Identification of the catalytic triad in tripeptidyl-peptidase II through site-directed mutagenesis

Biochimica et Biophysica Acta 1601 (2002) 149 – 154 www.bba-direct.com

Identification of the catalytic triad in tripeptidyl-peptidase II through site-directed mutagenesis Hubert Hilbi a,1, Emese Jozsa b, Birgitta Tomkinson b,* a

Department of Microbiology, The Skirball Institute, New York University School of Medicine, New York, NY 10016, USA b Department of Biochemistry, Biomedical Center, Uppsala University, Box 576, Uppsala SE-75123, Sweden Received 18 July 2002; received in revised form 17 September 2002; accepted 25 September 2002

Abstract Tripeptidyl-peptidase II (TPP II) is a 138-kDa subtilisin-like serine peptidase forming high molecular mass oligomers of >1000 kDa. The enzyme participates in general protein turnover and apoptotic pathways, and also has specific substrates such as neuropeptides. Here we report the site-directed mutagenesis of amino acids predicted to be involved in catalysis. The amino acids forming the putative catalytic triad (Asp-44, His-264, Ser-449) as well as the conserved Asn-362, potentially stabilizing the transition state, were replaced by alanine and the mutated cDNAs were transfected into human embryonic kidney (HEK) 293 cells. In clones stably expressing the mutant proteins, TPP II activity did not exceed the endogenous activity, thus confirming the essential role of the above amino acids in catalysis. Mutant and wild-type TPP II subunits co-eluted from a gel filtration column, suggesting that the subunits associate and that the native subunit conformation was retained in the mutants. Interestingly, the S449A and a H264A mutant enzyme affected the quaternary structure of the endogenously expressed TPP II, resulting in formation of an active, larger complex of >10,000 kDa. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Tripeptidyl-peptidase II; Serine peptidase; Catalytic triad; Mutagenesis; Subtilase

1. Introduction Tripeptidyl-peptidase II (TPP II; E.C.3.4.14.10) is an enzyme that removes tripeptides from a free N-terminus of oligopeptides [1]. TPP II is present in the cytosol of a number of different cells of several different species, from fruit flies to humans [2 –4]. The proposed physiological role for this enzyme is to participate in general protein turnover, probably in concert with the proteasome and other exopeptidases [5,6]. The formation of tripeptides as intermediates during degradation of proteins to free amino acids accelerates the process by increasing the concentration of potential substrates for other exopeptidases [5]. Besides its function in general protein turnover, TPP II plays a more specific role as a cholecystokininAbbreviations: TPP II, tripeptidyl-peptidase II; pNA, para-nitroanilide; DTT, dithiotreitol; DMEM, Dulbecco’s modified Eagle’s medium * Corresponding author. Tel.: +46-18-471-4659; fax: +46-18-55-8431. E-mail address: [email protected] (B. Tomkinson). 1 Current address: Institute of Microbiology, Swiss Federal Institute of Technology (ETH) Zu¨rich, LFV B36.2, Schmelzbergstrasse 7, CH-8092 Zu¨rich, Switzerland.

inactivating enzyme in rat brain [7], or in apoptotic pathways [8]. During apoptosis triggered by the enteropathogenic bacterium Shigella flexneri in macrophages, TPP II promotes the maturation of pro-caspase-1, and therefore seems to participate upstream of this caspase [8]. The 138 kDa subunits of TPP II form large, oligomeric complexes (>1000 kDa) that are localized in the cytoplasm [1,2,9,10] or the plasma membrane [7]. The enzyme has been classified as a serine peptidase of the subtilisin type [11,12]. The active site serine residue (Ser-449) was identified by labeling with 3H-diisopropyl fluorophosphate and isolation of labeled tryptic peptides [11]. The remaining amino acids of the catalytic triad (Asp-44 and His-264) as well as Asn-362, which stabilizes the oxyanion in the transition state, were assigned based on sequence similarities to other subtilases [12 – 14]. There has been some ambiguity regarding the potential catalytic Asp-residue, which was first reported as Asp-211 [12] and later as Asp-44 [13]. In order to unequivocally prove the importance of these residues for catalysis, Asp-44, His-264, Asn362 and Ser-449 were mutated to alanine and the mutants characterized. This work demonstrates that the above

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mutants are catalytically inactive, thereby supporting the essential role of these amino acids during catalysis. Endogenous TPP II and mutant enzyme specimens co-eluted from a gel filtration column, suggesting that the native subunit conformation was retained in these mutants. Interestingly, some mutants displayed unexpected effects on the quaternary structure of TPP II endogenously expressed in the 293cells.

2. Materials and methods 2.1. Construction of TPP II mutants The amino acid residues forming the putative catalytic triad of TPP II (D44, H264, S449) as well as a conserved asparagine residue (N362) were mutated to alanine residues using the PCR-based QuickChange site-directed mutagenesis kit according to the manufacturer’s instruction (Stratagene). As a template for the PCR reaction, the murine TPP II cDNA variant without the extra 39 bp, inserted into the pcDNA3 vector, was used [15]. To introduce the single amino acid substitutions, the following oligonucleotide primers were designed (alanineencoding codon bold, mutation(s) underlined): 5V-C GCC GTC CTG GCC ACA GGG GTC G-3V (D44Afwd), 5V-C GAC CCC TGT GGC CAG GAC GGC G-3V(D44Arev), 5V-CC AGC GGA GGA GCT GCT GGA ACC CAT GTA GC-3V(H264Afwd), 5V-GC TAC ATG GGT TCC AGC AGC TCC TCC GCT GG-3V (H264Arev), 5VGTT TCA AGT GCT GGA GCT AAT GGT CCA TGC C-3V (N362Afwd), 5V-G GCA TGG ACC ATT AGC TCC AGC ACT TGA AAC-3V(N362Arev), 5V-G CTA ATG AAT GGG ACA GCA ATG TCT TCC CCC-3V(S449Afwd), 5V-GGG GGA AGA CAT TGC TGT CCC ATT CAT TAG C-3V (S449Arev). The 9.4-kb plasmid template (50 ng) was amplified by PCR in a total of 50 Al using 125 ng each of two corresponding primers, 2.5 mM each of the dNTP and 2.5 U Pfu DNA polymerase (16 cycles, annealing temperature 60 jC, elongation time 18 min). The D44A, H264A, N362A or S449A mutations were verified by sequencing the corresponding TPP II cDNA region using the following primers: 5V-CGA AAT TAA TAC GAC TCA C-3V (oTPPII/0), 5VGGG ATC CAA TTC ACA GAG-3V (oTPPII/1), 5V-CTG AAC GGA ATG GAG TTG-3V(oTPPII/2), or 5V-CAA GTG CTG ATG GAG CCC-3V (oTPPII/3), respectively. About 50 –70% of the plasmids contained the expected mutations. The remaining cDNA of the TPP II mutants was sequenced using the primers 5V-CAT TGA GCC TGT ATT TCC-3V (oTPPII/4), 5V-GCA GTA CAG CTT GTG AAG-3V(oTPPII/ 5), 5V-GAG CGG AGA AGT AAC ACC-3V(oTPPII/6), 5VGCA GGC TCC TTG ACA TTG-3V(oTPPII/7), and 5V-CAG GCC TGA TGC AGC TAC-3V(oTPPII/8), respectively, and was found to be identical to the wild-type TPP II cDNA sequence.

2.2. Transfection of 293-cells with TPP II mutants The plasmids harboring TPP II mutants (pcDNA3TPPII-D44A, -H264A, -N362A or -S449A) were transfected into the human embryonic kidney (HEK) cell line 293 (ATCC CRL 1573) by using the FuGENE 6 transfection reagent as instructed by the manufacturer (Roche). As controls, the 293-cells were transfected with the pcDNA3 vector carrying wild-type TPP II in either sense or antisense direction [15]. For transfections, 1  105 cells/ well (24-well plate) were grown overnight and incubated with 0.5 Ag DNA at a FuGENE 6/DNA ratio (v/w) of 3:1. After 3 days, stable transfectants were selected by growing the cells for 1 –2 weeks in DMEM with 10% heat-inactivated fetal calf serum and 0.5 mg/ml geneticin (G418, Life Technologies). 2.3. Isolation of highly expressing clones An aliquot of cells from each of the different transformants was diluted with medium and seeded in a 96-well plate at a concentration of 0.5 – 5 cells/well or seeded on 10cm plates at a concentration of 100 cells/plate. The cells were maintained in DMEM:F-12 (Nutrient mix) (GibcoBRL) with 10% (v/v) heat-inactivated fetal calf serum and 0.4 mg/ml geneticin, at 37 jC in a humidified 5% CO2 atmosphere. After 10 – 14 days, colonies formed from single cells were harvested and expanded. Cell lysates were prepared from these colonies and analyzed for enzyme activity and immunoreactivity with TPP II-specific antibodies by Western blot. The clone expressing wild-type murine TPP II was isolated previously [15]. 2.4. Preparation of cell extracts Cells from stable transformants expressing mutant TPP II were harvested and lysed with 50 mM Tris-buffer, pH 7.5, containing 1% Triton X-100 (100 Al/106 cells). The lysate was centrifuged for 30 min at 4 jC and 14,500  g. The supernatant was collected and diluted 10-fold with 100 mM potassium phosphate buffer, pH 7.5, containing 30% (w/v) glycerol and 1 mM DTT. Diluted supernatants were used for activity assays and Western blot analysis. 2.5. Enzyme assay Enzyme aliquots (100 Al) were mixed with 50 Al 0.8 mM Ala-Ala-Phe-pNA (AAF-pNA, Bachem) and 50 Al 0.2 M potassium phosphate buffer, pH 7.5, containing 8 mM DTT and 400 AM bestatin and incubated at 37 jC [4]. The change in absorbance at 405 nm was measured in a Spectramax PLUS plate reader (Molecular Devices). A molar absorbance of 9600 M 1 cm 1 for pNA was used [16]. The activity was related to the total amount of protein in the sample, determined with a modified Bradford method [17,18], using BSA as the standard.

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2.6. Gel filtration Cell extracts were prepared essentially as described above, except that the cells were lysed at a concentration of 10 Al/106 cells. The diluted supernatant (1.8 ml, corresponding to 1 – 2  107 cells) was loaded onto a Sepharose CL-4B (Amersham Biosciences) column (1  86.5 cm). The column was equilibrated and eluted with 0.1 M potassium phosphate buffer, pH 7.5, containing 30% (w/v) glycerol and 1 mM DTT, at a flow rate of 6 ml/h. Fractions of 1 ml were collected. The void-volume (Vo = 26.6 ml) and total volume (Vt = 73.8 ml) of the column was determined using Blue dextran (Amersham Biosciences) and dinitrophenol-hAla (Sigma), respectively. Kav values were calculated with the formula Kav = Ve Vo/Vt Vo. Individual fractions were analyzed for TPP II activity and by Western blot.

Fig. 1. Western blot analysis of individual 293-cell clones expressing the different TPP II mutants, which were selected for further characterization. Cell lysates (corresponding to 3 Ag protein) were prepared and loaded onto an 8% polyacrylamide gel, and Western blot analysis was performed as described in Materials and methods. The sizes of prestained molecular weight markers (Bio-Rad) are indicated.

cells (harboring the empty vector) and cells expressing wildtype TPP II were included on all gels to ensure that the actual intensity (PDmm2) was in the same range.

2.7. Western blot analysis SDS/PAGE and Western blot analysis using affinitypurified chicken anti human TPP II antibodies was performed essentially as described previously [19]. Samples were mixed with SDS/PAGE sample buffer to give final concentrations of 2.3% (w/v) SDS, 5% (v/v) h-mercaptoethanol and 10% (w/v) glycerol, and heated for 5 min at 95 jC before they were loaded onto an 8% polyacrylamide gel. Following electrophoresis, the proteins were transferred from the gel to a nitrocellulose filter by wet transfer in a Mini Trans-blot cell (Bio-Rad) for 1 h at 100 V in 20 mM Tris –150 mM glycine buffer, pH 8.3, containing 20% (v/v) methanol and 0.01% SDS. ECL-detection was performed according to the manufacturer’s recommendations (Amersham Biosciences). For the experiment presented in Table 1 and Fig. 1, the immunoreactivity was quantified on scanned X-ray films using Molecular Analyst software (Bio-Rad). The amount of sample was adjusted to a total of 3 Ag of protein (as determined by the Bradford method [17,18]) in each lane. Further, three different gels were quantified, and control

3. Results and discussion 3.1. Isolation of clones expressing TPP II mutant enzymes Based on sequence similarities to subtilases, the amino acid residues of TPP II forming the putative catalytic triad (Asp-44, His-264 and Ser-449) and Asn-362, potentially stabilizing the transition state, were assigned [4,12 – 14]. To confirm the involvement of these amino acids in catalysis, they were mutated to alanine residues. Alanine substitutions were chosen to avoid introducing new charge interactions or hydrogen bonds and to minimize unfavorable steric contacts [20]. The mutated cDNA was used to transfect 293-cells, and pools of stable transformants expressing the different mutants were selected. The N362A or S449A mutants were highly expressed in these pools as judged from Western blot analysis. In contrast, the D44A and H264A mutants were expressed to a much lower extent (data not shown). To obtain 293-cells highly expressing TPP II mutants, individual clones were selected and the expression level was

Table 1 TPP II activity in cell lysates from individual 293-cell clones expressing wild-type and mutant TPP II TPP II mutant

Clone

(n)

Activity (DA/min/mg)

Immunoreactivity (10 3  PD  mm2/mg)

Specific activity (activity/immunoreactivity)

Control Wild-type D44A D44A H264A H264A S449A S449A N362A N362A

D3 21 A5 11 A6 H2 1 D10 3 H6

(6) (5) (5) (5) (5) (5) (7) (4) (5) (4)

0.103 F 0.035 0.855 F 0.231 0.081 F 0.021 0.115 F 0.038 0.090 F 0.023 0.073 F 0.022 0.076 F 0.045 0.083 F 0.017 0.102 F 0.047 0.089 F 0.022

0.063 0.694 0.820 0.388 0.163 0.466 0.753 0.824 0.479 1.092

1.64 1.23 0.10 0.30 0.55 0.16 0.10 0.10 0.21 0.08

Cell lysates (corresponding to 1 – 3  106 cells) were prepared, and enzyme activity was assayed using AAF-pNA as the substrate, as described under Materials and methods. (n) indicates number of activity measurements. The immunoreactivity was quantified from two or three different Western blot analyses. PD, pixel density.

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analyzed. All clones selected for further characterization (except H-A6) expressed TPP II mutants well above the level of endogenous TPP II expressed by control cells transfected with the vector alone (Fig. 1). In accordance with the results obtained from analyzing transfected cell pools, a much higher proportion of individual N362A or S449A mutant clones showed a high expression level (data not shown). 3.2. TPP II mutant enzymes are catalytically inactive To analyze TPP II activity, the chromogenic peptide substrate AAF-pNA was used. Bestatin was included in the assay mixture to ensure that the substrate was not sequentially cleaved by aminopeptidases [15]. First, TPP II activity was determined in pools of 293-cells transiently transfected for 24 h with wild-type or TPP II mutant proteins. These transiently transfected cells expressed similar levels of wild-type or TPP II mutant protein (data not shown). TPP II activity was about four times higher in cells expressing wild-type enzyme than in cells transfected with the (i) mutant enzymes, (ii) vector carrying TPP II in reverse orientation, or (iii) empty vector (data not shown). Next, individual clones expressing TPP II mutant proteins were selected and TPP II activity was determined. The proteolytic activity towards AAF-pNA of clones expressing mutant enzymes was similar to control cells harboring the empty vector (‘‘control’’, Table 1), indicating that the mutant enzymes are catalytically inactive. In contrast, 293-cells overexpressing similar amounts of the wild-type enzyme showed eight-fold higher TPP II activity (‘‘wild-type’’, Table 1 and Ref. [15]). To account for different expression levels of mutant enzymes, we quantified the enzyme amounts by immuno-densitometry and calculated a specific activity (Table 1). The overexpressed wild-type murine TPP II had a specific activity similar to endogenously expressed human TPP II. In all clones expressing the mutant proteins, however, the specific activity was considerably lower and inversely correlated to the protein amount, i.e., the higher the expression level, the lower the specific activity (cf. Fig. 1). Considering the sensitivity of the assay used, the catalytic activity of the TPP II mutants N362A, S449A, D44A and H264A is at least one order of magnitude lower than the wild-type enzyme, but could be considerably lower. Ser-449 has previously been identified as the catalytic residue [11], and therefore, the S449A mutant was expected to be inactive. Since the other mutants yielded essentially the same results, the amino acid residues selected for mutation are required for the catalytic activity of TPP II. 3.3. Complex formation by TPP II active site mutant enzymes The 138 kDa TPP II-subunits forms a large (>1000 kDa) oligomeric complex, which is necessary for full enzymatic activity [9,21]. Previous investigations have shown that one

reason for a low enzyme activity upon overexpression of TPP II is that the large oligomeric complexes are not formed [21,22]. Thus, the inactivity of the D44A, H264A N362A and S449A mutant enzymes might be due to either a direct role of the mutated amino acids in catalysis or impaired complex formation by the mutants. To investigate the oligomeric structure of the mutant enzymes, cell lysates were fractionated by size exclusion chromatography (Sepharose CL-4B), and the fractions were analyzed for TPP II activity and protein. Representative chromatograms and quantitative results from two chromatographies of each clone are shown in Fig. 2 and Table 2, respectively. Endogenous TPP II activity and protein (data not shown) from 293-cells harboring an empty vector eluted at a Kav of 0.3, which corresponds to an elution volume of 35 –40 ml and a molecular mass of 2 –4  106 Da (Fig. 2, Table 2; ‘‘control‘‘). In addition, about 10% of endogenous TPP II activity eluted in the void volume, and therefore seems to form a very large complex (>10,000 kDa). In the presence of the D44A-mutant (clone D-A5 and D-11), the TPP II activity and protein eluted in a pattern very similar to endogenous TPP II (Figs. 2A and 3; Table 2; clone DA5). Similarly, in cells expressing N362A mutant proteins

Fig. 2. Size exclusion chromatography of 293-cells expressing mutant (A,B) or wild-type TPP II. Lysates from cells stably transformed with TPP II mutants were prepared as described in Materials and methods, and the diluted supernatant (1.8 ml, corresponding to 1 – 2  107 cells) was loaded onto a Sepharose CL-4B column. After chromatography, TPP II activity was analysed by the standard assay as described in Materials and methods. The enzyme activity in extracts from control cells (vector only) is included as a reference.

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Table 2 Quantification of TPP II activity after gel filtration chromatography Clone

Control Wild-type D-A5 D-11 H-A6 H-H2 S-1 S-D10 N-3 N-H6

Peak

Vo Ve Vo Ve Vo Ve Vo Ve Vo Ve Vo Ve Vo Ve Vo Ve Vo Ve Vo Ve

Chromatography #1 (cf. Fig. 2)

Chromatography #2

Activity (DA/min/ml)

Volume (ml)

Total activity (DA/min)

Percentage (%)

Total activity (DA/min)

Percentage (%)

0.008 0.021 0.012 0.123 0.003 0.022 0.004 0.016 0.025 0.008 0.025 0.021 0.029 0.007 0.034 0.005 0.011 0.015 0.013 0.017

3.6 10.2 1.8 12.7 5.5 13.8 4.3 11.9 6.4 13.8 5.4 14.4 4.6 8.2 6.3 13.5 4.5 13.5 5.6 15.0

0.03 0.22 0.02 1.56 0.02 0.30 0.02 0.19 0.16 0.12 0.14 0.30 0.13 0.05 0.21 0.07 0.05 0.20 0.08 0.26

12 88 1 99 6 94 9 91 58 42 31 69 71 29 76 24 19 81 23 77

0.02 0.25 0.02 1.25 0.02 0.23 0.02 0.16 0.11 0.04 0.02 0.12 0.07 0.03 0.18 0.06 0.08 0.22 0.06 0.12

7 93 1 99 9 91 13 87 71 29 15 85 67 33 73 27 26 74 33 67

Gel filtration of 293-cell extracts was performed as described in the legend of Fig. 2. The fractions of the activity peak eluting in the void volume (Vo) or at Kav c 0.3 (Ve) were pooled and analyzed as described in Materials and methods. The distributions between the two peaks are shown for two chromatographies (#1, #2) for each clone.

(clone N-3 and N-H6), most TPP II activity and protein eluted at a Kav of 0.3 (Figs. 2A and 3; Table 2; clone N-H6). In cells expressing H264A (clone H-A6 and H-H2) and S449A mutant proteins (clone S-D10 and S-1), larger amounts of activity and protein eluted in the void volume (Figs. 2B and 3; Table 2; H-A6 and H-A2, or S-D10 and S1, respectively). However, in both cases, part of the activity

Fig. 3. Western blot analysis of fractions after size exclusion chromatography. Gel filtration of cell extracts was performed as described in Fig. 2. The numbers above each lane indicate elution volume (ml). Only data for the clone with the highest expression of each TPP II mutant is shown.

still eluted at a Kav of 0.3. Upon overexpression of wild-type TPP II, 99% of the active enzyme eluted as the 2 –4  106 Da complex (Fig. 2C). As the D44A and N362A mutant enzymes elute with patterns similar to wild-type TPP II and the H264A and S449A mutant enzymes form significant amounts of the native complex, we conclude that inactivity of these mutants is not due to impaired complex formation. Western blot analysis of the column fractions showed that most of the TPP II protein eluted with TPP II activity (Fig. 3, and data not shown). Therefore, the mutant proteins seem to form complexes with endogenous active TPP II, suggesting that the structure of the mutant subunits is not altered. A possible exception is TPP II mutant H264A clone H-H2. In this case, most TPP II protein eluted in the void volume (Fig. 3), even though a substantial part of the TPP II activity eluted at a Kav of 0.3 (Fig. 2B). The finding that the TPP II active site mutations do not seem to affect the tertiary structure is consistent with the effects of the corresponding mutations in other subtilases [20,23]. Replacing the catalytic Ser-221, His-64, Asp-32 or Asn-155 with alanine in subtilisin reduces kcat 2  106-, 2  106-, 3  104-, or 102 – 103fold, respectively [20]. In all cases, however, the changes of Km are small, thus indicating that the overall structure of the enzyme is unchanged [20,23]. Formation of the >1000 kDa complex is required for full enzymatic activity of TPP II [9,22]. In contrast, formation of the >10,000 kDa complex did not seem to alter TPP II activity, as the total activity in the cell extracts remained constant in the presence of different amounts of the highmolecular weight complex (Tables 1 and 2). Formation of

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this complex is not a mere consequence of TPP II protein overexpression, since it was not formed by wild-type TPP II (Fig. 2C; Table 2), which was expressed at high levels (Fig. 1). Likewise, the low amounts of H264A mutant protein expressed by clone H-A6 formed more of the >10,000 kDa complex compared to the higher levels of mutant protein expressed by clone H –H2. The physiological significance of the formation of the >10,000 kDa complex is unclear. A splice variant of TPP II, containing 13 extra amino acids in the C-terminal part of the subunit, has been identified [13]. Interestingly, upon expression of this protein in 293-cells, a high molecular weight complex, eluting in the void volume of the Sepharose CL4B column, was obtained [15]. Formation of this complex seems to be facilitated by the C-terminal insert and might also be triggered by some of the TPP II mutant proteins described above. It remains to be determined whether the >10,000 kDa complexes formed by the wild-type TPP II splice variant or by TPP II mutant proteins share similarities besides their size. The >10,000 kDa complex might be physiologically relevant rather than a nonfunctional aggregate because it is formed by endogenous TPP II (Fig. 2A and Ref. [15]), and is active regardless of whether it is formed by endogenous TPP II, in presence of TPP II mutants (Fig. 2A,B) or possibly by the splice variant of TPP II [15]. At this point, it is not known if the >10,000 kDa complex involves other proteins than TPP II. One possibility is that the high-molecular form could be a complex between the mutated enzymes and a high molecular weight inhibitor. If the mutations affect kcat but not Km it is possible that, due to lack of cleavage by the mutant enzymes, an inhibitor can bind irreversibly to mutated TPP II. In conclusion, the mutation of Asp-44, His-264, Ser-449 and Asn-362 to alanine abolishes TPP II activity in accordance with the prediction that these amino acid residues function in catalysis. The mutants associate with endogenously expressed TPP II, indicating that their tertiary structure is not substantially altered. Unexpectedly, some mutants affect the quaternary structure of endogenously expressed TPP II and form large (>10,000 kDa), active complexes with currently unknown function. Acknowledgements We wish to thank Arturo Zychlinsky for encouraging and facilitating this work. H.H. and B.T. were supported by a fellowship from the Swiss National Science Foundation and a grant from the Swedish Medical Research Council (project 09914), respectively. References ¨ . Zetterqvist, Tripeptidyl aminopepti[1] R.-M. Ba˚lo¨w, U. Ragnarsson, O dase in the extralysosomal fraction of rat liver, J. Biol. Chem. 258 (1983) 11622 – 11628.

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