One Site Mutation Disrupts Dimer Formation in Human DPP-IV Proteins
2004; Elsevier BV; Volume: 279; Issue: 50 Linguagem: Inglês
10.1074/jbc.m406185200
ISSN1083-351X
AutoresChia‐Hui Chien, Li‐Hao Huang, Chi‐Yuan Chou, Yuan‐Shou Chen, Yu‐San Han, Gu‐Gang Chang, Po‐Huang Liang, Xin Chen,
Tópico(s)Diabetes Treatment and Management
ResumoDPP-IV is a prolyl dipeptidase, cleaving the peptide bond after the penultimate proline residue. It is an important drug target for the treatment of type II diabetes. DPP-IV is active as a dimer, and monomeric DPP-IV has been speculated to be inactive. In this study, we have identified the C-terminal loop of DPP-IV, highly conserved among prolyl dipeptidases, as essential for dimer formation and optimal catalysis. The conserved residue His750 on the loop contributes significantly for dimer stability. We have determined the quaternary structures of the wild type, H750A, and H750E mutant enzymes by several independent methods including chemical cross-linking, gel electrophoresis, size exclusion chromatography, and analytical ultracentrifugation. Wild-type DPP-IV exists as dimers both in the intact cell and in vitro after purification from human semen or insect cells. The H750A mutation results in a mixture of DPP-IV dimer and monomer. H750A dimer has the same kinetic constants as those of the wild type, whereas the H750A monomer has a 60-fold decrease in kcat. Replacement of His750 with a negatively charged Glu (H750E) results in nearly exclusive monomers with a 300-fold decrease in catalytic activity. Interestingly, there is no dynamic equilibrium between the dimer and the monomer for all forms of DPP-IVs studied here. This is the first study of the function of the C-terminal loop as well as monomeric mutant DPP-IVs with respect to their enzymatic activities. The study has important implications for the discovery of drugs targeted to the dimer interface. DPP-IV is a prolyl dipeptidase, cleaving the peptide bond after the penultimate proline residue. It is an important drug target for the treatment of type II diabetes. DPP-IV is active as a dimer, and monomeric DPP-IV has been speculated to be inactive. In this study, we have identified the C-terminal loop of DPP-IV, highly conserved among prolyl dipeptidases, as essential for dimer formation and optimal catalysis. The conserved residue His750 on the loop contributes significantly for dimer stability. We have determined the quaternary structures of the wild type, H750A, and H750E mutant enzymes by several independent methods including chemical cross-linking, gel electrophoresis, size exclusion chromatography, and analytical ultracentrifugation. Wild-type DPP-IV exists as dimers both in the intact cell and in vitro after purification from human semen or insect cells. The H750A mutation results in a mixture of DPP-IV dimer and monomer. H750A dimer has the same kinetic constants as those of the wild type, whereas the H750A monomer has a 60-fold decrease in kcat. Replacement of His750 with a negatively charged Glu (H750E) results in nearly exclusive monomers with a 300-fold decrease in catalytic activity. Interestingly, there is no dynamic equilibrium between the dimer and the monomer for all forms of DPP-IVs studied here. This is the first study of the function of the C-terminal loop as well as monomeric mutant DPP-IVs with respect to their enzymatic activities. The study has important implications for the discovery of drugs targeted to the dimer interface. Dipeptidyl peptidase IV (DPP-IV, 1The abbreviations used are: DPP-IV, DPP-IV protein purified from human semen; rDPP-IV, human recombinant DPP-IV protein purified from baculoviral-infected insect cells; DTSP, dithiobis-succinimidyl propionate; H-Gly-Pro-pNA, H-Gly-Pro-p-nitroanilide; DTT, dithiothreitol; AUC, analytical ultracentrifugation; GLP-1, glucagon-like peptide-1; GIP, glucose-dependent insulinotropic polypeptide; POP, prolyl oligopeptidase; FAP, fibroblast activation protein; ADA, adenosine deaminase; CMV, cytomegalovirus; TCID50, 50% tissue-culture infectious dose; PBS, phosphate-buffered saline.1The abbreviations used are: DPP-IV, DPP-IV protein purified from human semen; rDPP-IV, human recombinant DPP-IV protein purified from baculoviral-infected insect cells; DTSP, dithiobis-succinimidyl propionate; H-Gly-Pro-pNA, H-Gly-Pro-p-nitroanilide; DTT, dithiothreitol; AUC, analytical ultracentrifugation; GLP-1, glucagon-like peptide-1; GIP, glucose-dependent insulinotropic polypeptide; POP, prolyl oligopeptidase; FAP, fibroblast activation protein; ADA, adenosine deaminase; CMV, cytomegalovirus; TCID50, 50% tissue-culture infectious dose; PBS, phosphate-buffered saline. also known as CD26) (EC 3.4.14.5) is a well documented drug target for the treatment of type II diabetes (1Drucker D.J. Exp. Opin. Investig. Drugs. 2003; 12: 87-100Crossref PubMed Scopus (232) Google Scholar). It is a serine protease involved in the in vivo degradation of two insulin-sensing hormones, glucagonlike peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) (2Mentlein R. Regul. Pept. 1999; 85: 9-24Crossref PubMed Scopus (1150) Google Scholar, 3Pauly R.P. Rosche F. Wermann M. McIntosh C.H. Pederson R.A. Demuth H.U. J. Biol. Chem. 1996; 271: 23222-23229Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Either inhibiting the enzymatic activity of DPP-IV in various animal models or knocking out DPP-IV in mice and rats prolongs the half-lives of these two insulinsensing hormones, increases insulin secretion and improves glucose tolerance (4Pospisilik J.A. Stafford S.G. Demuth H.U. McIntosh C.H. Pederson R.A. Diabetes. 2002; 51: 2677-2683Crossref PubMed Scopus (137) Google Scholar, 5Ahren B. Holst J.J. Martensson H. Balkan B. Eur. J. 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Thornberry N.A. Zhang B.B. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6825-6830Crossref PubMed Scopus (301) Google Scholar). Hence inhibition of DPP-IV may be effective in the treatment of type II diabetes. Understanding the catalytic mechanism of DPP-IV is thus essential to discovering inhibitors for the treatment of the disease. DPP-IV belongs to the prolyl oligopeptidase (POP) family, a subfamily of serine proteases (12Polgar L. Cell Mol. Life Sci. 2002; 59: 349-362Crossref PubMed Scopus (270) Google Scholar, 13Rosenblum J.S. Kozarich J.W. Curr. Opin. Chem. Biol. 2003; 7: 496-504Crossref PubMed Scopus (263) Google Scholar). This class of prolyl peptidases includes DPP-IV, prolyl oligopeptidase (POP), DPP-II, DPP8, DPP9, and fibroblast activation protein (FAP) (12Polgar L. Cell Mol. Life Sci. 2002; 59: 349-362Crossref PubMed Scopus (270) Google Scholar, 13Rosenblum J.S. Kozarich J.W. Curr. Opin. Chem. Biol. 2003; 7: 496-504Crossref PubMed Scopus (263) Google Scholar). Unlike classic serine proteases, the POP family of enzymes is highly selective toward peptides that have a proline residue at the penultimate position (18De Meester I. Durinx C. Bal G. Proost P. Struyf S. Goossens F. Augustyns K. Scharpe S. Adv. Exp. Med. Biol. 2000; 477: 67-87Crossref PubMed Google Scholar). The x-ray structures of DPP-IV and POP have shed light on the catalytic mechanisms, which differ significantly from those of the classic serine proteases, such as trypsin and subtilisin (12Polgar L. Cell Mol. Life Sci. 2002; 59: 349-362Crossref PubMed Scopus (270) Google Scholar, 14Rasmussen H.B. Branner S. Wiberg F.C. Wagtmann N. Nat. Struct. Biol. 2003; 10: 19-25Crossref PubMed Scopus (360) Google Scholar, 15Fulop V. Bocskei Z. Polgar L. Cell. 1998; 94: 161-170Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 16Engel M. Hoffmann T. Wagner L. Wermann M. Heiser U. Kiefersauer R. Huber R. Bode W. Demuth H.U. Brandstetter H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5063-5068Crossref PubMed Scopus (287) Google Scholar, 17Thoma R. Loffler B. Stihle M. Huber W. Ruf A. Hennig M. Structure (Camb.). 2003; 11: 947-959Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 18De Meester I. Durinx C. Bal G. Proost P. Struyf S. Goossens F. Augustyns K. Scharpe S. Adv. Exp. Med. Biol. 2000; 477: 67-87Crossref PubMed Google Scholar). DPP-IV consists of two domains, the α/β hydrolase domain and the β-propeller domain, with the active site inbetween (14Rasmussen H.B. Branner S. Wiberg F.C. Wagtmann N. Nat. Struct. Biol. 2003; 10: 19-25Crossref PubMed Scopus (360) Google Scholar, 15Fulop V. Bocskei Z. Polgar L. Cell. 1998; 94: 161-170Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 16Engel M. Hoffmann T. Wagner L. Wermann M. Heiser U. Kiefersauer R. Huber R. Bode W. Demuth H.U. Brandstetter H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5063-5068Crossref PubMed Scopus (287) Google Scholar, 17Thoma R. Loffler B. Stihle M. Huber W. Ruf A. Hennig M. Structure (Camb.). 2003; 11: 947-959Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). The substrate specificity of DPP-IV is dictated by a proline-binding pocket and a Glu205-Glu206 motif at the active site (14Rasmussen H.B. Branner S. Wiberg F.C. Wagtmann N. Nat. Struct. Biol. 2003; 10: 19-25Crossref PubMed Scopus (360) Google Scholar, 15Fulop V. Bocskei Z. Polgar L. Cell. 1998; 94: 161-170Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 16Engel M. Hoffmann T. Wagner L. Wermann M. Heiser U. Kiefersauer R. Huber R. Bode W. Demuth H.U. Brandstetter H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5063-5068Crossref PubMed Scopus (287) Google Scholar, 17Thoma R. Loffler B. Stihle M. Huber W. Ruf A. Hennig M. Structure (Camb.). 2003; 11: 947-959Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Only small size peptides are hydrolyzed by this class of enzymes because of the unique propeller structure and/or side opening substrates used to access the active site (14Rasmussen H.B. Branner S. Wiberg F.C. Wagtmann N. Nat. Struct. Biol. 2003; 10: 19-25Crossref PubMed Scopus (360) Google Scholar, 15Fulop V. Bocskei Z. Polgar L. Cell. 1998; 94: 161-170Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 16Engel M. Hoffmann T. Wagner L. Wermann M. Heiser U. Kiefersauer R. Huber R. Bode W. Demuth H.U. Brandstetter H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5063-5068Crossref PubMed Scopus (287) Google Scholar, 17Thoma R. Loffler B. Stihle M. Huber W. Ruf A. Hennig M. Structure (Camb.). 2003; 11: 947-959Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Among the shared properties, the most obvious difference between POP and DPP-IV is the relationship of catalytic activity with respect to its quaternary structure. POP exists in solution as a monomer and is active in such a form (15Fulop V. Bocskei Z. Polgar L. Cell. 1998; 94: 161-170Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar). In contrast, DPP-IV is active only as a dimer or oligomer, and monomeric DPP-IV is speculated to be inactive, even though DPP-IV monomer has never been isolated and demonstrated to be inactive (14Rasmussen H.B. Branner S. Wiberg F.C. Wagtmann N. Nat. Struct. Biol. 2003; 10: 19-25Crossref PubMed Scopus (360) Google Scholar, 19Lambeir A.M. Diaz Pereira J.F. Chacon P. Vermeulen G. Heremans K. Devreese B. Van Beeumen J. De Meester I. Scharpe S. Biochim. Biophys. Acta. 1997; 1340: 215-226Crossref PubMed Scopus (50) Google Scholar). Based on the crystal structures of DPP-IV, two loops are located in the dimer interface and were proposed to be involved in dimer interaction, the C-terminal loop at the α/β hydrolase domain and the propeller loop extended from strand 2 of the fourth blade in the β-propeller domain (14Rasmussen H.B. Branner S. Wiberg F.C. Wagtmann N. Nat. Struct. Biol. 2003; 10: 19-25Crossref PubMed Scopus (360) Google Scholar, 16Engel M. Hoffmann T. Wagner L. Wermann M. Heiser U. Kiefersauer R. Huber R. Bode W. Demuth H.U. Brandstetter H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5063-5068Crossref PubMed Scopus (287) Google Scholar, 17Thoma R. Loffler B. Stihle M. Huber W. Ruf A. Hennig M. Structure (Camb.). 2003; 11: 947-959Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar) (Fig. 1A). The C-terminal loop of DPP-IV consists of the last 50 amino acid residues with two α-helices (amino acids 713-725 and 745-763) and one β-sheet (amino acids 726-744) interacting with the same region from the other monomer across a 2-fold axis (Fig. 1B). Both hydrophobic and hydrophilic interactions have been proposed to be responsible for dimer formation (12Polgar L. Cell Mol. Life Sci. 2002; 59: 349-362Crossref PubMed Scopus (270) Google Scholar, 13Rosenblum J.S. Kozarich J.W. Curr. Opin. Chem. Biol. 2003; 7: 496-504Crossref PubMed Scopus (263) Google Scholar, 14Rasmussen H.B. Branner S. Wiberg F.C. Wagtmann N. Nat. Struct. Biol. 2003; 10: 19-25Crossref PubMed Scopus (360) Google Scholar, 15Fulop V. Bocskei Z. Polgar L. Cell. 1998; 94: 161-170Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar). This loop is highly conserved among the family of DPP-IV-containing prolyl dipeptidases (Fig. 2), though the functional importance of the loop has not been addressed experimentally.Fig. 2Conservation of the C-terminal loop among prolyl dipeptidases. The sequences are from the following GenBank™ accession numbers: NP_001926 (DPP-IV), Q12884 (FAP), NP_001927 (DPP6), AAG29766 (DPP8), AAL47179 (DPP9), and P42658 (DPP10). The conserved His (His750 for DPP-IV) is indicated by a green triangle and the catalytic triads by red stars, respectively. The alignment was done using ClustalW and TEXSHADE programs.View Large Image Figure ViewerDownload (PPT) In this article, the quaternary structures and catalytic activities were studied and compared among endogenous DPP-IV from human semen, recombinant wild-type and mutant DPP-IVs expressed in baculoviral-infected insect cells. The role of the highly conserved C-terminal loop for dimerization was investigated. For the first time, we have isolated and characterized the biochemical properties of monomeric mutant DPP-IV proteins altered at residue His750 of the loop, which is highly conserved among DPP-IV-containing prolyl dipeptidases (Fig. 2). Materials—The enzyme substrate H-Gly-Pro-pNA and dipeptide Gly-Pro were purchased from Bachem. Fetal bovine serum was from Hyclone. Lipofectin and the insect culture media, Grace and Express Five media, were from Invitrogen. Human liver cDNA library and linear viral vector were from Clontech. The ECL Western detection kit was from PerkinElmer Life Sciences. Q Sepharose™ High Performance, CNBr-activated Sepharose 4B and Superdex 200 prepacked-columns were from Amersham Biosciences. The chemical cross-linker dithiobis-succinimidyl propionate (DTSP) was from Pierce. Bovine adenosine deaminase (ADA) was from Roche Applied Science. Construction of the Secreted DPP-IV Expression Plasmid—The baculovirus expression plasmid pBac8-CD5 was constructed with the secretion tag CD5. Vector pBac-PAK8 (Clontech) was modified by inserting an MT-EGFP cassette at EcoRV site to facilitate the selection of the virus expressing EGFP (20Lee D.F. Chen C.C. Hsu T.A. Juang J.L. J. Virol. 2000; 74: 11873-11880Crossref PubMed Scopus (50) Google Scholar). CD5 coding sequence was amplified by PCR from human Jurkat cell cDNA with the following primers: 5′-CGGGATCCATGCCCATGGGGTCTCT-3′ and 5′-CCGCTCGAGCCGAGGCAGGAAGC-3′. The CD5 cDNA fragment was released by digestion with BamH1 and XhoI before ligation into pBac-PAK8, resulting in pBac8-CD5. The expression plasmid of DPP-IV with the secretion tag CD5 is constructed as follows. The human cDNA fragment of DPP-IV containing amino acids 39-766 was amplified by PCR from a human liver cDNA library with the primers 5′-CCGCTCGAGAAAAACTTACACTCTA-3′ and 5′-GCGTCGACCTAAGGTAAAGAGAAACATTG-3′, and cloned into pCR®-Blunt II-Topo vector (Invitrogen). The DPP-IV cDNA was then released by digestion with XhoI and EcoRI before ligation into the vector pBac8-CD5. Site-directed mutagenesis of DPP-IV was carried out using Pfu Turbo DNA Polymerase (Stratagene). The primers used for generating H750A and H750E mutants are 5′-AGCACACCAAGAAATATATACCCAC-3′ and 5′-GTGGGTATATATTTCTTGGTGTGCT-3′ for H750E, and 5′-AGCACACCAAGCTATATATACCCAC-3′ and 5′-GTGGGTATATATAGCTTGGTGTGCT-3′ for H750A, respectively. All DPP-IV cDNA fragments cloned were sequenced to verify that they contain no additional mutations other than those desired. Insect Cell Culture, DNA Transfection, Virus Selection, and Amplification—Sf9 cells were grown in Grace medium supplemented with 10% fetal bovine serum at 27 °C. The transfection of DNA to Sf9 cells, and the selection and amplification of the recombinant virus were carried out as described (21Chen Y.S. Chien C.H. Goparaju C.M. Hsu J.T. Liang P.H. Chen X. Protein Expr. Purif. 2004; 35: 142-146Crossref PubMed Scopus (30) Google Scholar). For expression and purification purposes, Hi5 cells, instead of Sf9 cells, were used. Hi5 cells were infected at a multiplicity of infection of 1.0 TCID50 unit/cell (TCID50 is 50% tissue culture infectious dose), determined to be the optimal condition for protein expression as described (21Chen Y.S. Chien C.H. Goparaju C.M. Hsu J.T. Liang P.H. Chen X. Protein Expr. Purif. 2004; 35: 142-146Crossref PubMed Scopus (30) Google Scholar), and the cells were harvested at 72-h post-transfection. Purification of DPP-IV Proteins from Hi5 Insect Cells and Human Semen—The purification of wild-type recombinant DPP-IV was carried out as described (14Rasmussen H.B. Branner S. Wiberg F.C. Wagtmann N. Nat. Struct. Biol. 2003; 10: 19-25Crossref PubMed Scopus (360) Google Scholar). ADA affinity columns were prepared as described (22de Meester I. Vanhoof G. Lambeir A.M. Scharpe S. J. Immunol. Methods. 1996; 189: 99-105Crossref PubMed Scopus (92) Google Scholar). For the purification of both H750A and H750E mutant proteins, only the ADA column was used with the omission of Triton X-100 in both the washing and elution solutions. Human semen DPP-IV protein was purified from healthy Asian male donors as described (22de Meester I. Vanhoof G. Lambeir A.M. Scharpe S. J. Immunol. Methods. 1996; 189: 99-105Crossref PubMed Scopus (92) Google Scholar). The elution buffer for protein bound on an ADA column did not contain Triton X-100. Freezing at -80 °C does not change either the quaternary structure (determined by AUC) or the enzymatic activities of DPP-IV proteins described in this study. The purity of the protein was determined by SDS-PAGE, and proteins were visualized with Coomassie Blue. The amount of protein was determined by the method of Bradford using bovine serum albumin as the standard. Polyacrylamide Gel Electrophoresis and Western Blot Analysis—Purified proteins were run on a 4-20% gradient native polyacrylamide gel with the gel running system from Amersham Biosciences. SDS-PAGE and Western blot analysis were conducted as described (23Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: A8:40-A8:51Google Scholar). Rabbit anti-DPP-IV antibody was generated in house using purified semen DPP-IV as the antigen. Kinetic Constant Determinations—To measure the kinetic parameters, the chromogenic substrate H-Gly-Pro-pNA was utilized to initiate the reaction, which was monitored at OD405 nm as a function of time (21Chen Y.S. Chien C.H. Goparaju C.M. Hsu J.T. Liang P.H. Chen X. Protein Expr. Purif. 2004; 35: 142-146Crossref PubMed Scopus (30) Google Scholar). The enzyme concentrations used in the reaction were 10 nm for wild-type and H750A proteins, and 100 nm for the H750E protein, respectively. The initial rate was measured with less than 10% substrate depletion for the first 10 to 300 s. The steady state parameters, kcat and Km, were determined from initial velocity measurements at 0.5 to 5 Km of the substrate concentrations. Lineweaver Burk plots were analyzed using non-linear regression of the Michaelis-Menten equation. Correlation coefficients greater than 0.99 were obtained. Chemical Cross-linking in Intact Cells and Size Exclusion Chromatography—Chemical cross-linking in intact cells was conducted as described (24Kreis T.E. Lodish H.F. Cell. 1986; 46: 929-937Abstract Full Text PDF PubMed Scopus (248) Google Scholar). Size exclusion chromatography was conducted at 4 °C. Purified proteins (0.5 ml at a concentration of 5 μm) were applied to a Superdex 200 10/30 column (10 × 300-310 mm) pre-equilibrated with PBS. The sample was eluted with the same buffer at 0.3 ml/min and 0.25 ml fractions were collected. The Superdex 200 10/30 column was calibrated with the Stokes Radii of ferritin (6.1 nm), catalase (5.22 nm), aldolase (4.81 nm), albumin (3.55 nm), ovalbumin (3.05 nm), and chymotrypsinogen A (2.09 nm) from Amersham Biosciences. Analytical Ultracentrifugation—DPP-IV proteins at concentrations of around 0.1 to 0.2 mg/ml (1.2-2.3 μm) were used for AUC analysis with either PBS, high salt (100 mm Tris-HCl, 50 mm NaCl, 0.5 m Na2SO4, pH 7.5) or low salt (100 mm Tris-HCl, 50 mm NaCl, pH 7.5) buffers as indicated. Buffer was changed using an Amicon device and DPP-IV proteins were allowed to equilibrate for at least 4 h or longer as indicated in the text at 25 °C after buffer changes. The sedimentation coefficients (S) of the enzyme were estimated by a Beckman-Coulter XL-A analytical ultracentrifuge with an An60Ti rotor as described (25Chang H.C. Chang G.G. J. Biol. Chem. 2003; 278: 23996-24002Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Sedimentation velocity analysis was performed at 40,000 rpm at 25 °C with standard double sector aluminum centerpieces. The UV absorption of the cells was scanned every 5 min for 4 h. Sedimentation equilibrium was performed at 20 °C with six-channel open centerpieces and then centrifuged at 12,000 rpm for 12 h. The data from both sedimentation velocity and sedimentation experiments were analyzed with the SedFit version 8.7 program to obtain molecular weights and sedimentation coefficients (25Chang H.C. Chang G.G. J. Biol. Chem. 2003; 278: 23996-24002Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Sednterp version 1.07 program is used to obtain solvent density, viscosity, Stokes' radius (Rs) and anhydrous frictional ratio (f/fo). Dilution Experiment—Enzyme concentrations ranging from 200 to 1.6 nm were used in the dilution experiments. The experiments were carried out with consecutive 2-fold dilutions in PBS containing 0.1% bovine serum albumin and 1 mm DTT. The solution after dilution was incubated at 25 °C for 16 h to ensure attainment of dimer-monomer equilibrium. The reaction was initiated by adding the substrate H-Gly-Pro-pNA at a final concentration of 10 μm for both wild-type DPP-IV and H750A proteins. The initial rate of the reaction was recorded and converted to specific activity. Human DPP-IV Protein Is a Dimer in Intact Cells and in Vitro—From the crystal structures, the human recombinant DPP-IV was shown to be a homodimer whereas DPP-IV purified from porcine kidney is a homotetramer (14Rasmussen H.B. Branner S. Wiberg F.C. Wagtmann N. Nat. Struct. Biol. 2003; 10: 19-25Crossref PubMed Scopus (360) Google Scholar, 16Engel M. Hoffmann T. Wagner L. Wermann M. Heiser U. Kiefersauer R. Huber R. Bode W. Demuth H.U. Brandstetter H. Proc. Natl. Acad. Sci. U. S. 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Next we determined whether endogenous DPP-IV purified from human semen (sDPP-IV) forms dimers in vitro. The purified protein was quite pure as demonstrated by SDS-PAGE (Fig. 3B, lane 1). By measuring its kinetic constants (kcat and Km values), we confirmed that purified sDPP-IV was as active as reported previously (Table I) (31Lambeir A.M. Proost P. Durinx C. Bal G. Senten K. Augustyns K. Scharpe S. Van Damme J. De Meester I. J. Biol. Chem. 2001; 276: 29839-29845Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). On a native gel, sDPP-IV runs predominantly as a dimer of about 200 kDa with the presence of minor but higher molecular mass species (Fig. 3C, lane 1). It elutes at a position corresponding to a 400 kDa protein with a Stokes' radius of 5.9 nm, determined by gel filtration chromatography (Fig. 4A and Table II). Cross-linking of the purified protein in vitro showed that the protein is dimeric with a mass of 250 kDa (data not shown). We then used AUC to determine the hydrodynamic properties of sDPP-IV. As shown in Fig. 5A, sDPP-IV is undoubtedly homodimeric with a sedimentation coefficient of 9.1 S (Table II) and a molecular mass of 225 kDa. Notably, there is only a single peak corresponding to the dimer in the AUC experiment, suggesting that the dimer is the predominant form under the conditions tested. For sDPP-IV, the value of the anhydrous frictional ratio f/fo is 1.4, indicating that the protein is non-spherical. Therefore, gel filtration does not provide an accurate measurement of sDPP-IV's quaternary structure and molecular weight, because of the protein's non-globular shape. The aberrant mobility in gel filtration was also observed in previous studies with DPP-IV proteins purified from either human fibroblast cells or urine (29Saison M. Verlinden J. Van Leuven F. Cassiman J.J. Van den Berghe H. Biochem. J. 1983; 216: 177-183Crossref PubMed Scopus (25) Google Scholar, 32Kato T. Hama T. Kojima K. Nagatsu T. Sakakibara S. Clin. Chem. 1978; 24: 1163-1166Crossref PubMed Scopus (31) Google Scholar).Table IKinetic constants of wild type and mutant DPP-IVs The experiments were repeated at least three times with similar results obtained using different batches of purified proteins. What is shown here is one representative set of the data. Substrate used for kinetic constant measurement is H-Gly-Pro-pNA. The experiments were carried out as described under "Experimental Procedures."PBS bufferHigh salt bufferkcatKmkcat/KmkcatKmkcat/Kms-1μmμm-1 s-1s-1μmμm-1 s-1sDPP-IV73960.76NDaND, not determinedNDNDrDPP-IV87900.97571810.31H750A31770.40161250.13H750A monomer1.4640.02NDNDNDH750A dimer73790.92NDNDNDH750E2.69560.0032.110920.002a ND, not determined Open table in a new tab Table IIHydrodynamic properties of wild type and mutant DPP-IVs The experiments were repeated at least twice with similar results obtained using different batches of purified proteins. What is shown here is one representative set of the data. The predicted monomeric Mr of sDPP-IV and rDPP-IV without glycosylation is 85,246 and 84,371, respectively.PBS bufferHigh salt buffersDPP-IVrDPP-IVH750A dimerH750A monomerH750ErDPP-IVH750A dimerH750A monomerH750EStokes radius (Rs) (nm)5.9aThe values of the Stokes' radii were obtained from gel filtration experiments5.6aThe values of the Stokes' radii were obtained from gel filtration experiments5.7aThe values of the Stokes' radii were obtained from gel filtration experiments4.6aThe values of the Stokes' radii were obtained from gel filtration experiments4.6aThe values of the Stokes' radii were obtained from gel filtration experiments4.9bThe values of the Stokes' radii were obtained from sedimentation velocity experiments4.9bThe values of the Stokes' radii were obtained from sedimentation velocity experiments3.7bThe values of the Stokes' radii were obtained from sedimentation velocit
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