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• W4253199600 abstract "Quiescent cell proline dipeptidase (QPP) is an intracellular serine protease that is also secreted upon cellular activation. This enzyme cleaves N-terminal Xaa-Pro dipeptides from proteins, an unusual substrate specificity shared with dipeptidyl peptidase IV (CD26/DPPIV). QPP is a 58-kDa protein that elutes as a 120–130-kDa species from gel filtration, indicating that it forms a homodimer. We analyzed this dimerization with in vivoco-immunoprecipitation assays. The amino acid sequence of QPP revealed a putative leucine zipper motif, and mutational analyses indicated that this leucine zipper is required for homodimerization. The leucine zipper mutants showed a complete lack of enzymatic activity, suggesting that homodimerization is important for QPP function. On the other hand, an enzyme active site mutant retained its ability to homodimerize. These data are the first to demonstrate a role for a leucine zipper motif in a proteolytic enzyme and suggest that leucine zipper motifs play a role in mediating dimerization of a diverse array of proteins. Quiescent cell proline dipeptidase (QPP) is an intracellular serine protease that is also secreted upon cellular activation. This enzyme cleaves N-terminal Xaa-Pro dipeptides from proteins, an unusual substrate specificity shared with dipeptidyl peptidase IV (CD26/DPPIV). QPP is a 58-kDa protein that elutes as a 120–130-kDa species from gel filtration, indicating that it forms a homodimer. We analyzed this dimerization with in vivoco-immunoprecipitation assays. The amino acid sequence of QPP revealed a putative leucine zipper motif, and mutational analyses indicated that this leucine zipper is required for homodimerization. The leucine zipper mutants showed a complete lack of enzymatic activity, suggesting that homodimerization is important for QPP function. On the other hand, an enzyme active site mutant retained its ability to homodimerize. These data are the first to demonstrate a role for a leucine zipper motif in a proteolytic enzyme and suggest that leucine zipper motifs play a role in mediating dimerization of a diverse array of proteins. quiescent cell proline dipeptidase dipeptidyl peptidase IV prolylcarboxypeptidase immunoprecipitate polyacrylamide gel electrophoresis hemagglutinin phosphate-buffered saline amino-4- trifluoromethylcoumarin Quiescent cell proline dipeptidase (QPP)1 is a 58-kDa protein that was recently isolated and cloned from human T cells (1Underwood R. Chiravuri M. Yardley K. Lee H. Schmitz T. Huber B.T. J. Biol. Chem. 1999; 274: 34053-34058Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Highly specific inhibitors of post-proline cleaving aminodipeptidases cause cell death in quiescent lymphocytes, and the search for the target of these inhibitors led to the cloning of QPP and its subsequent nomenclature (2Chiravuri M. Schmitz T. Underwood R. Yardley K. Huber B.T. J. Immunol. 1999; 163: 3092-3099PubMed Google Scholar). QPP is a serine protease that cleaves dipeptides off the N terminus of proteins when the penultimate amino acid is a proline or an alanine. Although the substrates of QPP have yet to be elucidated, there are a striking number of cytokines, chemokines, and other signal molecules with highly conserved Xaa-Pro and Xaa-Ala motifs on the N terminus, rendering them potential substrates for QPP. Dipeptidyl peptidase IV (CD26/DPPIV), which shares substrate specificity with QPP, cleaves N-terminal Xaa-Pro motifs from chemokines such as macrophage-derived chemokines, regulated on activation normal T cell expressed and secreted, and stromal-derived factor 1 (3Oravecz T. Pall M. Roderiquez G. Gorrell M.D. Ditto M. Nguyen N.Y. Boykins R. Unsworth E. Norcross M.A. J. Exp. Med. 1997; 186: 1865-1872Crossref PubMed Scopus (318) Google Scholar, 4Shioda T. Kato H. Ohnishi Y. Tashiro K. Ikegawa M. Nakayama E.E. Hu H. Kato A. Sakai Y. Liu H. Honjo T. Nomoto A. Iwamoto A. Morimoto C. Nagai Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6331-6336Crossref PubMed Scopus (160) Google Scholar, 5Proost P. Struyf S. Schols D. Opdenakker G. Sozzani S. Allavena P. Mantovani A. Augustyns K. Bal G. Haemers A. Lambeir A.M. Scharpe S. Van Damme J. De Meester I. J. Biol. Chem. 1999; 274: 3988-3993Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). This cleavage results in the functional inactivation of the three signal molecules, indicating that this may be an important site of regulation of signal molecules in vivo. Despite the large number of signal molecules with a conserved N-terminal Xaa-Pro motif, there are relatively few exopeptidases with the ability to cleave peptide bonds containing proline (6Vanhoof G. Goossens F. De Meester I. Hendriks D. Scharpe S. FASEB J. 1995; 9: 736-744Crossref PubMed Scopus (382) Google Scholar). These include the aminodipeptidases QPP and CD26/DPPIV and prolylcarboxypeptidase (PCP, angiotensinase), a post-proline cleaving carboxypeptidase that cleaves amino acids off the C terminus of proteins (7Tan F. Morris P.W. Skidgel R.A. Erdos E.G. J. Biol. Chem. 1993; 268: 16631-16638Abstract Full Text PDF PubMed Google Scholar). The post-proline cleaving enzymes are likely to emerge as an important protease family. As indicated above, CD26/DPPIV has been shown to modulate the function of several chemokines (3Oravecz T. Pall M. Roderiquez G. Gorrell M.D. Ditto M. Nguyen N.Y. Boykins R. Unsworth E. Norcross M.A. J. Exp. Med. 1997; 186: 1865-1872Crossref PubMed Scopus (318) Google Scholar, 4Shioda T. Kato H. Ohnishi Y. Tashiro K. Ikegawa M. Nakayama E.E. Hu H. Kato A. Sakai Y. Liu H. Honjo T. Nomoto A. Iwamoto A. Morimoto C. Nagai Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6331-6336Crossref PubMed Scopus (160) Google Scholar, 5Proost P. Struyf S. Schols D. Opdenakker G. Sozzani S. Allavena P. Mantovani A. Augustyns K. Bal G. Haemers A. Lambeir A.M. Scharpe S. Van Damme J. De Meester I. J. Biol. Chem. 1999; 274: 3988-3993Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar), whereas PCP is a candidate gene for mediating essential hypertension (8Watson Jr., B. Nowak N.J. Myracle A.D. Shows T.B. Warnock D.G. Genomics. 1997; 44: 365-367Crossref PubMed Scopus (25) Google Scholar). It is interesting to note that even though QPP and CD26/DPPIV share substrate specificity, they do not have homologous amino acid sequences. QPP shares a significant degree of homology with PCP (7Tan F. Morris P.W. Skidgel R.A. Erdos E.G. J. Biol. Chem. 1993; 268: 16631-16638Abstract Full Text PDF PubMed Google Scholar) at the amino acid level (41% sequence identity) but not at the nucleotide level. QPP, CD26/DPPIV, and PCP share common structural features that may be reflective of their convergent evolution to form efficient post-proline cleaving enzymes: 1) they have the same ordering of the catalytic triad: Ser, Asp, His (1Underwood R. Chiravuri M. Yardley K. Lee H. Schmitz T. Huber B.T. J. Biol. Chem. 1999; 274: 34053-34058Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 6Vanhoof G. Goossens F. De Meester I. Hendriks D. Scharpe S. FASEB J. 1995; 9: 736-744Crossref PubMed Scopus (382) Google Scholar); 2) they are glycoproteins, and glycosylation is essential for the enzymatic activity of at least two of them, CD26/DPPIV (9Fan H. Meng W. Kilian C. Grams S. Reutter W. Eur. J. Biochem. 1997; 246: 243-251Crossref PubMed Scopus (74) Google Scholar) and QPP 2M. Chiravuri, F. Agarraberes, S. L. Mathieu, H. Lee, and B. T. Huber, submitted for publication.2M. Chiravuri, F. Agarraberes, S. L. Mathieu, H. Lee, and B. T. Huber, submitted for publication.; and 3) CD26/DPPIV and PCP form homodimers, and we show here that QPP also oligomerizes. CD26/DPPIV forms homodimers through disulfide links (10Morimoto C. Schlossman S.F. Immunol. Rev. 1998; 161: 55-70Crossref PubMed Scopus (366) Google Scholar, 11von Bonin A. Huhn J. Fleischer B. Immunol. Rev. 1998; 161: 43-53Crossref PubMed Scopus (132) Google Scholar), whereas PCP forms homodimers through a poorly understood mechanism, believed to be mediated through serine repeats (12Skidgel R.A. Erdos E.G. Immunol. Rev. 1998; 161: 129-141Crossref PubMed Scopus (137) Google Scholar). Leucine zipper motifs are protein-protein dimerization motifs consisting of heptad repeats of leucine residues that form a coiled-coil structure (13Landschulz W.H. Johnson P.F. McKnight S.L. Science. 1988; 240: 1759-1764Crossref PubMed Scopus (2536) Google Scholar, 14O'Shea E.K. Rutkowski R. Kim P.S. Science. 1989; 243: 538-542Crossref PubMed Scopus (697) Google Scholar). These motifs have been well described in the context of transcription factors such as c-Fos and c-Jun where they mediate homo- and heterodimerization critical for the DNA binding properties of these transcription factors (15Junius F.K. O'Donoghue S.I. Nilges M. Weiss A.S. King G.F. J. Biol. Chem. 1996; 271: 13663-13667Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). However, an increasing body of literature indicates that leucine zipper motifs mediate dimerization in a variety of other proteins. These include enzymes such as mixed lineage kinase-3 and tyrosine hydroxylase (16Leung I.W. Lassam N. J. Biol. Chem. 1998; 273: 32408-32415Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 17Vrana K.E. Walker S.J. Rucker P. Liu X. J. Neurochem. 1994; 63: 2014-2020Crossref PubMed Scopus (49) Google Scholar), where the dimerization mediated by leucine zippers can be important for the activity of these enzymes. Mutagenesis has been used to analyze potential leucine zipper-mediated dimerization of a number of proteins (16Leung I.W. Lassam N. J. Biol. Chem. 1998; 273: 32408-32415Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 17Vrana K.E. Walker S.J. Rucker P. Liu X. J. Neurochem. 1994; 63: 2014-2020Crossref PubMed Scopus (49) Google Scholar, 18Inoue H. Takahashi S. Fukui K. Miyake Y. J. Biol. Chem. 1991; 266: 11896-11900Abstract Full Text PDF PubMed Google Scholar, 19Simmerman H.K. Kobayashi Y.M. Autry J.M. Jones L.R. J. Biol. Chem. 1996; 271: 5941-5946Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 20Schumacher B. Staeheli P. J. Biol. Chem. 1998; 273: 28365-28370Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). We show here that active QPP elutes from gel filtration as a 120–130-kDa species, even though its estimated molecular mass from SDS-PAGE is 58 kDa (1Underwood R. Chiravuri M. Yardley K. Lee H. Schmitz T. Huber B.T. J. Biol. Chem. 1999; 274: 34053-34058Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Primary sequence analysis of QPP revealed a putative leucine zipper motif upstream of the catalytic region (see Fig. 1). In this paper, we investigated the role of this putative leucine zipper in QPP homodimerization. We used an in vivoco-immunoprecipitation scheme to analyze the dimerization properties of QPP. Independent point mutations in the leucine zipper region result in a loss of QPP homodimerization. The active site of QPP does not affect homodimerization, and a QPP active site mutant retains its ability to dimerize with wild type QPP, without influencing the enzymatic activity of wild type QPP. On the other hand, the leucine zipper mutants showed a loss of enzymatic activity. These results suggest that QPP homodimerizes through its putative leucine zipper motif and that this homodimerization is required for its enzymatic activity. These results may reflect the general requirement for structural features such as dimerization for post-proline cleaving enzymes. Furthermore, this is the first reported case of a leucine zipper motif mediating dimerization of a proteolytic enzyme. The QPP constructs were cloned into the pCI-neo expression vector (Promega, Madison, WI), as described previously (1Underwood R. Chiravuri M. Yardley K. Lee H. Schmitz T. Huber B.T. J. Biol. Chem. 1999; 274: 34053-34058Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). The QPP-HA and QPP-Myc constructs were generated by polymerase chain reaction with the high fidelity DeepVent polymerase (New England Biolabs, Beverly, MA), using antisense primers incorporating either the Myc (EQLLISEEDL) or the HA (YPYDVPDYA) epitope tags. To generate QPP mutants, the QuikChange site-directed mutagenesis kit (Stratagene, CA) was used. Briefly, primers containing the desired mutation together with flanking regions (>10 base pairs) were designed. Using QPP-HA in pCI-neo as a template, one round of polymerase chain reaction was performed withPfu Turbo DNA polymerase. The products were subjected to digestion with DpnI, and the nicked vector DNA incorporating the mutation was transformed into XL1-Blue supercompetent cells. 293T cells were grown in general medium (Dulbecco's modified Eagle's medium (Life Technologies, Inc.) with 10% fetal calf serum, 100 IU penicillin, 10 mg/ml streptomycin,l-glutamine, 2-mercaptoethanol, and sodium pyruvate). Transfections of 293T fibroblasts were performed using the calcium phosphate precipitation method. Two million cells/10-cm plate were plated 24 h prior to transfection. During transfection, 62 μl of 2 molar CaCl2 was added to 438 μl of double distilled H2O containing the DNA (30 μg) to be transfected. This was added to 500 μl of 2× HBS by bubbling. The mixture was immediately added to the cells. The medium was replaced after 8 h. Cells were harvested 48 h later. Stable 293T lines were generated by co-transfection of a QPP-HA construct in pCI-neo and pBABE-puro at a ratio of 15:1, respectively. These transfectants were expanded into general medium supplemented with 1.5 μg/ml puromycin 48 h after transfection. Clones were selected and assayed for QPP expression. Stable QPP-HA expressing 293T cells were were resuspended in 2.5 ml of lysis buffer (0.02m phosphate buffer, pH 7.4, 4 μg/ml aprotinin, 8 μg/ml leupeptin, 8 μg/ml antipain) and lysed by Dounce homogenization. The homogenate was centrifuged at 1000 × g for 10 min at 4 °C. The supernatant was then spun at 45,000 × gfor 20 min and finally at 100,000 × g (S-110) for 1 h at 4 °C. The S-110 fraction was dialyzed for 16 h against 50 mm phosphate buffer + 150 mm NaCl at 4 °C. The S-110 fraction was loaded onto a Sephacryl S-200 column (Amersham Pharmacia Biotech) that was previously calibrated using a molecular mass marker kit (Sigma). The column was washed with 10 column volumes of column buffer (50 mm phosphate buffer + 150 mm NaCl). The column was run at 16 ml/h. 1.2-ml fractions were collected, assayed for Ala-Pro-AFC cleavage, and analyzed by Western blot analysis. 1–2 × 107 cells were resuspended in lysis buffer (20 mm Hepes, 1.5 mm MgCl2, 2 mm EDTA, 10 mm KCl, 0.1–1% Nonidet P-40, 5 μg/ml antipain, and 5 μg/ml leupeptin) for 30 min at 4 °C. The nuclei were spun out at 2000 rpm on a microcentrifuge for 10 min. Protein concentration was measured using the BCA protein estimation kit (Pierce). These samples were boiled for 5 min, subjected to SDS-PAGE, transferred onto polyvinylidene difluoride membranes, and blocked with 5% nonfat milk in PBS-T (PBS, 0.1% Tween-20y) for 1 h at room temperature. The primary anti-Myc (BD-Pharmingen, San Diego, CA) or anti-HA (BabCo, Richmond, CA) antibodies were incubated for 1 h at room temperature or overnight at 4 °C on an orbital shaker. The membrane was washed 3 × 10 min with PBS-T. The secondary antibody, conjugated to horseradish peroxidase (Amersham Pharmacia Biotech) was incubated for 1 h at room temperature. The membrane was then washed 3 × 15 min with PBS-T. The membrane was rinsed with PBS, followed by addition of chemiluminescence substrate and autoradiography. 1–2 × 107 cells were resuspended in lysis buffer (20 mm Hepes, 1.5 mm MgCl2, 2 mm EDTA, 10 mm KCl, 0.1% Nonidet P-40, 5 μg/ml antipain, and 5 μg/ml leupeptin) for 30 min at 4 °C. The nuclei were spun out at 2000 rpm on a microcentrifuge for 10 min. Protein concentration was measured, using the BCA protein estimation kit (Pierce). Lysates were added to a 96-well plate, followed by addition of the substrate solution (20 μm Ala-Pro-AFC (Enzyme Systems Products, CA) in 50 mm Hepes). The samples were analyzed on a Fmax fluorescence plate reader (Molecular Devices) (Excitation, 390 nm; emission, 510 nm). 48 h after transfection, cells were washed in cold PBS and lysed in IP buffer (20 mm Hepes, 1.5 mm MgCl2, KCl, EDTA, 1% Nonidet P-40, 5 μg/ml leupeptin, and 5 μg/ml phenylmethylsulfonyl fluoride) at 4 °C for 30 min. This was followed by a 300 × gcentrifugation at 4 °C. The post-nuclear supernatant was transferred to a fresh Eppendorf tube, and the protein concentration was measured by BCA analysis. Samples were equalized for amount of protein and volume and then precleared by incubation with 50 μl of protein G beads (Pierce) for 1 h at 4 °C. The post-nuclear supernatant was then treated with anti-HA antibody (6–8 μg) overnight at 4 °C with shaking. 100 μl of protein SG beads were added for 1 h at 4 °C, and these beads were washed 4–5 times in lysis buffer. Finally, the beads were resuspended in 60 μl of SDS loading buffer and boiled for 5 min. The samples were centrifuged, and the supernatants were run on SDS-PAGE and analyzed by Western blot. QPP is a 492-amino acid glycoprotein that is synthesized with a signal peptide (Fig.1 A). The active site serine is found in a consensus GXSXG sequence and together with Asp418 and His442 makes up the catalytic triad (1Underwood R. Chiravuri M. Yardley K. Lee H. Schmitz T. Huber B.T. J. Biol. Chem. 1999; 274: 34053-34058Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). These residues are conserved in the QPP homologue, PCP (1Underwood R. Chiravuri M. Yardley K. Lee H. Schmitz T. Huber B.T. J. Biol. Chem. 1999; 274: 34053-34058Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar,7Tan F. Morris P.W. Skidgel R.A. Erdos E.G. J. Biol. Chem. 1993; 268: 16631-16638Abstract Full Text PDF PubMed Google Scholar). When initially purified from T cells, QPP was observed to elute as a 120-kDa species, whereas SDS-PAGE revealed a 58-kDa species, suggesting that QPP exists as a dimer (1Underwood R. Chiravuri M. Yardley K. Lee H. Schmitz T. Huber B.T. J. Biol. Chem. 1999; 274: 34053-34058Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). To confirm these findings, QPP was analyzed by gel filtration chromatography (Fig. 2). 293T cells (2A5) stably expressing QPP were lysed and fractionated on a precalibrated gel filtration column (Fig. 2 A), and QPP was analyzed by both Western blot analysis and enzyme activity assays. As can be seen in Fig. 2 B, little QPP is found in those fractions corresponding to molecular masses of 50–60 kDa, while the majority of QPP is found in fractions corresponding to a molecular mass higher than 120 kDa. QPP requires glycosylation for its enzyme activity, and it migrates under SDS-PAGE as a 58-kDa species in its glycosylated form.2 This glycosylated form was found predominantly in fractions 63–67, which would include proteins with molecular masses of 117 to approximately 144 kDa. Additional analysis was performed by measuring QPP activity in each of the fractions. Enzyme activity analysis shows that the majority of QPP activity is found in fractions 65–68, with the peak activity found in fractions 66 and 67, corresponding to a molecular mass of between 120 and 130 kDa (126 kDa) (Fig. 2 C). These data show that the functionally active form of QPP exists as a dimer in vitro. To investigate the dimerization properties of QPP in vivo, we utilized a co-immunoprecipitation scheme employing different epitope-tagged forms of QPP. Two QPP expression constructs with either a C-terminal Myc epitope tag or an HA epitope tag were transfected individually or in combination into 293T cells. Following transfection, lysates from these cells were immunoprecipitated with an anti-HA antibody, followed by SDS-PAGE and Western blot analysis using an anti-Myc antibody. As can be seen in Fig.3 A, following anti-HA immunoprecipitation, QPP-Myc was only precipitated when cotransfected with QPP-HA. Anti-HA Western blot analysis on the immunoprecipitates (Fig. 3 B, lane 2) shows that the anti-HA antibody does not directly immunoprecipitate QPP-Myc and that the QPP-HA that is immunoprecipitated with the anti-HA antibody (Fig. 3 B,lane 1) is not detected by the anti-Myc antibody (Fig.3 A, lane 1). This indicates that the anti-Myc and anti-HA antibodies are highly specific and do not cross react with the “opposing” epitope tag. Western blot analyses of the pre-IP lysates (Fig. 3, C and D) indicate that both constructs were expressed individually and in the cotransfected samples. These data show that QPP homodimerizes in vivo when overexpressed in 293T human fibroblast cells. Given that QPP forms oligomers, we analyzed the primary sequence of QPP for potential dimerization motifs. This analysis revealed a putative leucine zipper coiled-coil motif, consisting of a heptad repeat of leucine residues upstream of the catalytic domain (Fig. 1, B and C). Leucine zipper motifs serve as protein-protein interaction domains and mediate homo- and heterodimerization of a number of proteins (15Junius F.K. O'Donoghue S.I. Nilges M. Weiss A.S. King G.F. J. Biol. Chem. 1996; 271: 13663-13667Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 16Leung I.W. Lassam N. J. Biol. Chem. 1998; 273: 32408-32415Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 17Vrana K.E. Walker S.J. Rucker P. Liu X. J. Neurochem. 1994; 63: 2014-2020Crossref PubMed Scopus (49) Google Scholar, 19Simmerman H.K. Kobayashi Y.M. Autry J.M. Jones L.R. J. Biol. Chem. 1996; 271: 5941-5946Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 20Schumacher B. Staeheli P. J. Biol. Chem. 1998; 273: 28365-28370Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 21Hufton S.E. Jennings I.G. Cotton R.G. Biochim. Biophys. Acta. 1998; 1382: 295-304Crossref PubMed Scopus (20) Google Scholar, 22Fry A.M. Arnaud L. Nigg E.A. J. Biol. Chem. 1999; 274: 16304-16310Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). To analyze the role of the putative leucine zipper in QPP, we designed two independent QPP leucine zipper mutants, QPP-F2 and QPP-L2. Both mutants were made using point mutations to minimize gross structural change in the mutant proteins. The first mutant, QPP-F2, was made by mutating Phe138 to a proline. This mutation would be expected to disrupt the formation of the secondary α-helical structure and thereby prevent formation of the quaternary coiled-coil structure. The second mutant, QPP-L2, involved the mutation of two leucine residues to two alanine residues (Leu141 and Leu142 to Ala). This more subtle mutation would not be expected to disrupt the secondary α-helical structure of QPP, but the shorter alanine side chain would not be able to form the quaternary leucine zipper-mediated coiled-coil structure. The same co-immunoprecipitation analyses as outlined above (Fig. 3) were carried out to test the ability of the leucine zipper mutants to form dimers. 293T cells were co-transfected with QPP-Myc and one of the following constructs: QPP-HA (wild type), QPP-F2-HA, or QPP-L2-HA. As seen in Fig. 4 A, even as wild type QPP-HA homodimerized with QPP-Myc, both the QPP-F2 and the QPP-L2 leucine zipper mutants showed an inability to homodimerize with the QPP-Myc construct. Anti-HA Western blot analysis of anti-HA immunoprecipitated lysates shows that the leucine zipper mutants immunoprecipitate at similar levels to wild type QPP (Fig.4 B), although the wild type QPP-Myc did not co-immunoprecipitate with the mutant constructs. Anti-Myc Western blot analysis of the preimmunoprecipitate lysates indicated that all three samples shown in Fig. 4 A had similar levels of QPP-Myc expression (Fig. 4 C). Anti-HA Western blot analyses revealed that the leucine zipper mutants were expressed at similar levels as wild type QPP (Fig. 4 D). QPP is a serine protease with an aminodipeptidase activity that cleaves N-terminal dipeptides when the penultimate amino acid is a proline or an alanine. QPP overexpressed in 293T fibroblasts shows full functional activity in terms of its ability to cleave the reporter substrate Ala-Pro-AFC (Fig.5 B). The putative active site serine of QPP is found within a GXSXG motif that is highly conserved between QPP and its homologues (1Underwood R. Chiravuri M. Yardley K. Lee H. Schmitz T. Huber B.T. J. Biol. Chem. 1999; 274: 34053-34058Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). A mutant was made that altered the active site serine into an alanine (GGAYG). This construct (QPP-SA) was expressed with a Myc epitope tag in 293T fibroblasts and was detected as a 58-kDa species by Western blot analysis using an anti-Myc antibody (Fig. 5 A). However, this mutant (QPP-SA) showed no detectable enzymatic activity compared with the wild type QPP (Fig. 5 B). We used the active site mutant to determine whether 1) a functional active site is necessary for QPP dimerization and 2) in the event the active site mutant (QPP-SA) dimerized with wild type QPP, it affected QPP enzymatic activity. To answer these questions, we used a stable 293T line (2A5) expressing a QPP-HA construct that allowed us to analyze dimerization properties of the QPP active site mutant and to detect any effect of the QPP-SA construct on a fixed level of QPP-HA enzymatic activity of the 2A5 line. The active site mutant QPP-SA with a Myc epitope tag was transfected into the 2A5 line (Fig. 6, Aand B). Lysates from these cells were immunoprecipitated as before using an anti-HA antibody and subjected to SDS-PAGE and anti-Myc immunoblot analysis. As can be seen in Fig. 6 A, the active site mutant retained its ability to dimerize with wild type QPP, indicating that a functional active site is not necessary for the dimerization to take place. 2A5 samples transfected with vector alone or the QPP-SA-Myc construct showed the same level of QPP-HA (Fig.6 C). We analyzed QPP enzyme activity in both of these samples, and, as seen in Fig. 6 D, the active site mutant did not affect the enzymatic activity of the wild type QPP, even though the mutant dimerized with the wild type. We previously observed that QPP enzymatic activity was highly sensitive to structural alterations such as deglycosylation.2 Given that homodimerization is a common feature of QPP, PCP, and CD26/DPPIV, we decided to investigate the effect of abolishing QPP dimerization on its activity. To determine the importance of the leucine zipper for QPP enzymatic activity, we measured enzymatic function of the QPP-F2 and QPP-L2 leucine zipper mutants of QPP that have mutations upstream of the catalytic triad. As seen in Fig.7 B, compared with vector transfected controls, the QPP-Myc and QPP-HA constructs showed full enzymatic activity. The QPP-F2 and QPP-L2 mutants, however, lacked enzymatic function. This was particularly interesting with the QPP-L2 mutant, which has a relatively subtle mutation of two leucines to two alanines. This construct, which also lacks the ability to homodimerize (Fig. 4), had no enzymatic activity over the vector transfected controls. To ensure equivalent expression levels in the samples tested, Western blot analysis was performed using an anti-HA antibody. As can be seen in Fig. 7 A, the leucine zipper mutants QPP-F2 and QPP-L2 were expressed at similar levels as the wild type QPP-HA construct. QPP is a glycoprotein of 58 kDa that assumes a mass of 53 kDa when deglycosylated.2 The fact that the QPP-F2 and QPP-L2 mutants migrate at 58 kDa (Fig. 7 A) on SDS-PAGE analysis indicates that these mutants were correctly routed through the trans-Golgi network and underwent core and terminal glycosylation in the absence of homodimerization. Leucine zipper motifs have been well described for transcription factors such as c-Jun where they mediate homo- and heterodimerization important for DNA binding (15Junius F.K. O'Donoghue S.I. Nilges M. Weiss A.S. King G.F. J. Biol. Chem. 1996; 271: 13663-13667Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). A number of reports, however, have shown that leucine zippers play a far more universal role as protein dimerization domains. For example, enzymes such as tyrosine hyroxylase (17Vrana K.E. Walker S.J. Rucker P. Liu X. J. Neurochem. 1994; 63: 2014-2020Crossref PubMed Scopus (49) Google Scholar), MxA GTPase (20Schumacher B. Staeheli P. J. Biol. Chem. 1998; 273: 28365-28370Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), phenylalanine hydroxylase (21Hufton S.E. Jennings I.G. Cotton R.G. Biochim. Biophys. Acta. 1998; 1382: 295-304Crossref PubMed Scopus (20) Google Scholar), human centrosomal kinase (Nek2) (22Fry A.M. Arnaud L. Nigg E.A. J. Biol. Chem. 1999; 274: 16304-16310Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), and mixed lineage kinase (16Leung I.W. Lassam N. J. Biol. Chem. 1998; 273: 32408-32415Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) have been shown to dimerize through leucine zippers. QPP has a molecular mass of 58 kDa, as seen in SDS-PAGE, but elutes as a 120–130-kDa species in gel filtration (1Underwood R. Chiravuri M. Yardley K. Lee H. Schmitz T. Huber B.T. J. Biol. Chem. 1999; 274: 34053-34058Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), indicative of homodimer formation. Analysis of the QPP primary sequence revealed a leucine zipper heptad repeat structure that has a predicted ability to form a coiled-coil structure. This dimerization motif is upstream of the catalytic region of QPP. Interestingly, comparison of the deduced amino acid sequences of QPP cDNA derived from human and mouse shows that the leucine zipper motif is conserved, suggesting that this region is important for QPP activity. In this report, in vivo co-immunoprecipitation assays were performed to analyze the dimerization properties of QPP. Two independent leucine zipper mutants lost their ability to homodimerize with wild type QPP. This suggests that the leucine zipper motif is important for homodimerization of QPP. The active site of QPP does not seem to play a role in homodimerization, because the active site mutant, QPP-SA, showed an ability to homodimerize with wild type QPP. Furthermore, the dimerization seems to be a structural rather than allosteric requirement, because dimerization with the QPP-SA mutant had no effect on the activity of wild type QPP. On the other hand, the leucine zipper mutants showed a complete loss of enzymatic function. We, therefore, conclude that QPP homodimerization is mediated through a leucine zipper and that this homodimerization is required for QPP enzymatic activity, either to assume its correct structural conformation and/or to recognize and cleave its substrate. Disulfide bonds do not appear to play a role in QPP homodimerization as QPP migrates as a 58-kDa species under both reducing and nonreducing conditions (data not shown). Two independent leucine zipper mutants yielded the same results in terms of dimerization and enzymatic activity; the QPP-F2 mutant is expected to lose secondary structure because of a kink introduced by the proline in the α-helical structure, thus preventing the formation of a coiled-coil structure. On the other hand, in the QPP-L2 construct, the mutation of two leucine residues to two alanine residues would not be expected to alter the secondary α-helical structure of QPP; thus, the results obtained with this mutant were particularly interesting. In both cases the mutations were introduced external to the catalytic domain and yet had profound effects on the enzymatic activity of QPP. The introduction of point mutations is not as drastic as a complete deletion of the entire leucine zipper, but we cannot entirely discount the possibility that such changes to the primary sequence cause changes in the folding pattern of QPP. The post-proline cleaving exopeptidases, QPP, PCP, and CD26/DPPIV, have common structural features such as N-linked glycosylation and homodimerization that are important for their enzymatic activity (1Underwood R. Chiravuri M. Yardley K. Lee H. Schmitz T. Huber B.T. J. Biol. Chem. 1999; 274: 34053-34058Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 6Vanhoof G. Goossens F. De Meester I. Hendriks D. Scharpe S. FASEB J. 1995; 9: 736-744Crossref PubMed Scopus (382) Google Scholar, 7Tan F. Morris P.W. Skidgel R.A. Erdos E.G. J. Biol. Chem. 1993; 268: 16631-16638Abstract Full Text PDF PubMed Google Scholar, 9Fan H. Meng W. Kilian C. Grams S. Reutter W. Eur. J. Biochem. 1997; 246: 243-251Crossref PubMed Scopus (74) Google Scholar, 10Morimoto C. Schlossman S.F. Immunol. Rev. 1998; 161: 55-70Crossref PubMed Scopus (366) Google Scholar, 12Skidgel R.A. Erdos E.G. Immunol. Rev. 1998; 161: 129-141Crossref PubMed Scopus (137) Google Scholar). Leucine zipper motifs play an important role in the dimerization of a wide array of proteins (15Junius F.K. O'Donoghue S.I. Nilges M. Weiss A.S. King G.F. J. Biol. Chem. 1996; 271: 13663-13667Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 16Leung I.W. Lassam N. J. Biol. Chem. 1998; 273: 32408-32415Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 17Vrana K.E. Walker S.J. Rucker P. Liu X. J. Neurochem. 1994; 63: 2014-2020Crossref PubMed Scopus (49) Google Scholar, 19Simmerman H.K. Kobayashi Y.M. Autry J.M. Jones L.R. J. Biol. Chem. 1996; 271: 5941-5946Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 20Schumacher B. Staeheli P. J. Biol. Chem. 1998; 273: 28365-28370Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 21Hufton S.E. Jennings I.G. Cotton R.G. Biochim. Biophys. Acta. 1998; 1382: 295-304Crossref PubMed Scopus (20) Google Scholar, 22Fry A.M. Arnaud L. Nigg E.A. J. Biol. Chem. 1999; 274: 16304-16310Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). In this report we have described the first reported case of a functional requirement for a leucine zipper motif for the enzymatic activity of a proteolytic enzyme. These results will elucidate the mechanism of QPP enzymatic activity and post-proline cleaving exopeptidases in general. These results also confirm that leucine zippers mediate dimerization of diverse protein families. We thank Dr. James Baleja for helpful discussions, Dr. Kurt Yardley, Nicole D'Avirro, and Sarada Tew for critical reading of the manuscript, and Lia Kim for excellent technical assistance." @default.
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