Mapping Part of the Functional Epitope for Ligand Binding on the Receptor for Urokinase-type Plasminogen Activator by Site-directed Mutagenesis
1999; Elsevier BV; Volume: 274; Issue: 53 Linguagem: Inglês
10.1074/jbc.274.53.37995
ISSN1083-351X
AutoresHenrik Gårdsvoll, Keld Danø, Michael Ploug,
Tópico(s)Peptidase Inhibition and Analysis
ResumoThe urokinase-type plasminogen activator receptor (uPAR) is a glycolipid anchored multidomain member of the Ly-6/uPAR protein domain superfamily. Studies by site-directed photoaffinity labeling, chemical cross-linking, and ligand-induced protection against chemical modification have highlighted the possible involvement of uPAR domain I and particularly loop 3 thereof in ligand binding (Ploug, M. (1998) Biochemistry 37, 16494–16505). Guided by these results we have now performed an alanine scanning analysis of this region in uPAR by site-directed mutagenesis and subsequently measured the effects thereof on the kinetics of uPA binding in real-time by surface plasmon resonance. Only four positions in loop 3 of uPAR domain I exhibited significant changes in the contribution to the free energy of uPA binding (ΔΔG ≥ 1.3 kcal mol−1) upon single-site substitutions to alanine (i.e. Arg53, Leu55, Tyr57, and Leu66). The energetic impact of these four alanine substitutions was not caused by gross structural perturbations, since all monoclonal antibodies tested having conformation-dependent epitopes on this domain exhibited unaltered binding kinetics. These sites together with a three-dimensional structure for uPAR may provide an appropriate target for rational drug design aimed at developing new receptor binding antagonists with potential application in cancer therapy. The urokinase-type plasminogen activator receptor (uPAR) is a glycolipid anchored multidomain member of the Ly-6/uPAR protein domain superfamily. Studies by site-directed photoaffinity labeling, chemical cross-linking, and ligand-induced protection against chemical modification have highlighted the possible involvement of uPAR domain I and particularly loop 3 thereof in ligand binding (Ploug, M. (1998) Biochemistry 37, 16494–16505). Guided by these results we have now performed an alanine scanning analysis of this region in uPAR by site-directed mutagenesis and subsequently measured the effects thereof on the kinetics of uPA binding in real-time by surface plasmon resonance. Only four positions in loop 3 of uPAR domain I exhibited significant changes in the contribution to the free energy of uPA binding (ΔΔG ≥ 1.3 kcal mol−1) upon single-site substitutions to alanine (i.e. Arg53, Leu55, Tyr57, and Leu66). The energetic impact of these four alanine substitutions was not caused by gross structural perturbations, since all monoclonal antibodies tested having conformation-dependent epitopes on this domain exhibited unaltered binding kinetics. These sites together with a three-dimensional structure for uPAR may provide an appropriate target for rational drug design aimed at developing new receptor binding antagonists with potential application in cancer therapy. uPA receptor amino-terminal fragment of uPA matrix-assisted laser desorption ionization mass spectrometry soluble uPAR urokinase-type plasminogen activator high performance liquid chromatography polymerase chain reaction polyacrylamide gel electrophoresis The urokinase-type plasminogen activator receptor (uPAR)1 is a multifunctional membrane glycoprotein primarily involved in the regulation of pericellular proteolysis due to its high affinity interaction with the growth factor-like module of urokinase-type plasminogen activator (uPA) (1Danø K. Behrendt N. Brünner N. Ellis V. Ploug P. Pyke C. Fibrinolysis. 1994; 8 Suppl. 1: 189-203Crossref Scopus (294) Google Scholar), but which has also been implicated in the promotion of cell adhesion due to its vitronectin and integrin binding properties (2Chapman H.A. Curr. Opin. Cell Biol. 1997; 9: 714-724Crossref PubMed Scopus (423) Google Scholar), in signal transduction (3Dear A.E. Medcalf R.E. Eur. J. Biochem. 1998; 252: 185-193Crossref PubMed Scopus (65) Google Scholar) and chemotaxis (4Resnati M. Guttinger M. Valcamonica S. Sidenius N. Blasi F. Fazioli F. EMBO J. 1995; 15: 1572-1582Crossref Scopus (303) Google Scholar, 5Fazioli F. Resnati M. Sidenius N. Higashimoto Y. Appella E. Blasi F. EMBO J. 1997; 16: 7279-7286Crossref PubMed Scopus (230) Google Scholar). However, the uPA-uPAR interaction per se is intimately coupled to the latter "non-proteolytic" functions of uPAR, since this interaction either elicits or modulates these events. Cell surfaces expressing uPAR constitutes favored microenvironments for uPA-mediated plasminogen activation (6Ellis V. Scully M.F. Kakkar V.V. J. Biol. Chem. 1989; 264: 2185-2188Abstract Full Text PDF PubMed Google Scholar). Consequently, the uPA-uPAR interaction represents an attractive molecular target for the development of small receptor binding antagonists that may prove useful during treatment of certain diseases in which uPAR has been implicated, i.e. cancer invasion and metastasis (7Yu W. Kim J. Ossowski L. J. Cell Biol. 1997; 137: 767-770Crossref PubMed Scopus (149) Google Scholar, 8Kim J. Yu W. Kovalski K. Ossowski L. Cell. 1998; 94: 353-362Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar, 9Min H.Y. Doyle L.V. Vitt C.R. Zandonella C.L. Stratton-Thomas J.R. Shuman M.A. Rosenberg S. Cancer Res. 1996; 56: 2428-2433PubMed Google Scholar, 10Kook Y.-H. Adamski J. Zelent A. Ossowski L. EMBO J. 1994; 13: 3983-3991Crossref PubMed Scopus (174) Google Scholar, 11Ossowski L. Russo-Payne H. Wilson E.L. Cancer Res. 1991; 51: 274-281PubMed Google Scholar, 12Crowley W.C. Cohen R.L. Lucas B.K. Liu G.H. Shuman M.A. Levinson A.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5021-5025Crossref PubMed Scopus (367) Google Scholar, 13Evans P.C. Elfman F. Parangi S. Conn M. Cunha G. Shuman M.A. Cancer Res. 1997; 57: 3594-3599PubMed Google Scholar). uPAR is a glycosylphosphatidylinositol-anchored plasma membrane protein (14Ploug M. Rønne E. Behrendt N. Jensen A. Blasi F. Danø K. J. Biol. Chem. 1991; 266: 1926-1936Abstract Full Text PDF PubMed Google Scholar) having an extracellular part composed of three homologous domains belonging to the Ly-6/uPAR protein domain family, 2Bork, P. and Bairoch, A. (1995) Trends Biochem. Sci. 20, poster C02. 2Bork, P. and Bairoch, A. (1995) Trends Biochem. Sci. 20, poster C02. as reviewed (15Ploug M. Ellis V. FEBS Lett. 1994; 349: 163-168Crossref PubMed Scopus (243) Google Scholar). This family is dominated by single domain proteins among which the glycolipid-anchored members are found primarily in mammalians (i.e. CD59, Ly-6, E48, and ThB) whereas the secreted members belong to either reptiles or amphibians (i.e.α-neurotoxins, fasiculins, cardiotoxins, and xenoxins). Intriguingly, a similar phylogenetic relationship exists for the few family members identified so far containing two Ly-6/uPAR domains, i.e. the glycolipid anchored RoBo-1 (16Noel L.S. Champion B.R. Holley C.L. Simmons C.J. Morris D.C. Payne J.A. Lean J.M. Chambers T.J. Zaman G. Lanyon L.E. Suva L.J. Miller L.R. J. Biol. Chem. 1998; 273: 3878-3883Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) and metastasis-associated C4.4 (17Rösel M. Claas C. Seiter S. Herlevsen M. Zöller M. Oncogene. 1998; 17: 1989-2002Crossref PubMed Scopus (55) Google Scholar) isolated from rat versus the secreted phospholipase A2 inhibitor isolated from cobra blood (18Ohkura N. Inoue S. Ikeda K. Hayashi K. Biochem. Biophys. Res. Commun. 1994; 204: 1212-1218Crossref PubMed Scopus (56) Google Scholar). Comparison of the three-dimensional protein structures available for several single domain members of this protein family reveals a "three finger" consensus structure consisting of 3 loops, a central 3-stranded β-sheet, and a globular, disulfide-rich core (19Ménez A. Toxicon. 1998; 36: 1557-1572Crossref PubMed Scopus (162) Google Scholar, 20Fletcher C.M. Harrison R.A. Lachmann P.J. Neuhaus D. Structure. 1994; 2: 185-199Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 21Dewan J.C. Grant G.A. Sacchettini J.C. Biochemistry. 1994; 33: 11315-13147Crossref PubMed Scopus (70) Google Scholar). The individual domains of uPAR (numbered I, II and III) are thought to adopt a similar "three finger fold" (15Ploug M. Ellis V. FEBS Lett. 1994; 349: 163-168Crossref PubMed Scopus (243) Google Scholar). The involvement of uPAR domain I (residues 1–87) 3Nucleotide numbering is according to the cDNA sequence published for human uPAR (22Roldan A.L. Cubellis M.V. Masucci M.T. Behrendt N. Lund L.R. Danø K. Appella E. Blasi F. EMBO J. 1990; 9: 467-474Crossref PubMed Scopus (541) Google Scholar) and amino acid numbering refers to the corresponding cDNA-derived protein sequence omitting the NH2-terminal signal peptide. 3Nucleotide numbering is according to the cDNA sequence published for human uPAR (22Roldan A.L. Cubellis M.V. Masucci M.T. Behrendt N. Lund L.R. Danø K. Appella E. Blasi F. EMBO J. 1990; 9: 467-474Crossref PubMed Scopus (541) Google Scholar) and amino acid numbering refers to the corresponding cDNA-derived protein sequence omitting the NH2-terminal signal peptide. in uPA binding is demonstrated by several lines of evidence. First, uPAR domain I can be specifically cross-linked to a receptor binding derivative of uPA using two different chemical cross-linking reagents (23Behrendt N. Ploug M. Patthy L. Houen G. Blasi F. Danø K. J. Biol. Chem. 1991; 266: 7842-7847Abstract Full Text PDF PubMed Google Scholar, 24Behrendt N. Rønne E. Danø K. J. Biol. Chem. 1996; 271: 22885-22894Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Second, the uPA-uPAR interaction can be inhibited competitively by monoclonal antibodies recognizing epitopes on uPAR domain I (25Rønne E. Behrendt N. Ellis V. Ploug M. Danø K. Høyer-Hansen G. FEBS Lett. 1991; 288: 233-236Crossref PubMed Scopus (179) Google Scholar, 26Luther T. Magdolen V. Albrecht S. Kasper M. Riemer C. Kessler H. Graeff H. Müller M. Schmitt M. Am. J. Pathol. 1997; 150: 1231-1244PubMed Google Scholar, 27Ploug M. Ellis V. Danø K. Biochemistry. 1994; 33: 8991-8997Crossref PubMed Scopus (110) Google Scholar). Third, uPAR domain I is also a target for the specific photoinsertion from a small peptide antagonist of uPA binding (28Ploug M. Østergård S. Hansen L.B.L. Holm A. Danø K. Biochemistry. 1998; 37: 3612-3622Crossref PubMed Scopus (80) Google Scholar). Specific proteolytic cleavage after Tyr87, situated in the linker region between domains I and II, is, however, also accompanied by a >1,500-fold reduction in the affinity for uPA (ΔΔG > 4 kcal/mol), which clearly emphasizes the necessity of the multidomain structure of uPAR for high affinity ligand binding (27Ploug M. Ellis V. Danø K. Biochemistry. 1994; 33: 8991-8997Crossref PubMed Scopus (110) Google Scholar, 28Ploug M. Østergård S. Hansen L.B.L. Holm A. Danø K. Biochemistry. 1998; 37: 3612-3622Crossref PubMed Scopus (80) Google Scholar). Previous attempts at dissecting the ligand interaction site on uPAR have highlighted the importance of loop 3 in uPAR domain I, since Arg53, Tyr57, and Leu66 reside at the receptor-ligand interface as assessed by either photoaffinity labeling (29Ploug M. Biochemistry. 1998; 37: 16494-16505Crossref PubMed Scopus (55) Google Scholar) or by ligand-induced protection against chemical modification with tetranitromethane (30Ploug M. Rahbek-Nielsen H. Ellis V. Roepstorff P. Danø K. Biochemistry. 1995; 34: 12524-12534Crossref PubMed Scopus (63) Google Scholar). Guided by these data we have performed an alanine scanning analysis of this region (residues 47–70) of uPAR by site-directed mutagenesis enabling us to discriminate betweenstructural and functional epitopes of ligand-binding, i.e. residues present at the interfaceversus those residues also making a productive contribution to the free energy of ligand-binding (31Clackson T. Wells J.A. Science. 1995; 267: 383-386Crossref PubMed Scopus (1779) Google Scholar, 32Clackson T. Ultsch M.H. Wells J.A. de Vos A.M. J. Mol. Biol. 1998; 277: 1111-1128Crossref PubMed Scopus (247) Google Scholar, 33Bogan A.A. Thorn K.S. J. Mol. Biol. 1998; 280: 1-9Crossref PubMed Scopus (1627) Google Scholar). Pfu DNA polymerase (EC2.7.7.7) was from Stratagene (La Jolla, CA). HPLC-purified DNA oligonucleotides were purchased from DNA Technology (Aarhus, Denmark). Active two-chain uPA (EC 3.4.21.31) was purchased from Serono (Aubonne, Switzerland), recombinant pro-uPA expressed in Escherichia coli was a generous gift from Dr. D. Saunders (Grünenthal, Germany), while the amino-terminal fragment of uPA (ATF) was kindly provided by Dr. A. Mazar (Ångstrom Pharmaceuticals, San Diego, CA). Murine anti-uPAR monoclonal antibodies were produced at the Finsen Laboratory (clones R2, R3, and R9) as described previously (25Rønne E. Behrendt N. Ellis V. Ploug M. Danø K. Høyer-Hansen G. FEBS Lett. 1991; 288: 233-236Crossref PubMed Scopus (179) Google Scholar). Recombinant Flavobacterium meningosepticum N-glycanase (EC 3.2.2.18) expressed in E. coli(25 units/μg) was from Roche Molecular Biochemicals (Germany) and α-chymotrypsin (EC 3.4.21.4) was from Worthington (Freehold, NJ). An expression vector, encoding a secreted truncated soluble form of human uPAR (denoted suPAR and encompassing amino acids 1–283),3 was constructed utilizing two separate PCR performed by standard PCR techniques usingPfu DNA polymerase (Fig. 1). In the first reaction, generating a domain I encoding fragment, a SpeI site was introduced in front of the non-coding 5′-end, and a XhoI site was introduced at the 3′-end by an A → G nucleotide shift at position 394.3 In the second reaction, generating a domain II + III fragment, a XhoI site was introduced into the 5′-end at position 394 as above, and a TAA stop codon, followed by anEcoRI site, was introduced at position 962 in the 3′-end. Finally, the two PCR products from these reactions were cut with the appropriate restriction enzymes, ligated, and cloned into theNheI (SpeI compatible) and EcoRI sites of the mammalian expression vector pCI-neo (Promega) as aSpeI-EcoRI fragment giving the plasmid pCI-neo/suPAR. Mutations were introduced into suPAR domain I by PCR using a one-tube based modification of the megaprimer procedure which employsPfu DNA polymerase (34Picard V. Ersdal-Badju E. Lu A. Bock S.C. Nucleic Acids Res. 1994; 22: 2587-2591Crossref PubMed Scopus (216) Google Scholar). In brief, the procedure requires three oligonucleotide primers: two flanking primers, which are upstream and downstream of the mutation site, and one mutagenic primer (Table I) designed to contain at least 10 perfectly matched bases at both the 5′- and 3′-end. To that end, uPAR domain I was flanked by SpeI and XhoI sites as above and cloned into pBluescript (Stratagene), as shown in Fig. 1. This plasmid (pBluescript/shuPAR-D1) was used as a template with T3 and T7 primers as flanking primers for the production of PCR products containing the desired mutation. Mutated domain I PCR fragments were cloned as cassettes into theNheI and XhoI sites of pCI-neo/suPAR depleted for wild-type domain I. The suPAR-W129A mutant in domain II was constructed using pCI-neo/suPAR wild-type DNA as template in a site-directed mutagenesis PCR using flanking primers residing in domain I and III, respectively. The W129A mutant PCR fragment was cloned as a cassette into XhoI and ApaI sites of pCI-neo/suPAR depleted for wild-type domain II. Prior to transfection PCR-generated sequences of all constructs were confirmed by DNA sequencing.Table IMutantMutagenic primersaSequences of the synthetic oligonucleotide primers are shown from 5′ to 3′ with the mutated codons highlighted by underlined bold letters.Molecular massbThe molecular masses were determined by MALDI-MS for chymotrypsin released domain I (residues 1–87) deglycosylated byN-glycanase. The mutants Asn52 → Gln and Thr54 → Ala were untreated since these point mutations compromise the sole N-linked glycosylation motif present in domain 1 (Asn-Arg-Thr54). The deviation between the determined mass and the calculated average molecular mass of deglycosylated domain I carrying the respective mutations are shown in brackets.Glycosylation of Asn52c+, denotes that the carbohydrate profile on Asn52determined by MALDI-MS for this mutant is comparable to that published previously for wild-type uPAR secreted by Chinese hamster ovary cells (38), while − indicates the absence of glycosylation on domain I.YielddThe amount of suPAR mutant protein secreted to the medium was measured by enzyme-linked immunosorbent assay.Dang/mlwt (1–283)None9,754.78 (0.7)+45W32AATCGTGCGCTTG GCG GAAGAAGGAGA9,638.66 (0.3)+24H47AAAAAGCTGTACC GCC TCAGAGAAGAC9,691.99 (3.0)+86E49AGTACCCACTCA GCG AAGACCAGGAC9,696.97 (0.9)+113K50AACCCACTCAGAG GCG ACCAACAGGAC9,700.97 (4.0)+146T51ACACTCAGAGAAG GCC AACAGGACCC9,721.83 (2.2)+53N52QTCAGAGAAGACC CAG AGGACCCTGAGC9,766.81 (0.3)−21R53AGAGAAGACCAAC GCG ACCCTGAGCTA9,666.64 (2.3)+40T54AAAGACCAACAGG GCC CTGAGCTATC9,721.39 (1.7)−72L55AACCAACAGGACC GCG AGCTATCGGAC9,710.80 (1.2)+68S56AAACAGGACCCTG GCC TATCGGACTGG9,736.55 (1.5)+128Y57AAGGACCCTGAGC GCT CGGACTGGCTT9,661.97 (3.0)+79R58AACCCTGAGCTAT GCG ACTGGCTTGAA9,668.96 (0.6)+76T59ACTGAGCTATCGG GCT GGCTTGAAGA9,725.14 (1.1)+24L61ATATCGGACTGGC GCG AAGATCACCAG9,709.54 (2.5)+174K62ACGGACTGGCTTG GCG ATCACCAGCCT9,698.43 (1.5)+42I63AACTGGCTTGAAG GCC ACCAGCCTTAC9,711.18 (0.8)+36T64AGGCTTGAAGATC GCC AGCCTTACCG9,724.40 (0.4)+59S65ATTGAAGATCACC GCC CTTACCGAGGT9,738.84 (0.8)+219L66AAAGATCACCAGC GCT ACCGAGGTTGT9,711.99 (1.6)+37T67AAAGATCACCAGC GCT ACCGAGGTTGT9,725.75 (1.7)+6E68ACCAGCCTTACC GCC GTTGTGTGTGGG9,696.03 (0.7)+15V69AGCCTTACCGAG GCT GTGTGTGGGTT9,728.06 (2.1)+115V70ATTACCGAGGTT GCC TGTGGGTTAGAC9,724.29 (1.7)+92W129ACTTCCTGGATC GCG TGGGTCACCANDeND, not determined.+51R53LGAGAAGACCAAC CTG ACCCTGAGCTA9,711.86 (0.8)+88R53KAGAAGACCAAC AAG ACCCTGAGCTA9,726.17 (1.2)+152L55MACCAACAGGACC ATG AGCTATCGGA9,770.98 (1.1)+81L55VACCAACAGGACC GTG AGCTATCGGA9,742.60 (2.6)+28Y57WGGACCCTGAGC TGG CGGACTGGCTTG9,779.90 (2.8)+279Y57FGGACCCTGAGC TTT CGGACTGGCTT9,737.07 (0.6)+179Y57KAGGACCCTGAGC AAA CGGACTGGCTTG9,721.09 (2.0)+318Y57EAGGACCCTGAGC GAA CGGACTGGCTTG9,720.80 (0.8)+8Y57HCAGGACCCTGAGT CAT CGGACTGGCT9,729.27 (1.2)+162a Sequences of the synthetic oligonucleotide primers are shown from 5′ to 3′ with the mutated codons highlighted by underlined bold letters.b The molecular masses were determined by MALDI-MS for chymotrypsin released domain I (residues 1–87) deglycosylated byN-glycanase. The mutants Asn52 → Gln and Thr54 → Ala were untreated since these point mutations compromise the sole N-linked glycosylation motif present in domain 1 (Asn-Arg-Thr54). The deviation between the determined mass and the calculated average molecular mass of deglycosylated domain I carrying the respective mutations are shown in brackets.c +, denotes that the carbohydrate profile on Asn52determined by MALDI-MS for this mutant is comparable to that published previously for wild-type uPAR secreted by Chinese hamster ovary cells (38Ploug M. Rahbek-Nielsen H. Nielsen P.F. Roepstorff P. Danø K. J. Biol. Chem. 1998; 273: 13933-13943Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), while − indicates the absence of glycosylation on domain I.d The amount of suPAR mutant protein secreted to the medium was measured by enzyme-linked immunosorbent assay.e ND, not determined. Open table in a new tab Chinese hamster ovary cells were transfected with expression vectors using a calcium phosphate precipitation procedure (Stratagene). After G418 selection, clones were picked, propagated, and the conditioned medium tested for the production of recombinant suPAR by enzyme-linked immunosorbent assay (35Rønne E. Høyer-Hansen G. Brünner N. Pedersen H. Rank F. Osborne C.K. Clark G.M. Danø K. Grøndahl-Hansen J. Breast Cancer Res. Treat. 1995; 33: 199-207Crossref PubMed Scopus (86) Google Scholar) using anti-uPAR monoclonal antibody R2 recognizing domain III as detecting antibody. The cells were maintained as monolayers at 37 °C in a humid atmosphere with 5% CO2 in minimum essential α-medium with ribo- and deoxyribonucleotides and GlutaMAX I (Life Technologies, Inc.) supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 800 μg/ml G418. The harvest fluid contained between 0.01 and 0.5 μg/ml recombinant protein (Table I). The suPAR mutants were purified by immunoaffinity chromatography from the conditioned media as described previously (36Ploug M. Kjalke M. Rønne E. Weidle U. Høyer-Hansen G. Danø K. J. Biol. Chem. 1993; 268: 17539-17546Abstract Full Text PDF PubMed Google Scholar) followed by reversed-phase HPLC using a Brownlee Aquapore C4 column and a linear gradient (40 min) from 0 to 70% (v/v) acetonitrile in 0.1% (v/v) trifluoroacetic acid at a flow rate 250 μl min−1. Purified mutants were dissolved in phosphate-buffered saline after solvent evaporation and were either quantified spectrophotometrically using E280 nm1% = 9.2 (37Rønne E. Behrendt N. Ploug M. Nielsen H.J. Wölisch E.W. Weidle U. Danø K. Høyer-Hansen G. J. Immunol. Methods. 1994; 167: 91-101Crossref PubMed Scopus (53) Google Scholar) or by quantitative amino acid analysis (mutants containing tryptophan substitutions). Purity was assessed by SDS-PAGE followed by silver staining and judged to be >95%. To ascertain that the desired mutation in suPAR domain I had occurred at the protein level and to analyze the consequence of the mutations on the processing of the glycan moiety attached to Asn52 (38Ploug M. Rahbek-Nielsen H. Nielsen P.F. Roepstorff P. Danø K. J. Biol. Chem. 1998; 273: 13933-13943Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), the NH2-terminal domain I (residues 1–87) was liberated from suPAR by limited proteolysis using chymotrypsin and subsequently purified by size exclusion chromatography using 0.1 mNH4HCO3 as solvent (36Ploug M. Kjalke M. Rønne E. Weidle U. Høyer-Hansen G. Danø K. J. Biol. Chem. 1993; 268: 17539-17546Abstract Full Text PDF PubMed Google Scholar). The mass of purified domain I was determined by MALDI-MS before and after treatment withN-glycanase (5 milliunits/μl for 60 min at 37 °C) and compared with that of wild-type uPAR domain I (38Ploug M. Rahbek-Nielsen H. Nielsen P.F. Roepstorff P. Danø K. J. Biol. Chem. 1998; 273: 13933-13943Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), see Table I. The kinetics of the interaction between suPAR (wild-type as well as mutants) and immobilized uPA, pro-uPA, or various monoclonal anti-uPAR antibodies were measured in real-time by surface plasmon resonance using a BIAcore 2000™ equipment (Pharmacia Biosensor, Uppsala, Sweden). A carboxymethylated dextran matrix (CM5 sensor chip) was preactivated withN-hydroxysuccinimide/N-ethyl-N′-[3-(diethylamino)propyl)carbodiimide according to the manufacturers recommendations. Random amine coupling of the respective ligands was achieved by subsequent injection of uPA, pro-uPA (each 20 μg/ml), or a monoclonal anti-uPAR antibody (50 μg/ml) in 10 mm sodium acetate, pH 5.0, at a flow rate of 5 μl min−1 for 6 min. Sensorgrams (resonance unitsversus time) were recorded by the BIAcore 2000TMat a flow rate of 10 μl min−1 at 5 °C, using 8 different concentrations of suPAR in the range of 2 to 200 nm in running buffer (10 mm HEPES, 150 mm NaCl, pH 7.4, including 0.005% surfactant P-20). All analyses were performed at least in triplicate using several purified preparations of the individual uPAR mutants and different sensor chips coupled with uPA (ranging from 1000 to 3000 resonance units). Sensor chips were regenerated at the end of each run by injection of 0.1m acetic acid containing 0.5 m NaCl. Data obtained from parallel mock coupled flow cells (derivatized in the presence of buffer only) served as blank sensorgrams for subtraction of changes in the bulk refractive index. The sensorgrams obtained were analyzed by nonlinear least squares curve fitting using BIAevaluation 3.0 software (Pharmacia Biasensor) assuming single-site association and dissociation models. In brief, values for k offwere determined from data collected during the dissociation phase (dR/dt = −k off R) after which the corresponding values for k on were derived from the following equation assuming pseudo-first order conditions:dR/dt =k on[suPAR](R max −R) − k off R, as described in detail (39Karlsson R. Michaelsson A. Mattsson L. J. Immunol. Methods. 1991; 145: 229-240Crossref PubMed Scopus (1011) Google Scholar), where R is the recorded surface plasmon resonance signal. Differences in the Gibbs free energy (ΔΔG) of uPA binding between mutant uPAR (ΔG mut) and wild-type uPAR (ΔG wt) were calculated by the following equation, ΔΔG=ΔGmut−ΔGwt=RT1n(Kd(mut)/Kd(wt))(Eq. 1) where R is the gas constant (R = 1.99 cal/mol K), T is the absolute temperature (T= 278 K), and K d is the equilibrium binding constant derived from the kinetic rate constants determined by surface plasmon resonance (K d =k off/k on). Matrix-assisted laser desorption mass spectrometry was performed on a linear time of flight instrument (VoyagerTM, Perseptive Biosystems, MA) equipped with a 1.2-m flight tube and a 337-nm nitrogen laser. Sample was desorbed from an α-cyano-4-hydroxycinnaminic acid matrix after sample deposition by the sandwich method (40Kussmann M. Nordhoft E. Rahbek-Nielsen H Haebel S. Rousel-Larsen L. Roepstorff P J. Mass Spectrom. 1997; 32: 593-601Crossref Scopus (434) Google Scholar). Spectra were calibrated internally by addition of 0.5 pmol of α-cobratoxin (7,821.04 Da), which yielded mass assignments of deglycosylated, wild-type domain I (9,754.07 Da) with a mass accuracy better than 0.05% (±5 Da). Qualitative assessments of the relative ligand-binding affinities of selected suPAR single-site mutants were obtained after preincubation of 0.5 nm suPAR mutant with 1.0 nm125I-labeled ATF followed by covalent cross-linking of the preformed complexes using N,N′-disuccinimidyl suberate (41Nielsen L.S. Kellerman G.M. Behrendt N. Picone R. Danø K. Blasi F. J. Biol. Chem. 1988; 263: 2358-2363Abstract Full Text PDF PubMed Google Scholar). The complex formation was visualized by autoradiography after SDS-PAGE of reduced and alkylated samples. In the present study we have selected non-glycine residues in loop 3 of uPAR domain I (residues 47–70) as the primary target for site-directed mutagenesis, since this region previously has been implicated in ligand-binding by either photoaffinity labeling (Arg53 and Leu66) (28Ploug M. Østergård S. Hansen L.B.L. Holm A. Danø K. Biochemistry. 1998; 37: 3612-3622Crossref PubMed Scopus (80) Google Scholar, 29Ploug M. Biochemistry. 1998; 37: 16494-16505Crossref PubMed Scopus (55) Google Scholar), ligand-induced protection against chemical modification (Tyr57) (30Ploug M. Rahbek-Nielsen H. Ellis V. Roepstorff P. Danø K. Biochemistry. 1995; 34: 12524-12534Crossref PubMed Scopus (63) Google Scholar) or enzymatic deglycosylation (Asn52) (38Ploug M. Rahbek-Nielsen H. Nielsen P.F. Roepstorff P. Danø K. J. Biol. Chem. 1998; 273: 13933-13943Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). In addition, the only two tryptophan residues present in uPAR (Trp32 and Trp129) were also mutagenized, since tryptophan residues generally participate so prevalently in protein-protein interactions (33Bogan A.A. Thorn K.S. J. Mol. Biol. 1998; 280: 1-9Crossref PubMed Scopus (1627) Google Scholar, 42Jones S. Thornton J.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13-20Crossref PubMed Scopus (2272) Google Scholar), and a surface exposed aromatic/hydrophobic patch on uPAR correlates to the vacancy of the high-affinity uPA-binding site as demonstrated by 8-anilino-1-naphthalene sulfonate fluorescence spectroscopy (27Ploug M. Ellis V. Danø K. Biochemistry. 1994; 33: 8991-8997Crossref PubMed Scopus (110) Google Scholar). We have chosen to perform the present alanine scanning mutagenesis analysis using a truncated, secreted uPAR variant (denoted suPAR), having uPA binding properties indistinguishable from those of the wild-type glycolipid-anchored receptor present on the cell surface (27Ploug M. Ellis V. Danø K. Biochemistry. 1994; 33: 8991-8997Crossref PubMed Scopus (110) Google Scholar). This truncated receptor can conveniently be produced by Chinese hamster ovary cells after transfection with a DNA carrying a deletion corresponding to the COOH-terminal signal peptide responsible for the attachment of the glycolipid anchor (14Ploug M. Rønne E. Behrendt N. Jensen A. Blasi F. Danø K. J. Biol. Chem. 1991; 266: 1926-1936Abstract Full Text PDF PubMed Google Scholar, 37Rønne E. Behrendt N. Ploug M. Nielsen H.J. Wölisch E.W. Weidle U. Danø K. Høyer-Hansen G. J. Immunol. Methods. 1994; 167: 91-101Crossref PubMed Scopus (53) Google Scholar, 43Masucci M.T. Pedersen N. Blasi F. J. Biol. Chem. 1991; 266: 8655-8658Abstract Full Text PDF PubMed Google Scholar). A cassette expression vector was therefore constructed in such a manner that single-site mutations in suPAR domain I could be introduced without the necessity to confirm the DNA sequence encoding the entire receptor for each mutant created (Fig. 1). Expression of recombinant "wild-type" suPAR (encompassing residues 1–283) as well as mutants thereof were accomplished after stable transfection of Chinese hamster ovary cells and the secreted receptor proteins were isolated from the harvest fluid by immunoaffinity chromatography using the high-affinity monoclonal antibody R2 specific for uPAR domain III (25Rønne E. Behrendt N. Ellis V. Ploug M. Danø K. Høyer-Hansen G. FEBS Lett. 1991; 288: 233-236Crossref PubMed Scopus (179) Google Scholar) followed by reversed-phase HPLC. The purity of these protein preparations were >95% as judged from silver-stained gels after SDS-PAGE of reduced and alkylated samples (data not shown). To reassure that the correct mutation had been introduced into the purified protein and to reveal any impact thereof on the carbohydrate processing of Asn52, suPAR domain I of each individual mutant was excised by limited proteolysis (36Ploug M. Kjalke M. Rønne E. Weidle U. Høyer-Ha
Referência(s)