after mixing with ten volumes of 0.1 M buffers of the indicated pH (pH 2.5, formate; pH 4.5– 6.0, acetate; and pH 6.5– 6.0, phosphate) After adjusting the pH to 4.0, the remaining activities were measured using Ala-Ala-Phe-MCA (F) and MOCAc-Gly-Lys-Pro-Ile-ProPhe-Phe-Arg-Leu-Lys(Dnp)r-NH (E) as substrates as described under Materials and Methods than 15% of its maximal activity at pH 4.5 On the other hand, the endopeptidase activity of Cln2p/TPP-I with respect to substrate-1 showed a pH optimum of approximately pH 3.0, and the activity at pH 4.5 was less than 30% of its maximum activity at pH 3.0 At pH 4.0, at which the kinetic parameters of the enzyme shown in Table was obtained, Cln2p/TPP-I showed about 50% of its maximal activity The optimum pH for the hemoglobin hydrolysis activity was observed at around pH 3.5, using formate buffer and at around pH 4.0, using an acetate buffer (Fig 1A), in agreement with the literature data (12) Thus, the optimum pH of CLN2p/TPP-I is dependent on the substrates used The stability of tripeptidyl peptidase activity and endopeptidase activity of CLN2p/TPP-I was analyzed by incubation over a wide range of pH at 37°C for 30 Both activities showed a similar tendency with respect to the stability at different pH, except pH 2.5 The enzyme was most stable at pH 3– and became labile near the neutral pH Both activities were essentially lost above pH 6.0 (Fig 1B) Ala-Ala-Phe-CH 2Cl, a chloromethyl ketone analogue is known to be a potent inhibitor of TPP-I (14) As shown in Table 2, three peptidase activities of CLN2p/ TPP-I, tripeptidyl peptidase activity, endopeptidase activity using the synthetic fluorogenic substrate (substrate-1) and hemoglobin hydrolyzing activity (not shown) were all strongly inhibited by, Ala-Ala-PheCH 2Cl At M of Ala-Ala-Phe-CH 2Cl, 94% of the tripeptidyl peptidase activity, 84% endopeptidase activity, and 84% of hemoglobin hydrolyzing activity (not shown) were inhibited The issue of whether CLN2p/ TPP-I is inhibited by tyrostatin, a potent inhibitor of PCP and XCP (17) is an interesting one This inhibitor showed no effect on both tripeptidyl peptidase activity 906 Vol 268, No 3, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE Tripeptidyl Peptidase and Pepstatin-Insensitive Endopeptidase Activities in Normal and Patient Fibroblasts with NCLs Specific activity (nmol/min/mg protein) ϫ 10 Ϫ1 AAF-MCA Cell line Description NFB3 NFB4 11C WG277 6B UA1 UA2 10C WG308 WG305 Normal Normal Carrier JNCL JNCL LINCL LINCL LINCL LINCL LINCL Substrate-1 ϩAAFCH 2Cl ϩAAFCH 2Cl 23.34 Ϯ 1.52 18.18 Ϯ 1.04 18.12 Ϯ 0.90 23.52 Ϯ 0.44 25.94 Ϯ 1.22 0.58 Ϯ 0.12 0.34 Ϯ 0.04 0.94 Ϯ 0.10 0.58 Ϯ 0.10 0.62 Ϯ 0.10 0.27 0.17 0.69 0.26 0.20 0.12 0.14 0.47 0.15 0.15 9.37 Ϯ 2.09 8.05 Ϯ 1.97 10.57 Ϯ 1.35 6.64 Ϯ 1.17 10.55 Ϯ 0.99 0.33 Ϯ 0.05 0.28 Ϯ 0.08 0.40 Ϯ 0.10 0.25 Ϯ 0.07 0.23 Ϯ 0.08 0.41 0.21 0.30 0.25 0.24 0.21 0.21 0.36 0.19 0.18 Note Results are means Ϯ standard errors of five determinations or the mean of two determinations (in the presence of AAF 2Cl) Tripeptidyl peptidase activity was measured at pH 4.0 and endopeptidase activity was measured at pH 3.5 in the presence of M pepstatin and 0.1 mM E-64c Substrate-1 is MOCAc-GKPIPFFRLK(Dnp)r-NH and endopeptidase activity of CLN2p/TPP-I (Table 2), which is possibly related to the substrate specificity of CLN2p/TPP-I (Table 1) CLN2p/TPP-I preferably cleaves substrate-1 from Bacillus coagulans J-4 but is less specific for the hydrolysis of substrate-2 of PCP PCP and XCP are strongly inhibited by tyrostatin, whereas J-4 is not (K Oda, unpublished results) Pepstatin, an inhibitor of aspartic proteinases and inhibitors of serine proteinases, DFP (diisopropyl fluoride phosphate) and PMSF (phenylmethylsulfonyl fluoride) had no effect on either tripeptidyl peptidase activity or endopeptidase activity (Table 2) Tripeptidyl peptidase and endopeptidase activities in fibroblasts from LINCL patients Mitochondriallysosomal fractions prepared from fibroblasts of two normal, two JNCL, five LINCL and one LINCL carrier were assayed for tripeptidyl peptidase activity and endopeptidase activity using substrate-1 In this experiment, endopeptidase activities were determined in the presence of pepstatin and E-64c ((ϩ)-(2S,3S)-3-[(S)Methyl-1-(3-methylbutylcarbamoyl)-butylcarbamoyl]2-oxiranecarboxylic acid) in order to avoid the effect of other lysosomal endopeptidases (Table 3) A dramatic reduction in tripeptidyl peptidase activity was observed in the case of LINCL fibroblasts, in agreement with results obtained by Vines and Warburton (15) Activities in normal and LINCL fibroblasts were sensitive to Ala-Ala-Phe-CH 2Cl, but were not inhibited by pepstatin (not shown), indicating that the determination of tripeptidyl peptidase activity is reliable Endopeptidase activities were also detected in the mitochondrial-lysosomal fractions of the control cells and cells from JNCL patients, whereas they were devoid in the cells with LINCL These activities were inhibited in the presence of Ala-Ala-Phe-CH 2Cl The above results indicate that Cln2p/TPP-I with both tripeptidyl peptidase and pepstatin insensitive endopeptidase activities is defective in LINCL We recently showed that lysosomal content which were depleted of Cln2p/TPP-I lost capacity to degrade subunit c of ATP synthase (13) It would be of interest to know which activity, i.e., the tripeptidyl peptidase activity or the endopeptidase activity is responsible for the lysosomal degradation of subunit c In the present study, the issue of whether Cln2p/ TPP-I is a carboxyl proteinase or not is not known For identifying the catalytic residues of Cln2p/TPP-I, site directed mutagenesis studies based on its sequence similarities to PCP and XCP, or the isolation and sequencing of peptides labeled with a radioactive AAFCH 2Cl would be required Recently, a pair of catalytic aspartic acids for PCP and XCP were determined to be catalytic residues by a molecular biological approach (23) The results suggest (23) that in the case of Cln2p, a pair of catalytic aspartic acid residues is conserved, and that the distance between the residues is close to that of PCP An analysis of the catalytic residues of Cln2p/TPP-I using a site directed mutagenesis approach is currently under investigation ACKNOWLEDGMENTS We thank Dr L S Wolfe of the Montreal Neurological Institute, Dr B M Bunn of University of Florida, and Drs T Ueno, K Ishidoh, D Muno, and I Tanida (Juntendo University) for helpful discussion We are grateful to Dr F Andermann, Dr S Carpenter, and Dr H Durham (Montreal Neurological Institute), Dr S Naidu (Kennedy Krieger Institute, Baltimore, MD), and Dr P Ferreira (Edmonton Genetics Clinic, University of Alberta) for their help in acquiring skin fibroblasts from patients and member of Central Laboratory for Medical Science (Juntendo University) for technical advice This work was supported, in part, by a Grant-in Aid for 907 Vol 268, No 3, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Scientific Research (Intracellular Proteolysis, 08278103 and 10670144) from the Ministry of Education, Science, and Culture of Japan and by the Science Research Promotion Foundation Fund from the Japan Private School Promotion Foundation REFERENCES Rider, J A., and Rider, D L (1988) Am J Med Genet Suppl 5, 21–26 Mole, S E (1999) Lancet 354, 443– 445 Palmer, D N., Martinus, R D., Cooper, S M., Midwinter, G G., Reid, J C., and Jolly, R D (1989) J Biol Chem 264, 5736 – 5740 Hall, N A., Lake, B D., Dewji, N N., and Patrick, A D (1991) Biochem J 275, 269 –272 Kominami, E., Ezaki, J., Muno, D., Ishido, K., Ueno, T., and Wolfe, L S (1992) J Biochem (Tokyo) 111, 278 –282 Ezaki, J., Wolfe, L S., Higuti, T., Ishidoh, K., and Kominami, E (1995) J Neurochem 64, 733–741 Ezaki, J., Wolfe, L S., and Kominami, E (1996) J Neurochem 67, 1677–1687 Ezaki, J., Wolfe, L S., and Kominami, E (1997) 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Y., and Yamamoto, K (1999) J Biochem 125, 1137–1143 20 Takeda-Ezaki, M., and Yamamoto, K (1993) Arch Biochim Biophys 304, 352–358 21 Narutataki, S., Dunn, B M., and Oda, K (1995) J Biochem 125, 75– 81 22 Shibata, M., Dunn, B M., and Oda, K (1998) J Biochem 124, 642– 647 23 Oyama, H., Abe, S., Ushiyama, S., Takahashi, S., and Oda, K (1999) J Biol Chem 274, 27815–27822 908 ...Vol 268, No 3, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE Substrate Specificities of Cln2p/TPP-I and Some Kinetic Parameters for the Hydrolysis of... Central Laboratory for Medical Science (Juntendo University) for technical advice This work was supported, in part, by a Grant-in Aid for 907 Vol 268, No 3, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH... described (17) All chemicals were of reagent grade and were purchased from a variety of chemical sources Assays The tripeptidyl peptidase activity was determined by using Ala-Ala-Phe-MCA The