Structure and Molecular Interactions of a Unique Antitumor Antibody Specific for N-Glycolyl GM3
2004; Elsevier BV; Volume: 279; Issue: 7 Linguagem: Inglês
10.1074/jbc.m311693200
ISSN1083-351X
AutoresUte Krengel, Lise‐Lotte Olsson, Carlos Martínez, Ariel Talavera, Gertrudis Rojas, Elin S Mier, Jonas Ångström, Ernesto Moreno,
Tópico(s)Galectins and Cancer Biology
ResumoN-Glycolyl GM3 ganglioside is an attractive target antigen for cancer immunotherapy, because this epitope is a molecular marker of certain tumor cells and not expressed in normal human tissues. The murine monoclonal antibody 14F7 specifically recognizes N-glycolyl GM3 and shows no cross-reactivity with the abundant N-acetyl GM3 ganglioside, a close structural homologue of N-glycolyl GM3. Here, we report the crystal structure of the 14F7 Fab fragment at 2.5 Å resolution and its molecular model with the saccharide moiety of N-glycolyl GM3, NeuGcα3Galβ4Glcβ. Fab 14F7 contains a very long CDR H3 loop, which divides the antigen-binding site of this antibody into two subsites. In the docking model, the saccharide ligand is bound to one of these subsites, formed solely by heavy chain residues. The discriminative feature of N-glycolyl GM3 versus N-acetyl GM3, its hydroxymethyl group, is positioned in a hydrophilic cavity, forming hydrogen bonds with the carboxyl group of Asp H52, the indole NH of Trp H33 and the hydroxyl group of Tyr H50. For the hydrophobic methyl group of N-acetyl GM3, this environment would not be favorable, explaining why the antibody specifically recognizes N-glycolyl GM3, but not N-acetyl GM3. Mutation of Asp H52 to hydrophobic residues of similar size completely abolished binding. Our model of the antibodycarbohydrate complex is consistent with binding data for several tested glycolipids as well as for a variety of 14F7 mutants with replaced VL domains. N-Glycolyl GM3 ganglioside is an attractive target antigen for cancer immunotherapy, because this epitope is a molecular marker of certain tumor cells and not expressed in normal human tissues. The murine monoclonal antibody 14F7 specifically recognizes N-glycolyl GM3 and shows no cross-reactivity with the abundant N-acetyl GM3 ganglioside, a close structural homologue of N-glycolyl GM3. Here, we report the crystal structure of the 14F7 Fab fragment at 2.5 Å resolution and its molecular model with the saccharide moiety of N-glycolyl GM3, NeuGcα3Galβ4Glcβ. Fab 14F7 contains a very long CDR H3 loop, which divides the antigen-binding site of this antibody into two subsites. In the docking model, the saccharide ligand is bound to one of these subsites, formed solely by heavy chain residues. The discriminative feature of N-glycolyl GM3 versus N-acetyl GM3, its hydroxymethyl group, is positioned in a hydrophilic cavity, forming hydrogen bonds with the carboxyl group of Asp H52, the indole NH of Trp H33 and the hydroxyl group of Tyr H50. For the hydrophobic methyl group of N-acetyl GM3, this environment would not be favorable, explaining why the antibody specifically recognizes N-glycolyl GM3, but not N-acetyl GM3. Mutation of Asp H52 to hydrophobic residues of similar size completely abolished binding. Our model of the antibodycarbohydrate complex is consistent with binding data for several tested glycolipids as well as for a variety of 14F7 mutants with replaced VL domains. The interest in antibodies that specifically recognize tumor markers has revived during the last few years, boosted by the approval, in 1997, of the first monoclonal antibody for cancer treatment by the United States Food and Drug Administration (FDA). Today, a total of six antibodies have been approved by the FDA for the therapy of several types of malignancies, and dozens of different antibodies with potential therapeutic effects in cancer are in clinical trials. One attractive target antigen for cancer immunotherapy is the N-glycolyl GM3 ganglioside (1Malykh Y.N. Schauer R. Shaw L. Biochimie (Paris). 2001; 83: 623-634Crossref PubMed Scopus (239) Google Scholar), a membrane-associated glycosphingolipid with a terminal N-glycolylated sialic acid residue (NeuGc). 1The abbreviations used are: NeuGc5′-N-glycolylneuraminidateFabantigen-binding fragments of immunoglobulinsCDRcomplementarity determining regionCLconstant light chainCHconstant heavy chainFvFab variable domainsGalgalactoseGlcglucosemAbmonoclonal antibodyNeuAc5′-N-acetylneuraminidatePDBProtein Data BankR.m.s.d.root mean square differencesr.s.c.c.real space correlation coefficientVLvariable light chainVHvariable heavy chainPEGpolyethylene glycolELISAenzyme-linked immunosorbent assayPBSphosphate-buffered saline.1The abbreviations used are: NeuGc5′-N-glycolylneuraminidateFabantigen-binding fragments of immunoglobulinsCDRcomplementarity determining regionCLconstant light chainCHconstant heavy chainFvFab variable domainsGalgalactoseGlcglucosemAbmonoclonal antibodyNeuAc5′-N-acetylneuraminidatePDBProtein Data BankR.m.s.d.root mean square differencesr.s.c.c.real space correlation coefficientVLvariable light chainVHvariable heavy chainPEGpolyethylene glycolELISAenzyme-linked immunosorbent assayPBSphosphate-buffered saline. This tumor marker is expressed in certain tumor cells, such as melanoma and breast tumors (2Müthing J. Stener H. Peter-Katalinic J. Marx U. Bethke V. Neuman V. Lehmann J. J. Biochem. 1994; 116: 64-73Crossref PubMed Scopus (38) Google Scholar, 3Marquina G. Waki H. Fernández L.E. Kon K. Carr A. Valiente O. Pérez R. Ando S. Cancer Res. 1996; 56: 5165-5171PubMed Google Scholar), but is otherwise absent from normal human tissues (4Varki A. Biochimie. (Paris). 2001; 83: 615-622Crossref PubMed Scopus (87) Google Scholar), which opens up the possibility of using antibodies specific toward these molecules both for diagnosis and immunotherapy. 5′-N-glycolylneuraminidate antigen-binding fragments of immunoglobulins complementarity determining region constant light chain constant heavy chain Fab variable domains galactose glucose monoclonal antibody 5′-N-acetylneuraminidate Protein Data Bank root mean square differences real space correlation coefficient variable light chain variable heavy chain polyethylene glycol enzyme-linked immunosorbent assay phosphate-buffered saline. 5′-N-glycolylneuraminidate antigen-binding fragments of immunoglobulins complementarity determining region constant light chain constant heavy chain Fab variable domains galactose glucose monoclonal antibody 5′-N-acetylneuraminidate Protein Data Bank root mean square differences real space correlation coefficient variable light chain variable heavy chain polyethylene glycol enzyme-linked immunosorbent assay phosphate-buffered saline. However, targeting N-glycolyl GM3 gangliosides is no easy task, as this glycosphingolipid shares common structural features with many other gangliosides expressed on the cell surface. In particular, N-glycolyl GM3 is highly similar to N-acetyl GM3, which is present in most human tissues. In fact, what should be targeted corresponds to an extremely subtle chemical modification: the difference between the N-glycolyl group in NeuGc and the N-acetyl group of NeuAc consists of the addition of a single oxygen atom to the N-acetyl moiety (CH2OH instead of CH3 in the context of a trisaccharide) (see Fig. 1). A murine monoclonal antibody (mAb), termed 14F7, that has this singular property has recently been described (5Carr A. Mullet A. Mazorra Z. Vázquez A.M. Alfonso M. Mesa C. Rengifo E. Pérez R. Fernández L.E. Hybridoma. 2000; 19: 241-247Crossref PubMed Scopus (100) Google Scholar, 6Carr A. Mesa C. del Carmen Arango M. Vázquez A.M. Fernández L.E. Hybrid. Hybridomics. 2002; 21: 463-468Crossref PubMed Scopus (36) Google Scholar). It exhibits high specificity toward N-glycolyl GM3 and strongly recognizes human melanoma and breast cancer tissues. 14F7 is quite unique, not only because of its binding specificity and rather strong binding affinity (in the nanomolar range) 2G. Rojas, A. Talavera, Y. Munoz, E. Rengifo, U. Krengel, J. Ångström, J. Gavilondo, and E. Moreno, submitted manuscript.2G. Rojas, A. Talavera, Y. Munoz, E. Rengifo, U. Krengel, J. Ångström, J. Gavilondo, and E. Moreno, submitted manuscript. for N-glycolyl GM3, but also because of several other properties. In particular, 14F7 is an IgG antibody, belonging to the IgG1 subclass, whereas most other anti-ganglioside antibodies are IgMs, as a result of only a primary immune response to carbohydrate antigens (7Portoukalian J. Clin. Rev. Allergy Immunol. 2000; 19: 73-78Crossref PubMed Scopus (8) Google Scholar). Furthermore, in experiments carried out in mice, the 14F7 monoclonal antibody showed a remarkable inhibition of the growth of solid tumors (6Carr A. Mesa C. del Carmen Arango M. Vázquez A.M. Fernández L.E. Hybrid. Hybridomics. 2002; 21: 463-468Crossref PubMed Scopus (36) Google Scholar). Recently, a pilot clinical trial with radiolabeled 14F7 has been completed, with very encouraging results showing that this antibody is able to specifically target breast tumors and their metastases in vivo. 3A. Casaco, personal communication.3A. Casaco, personal communication. To date, only very few structures of carbohydrate-binding antibodies have been reported (8Cygler M. Rose D.R. Bundle D.R. Science. 1991; 253: 442-445Crossref PubMed Scopus (247) Google Scholar, 9Cygler M. Wu S. Zdanov A. Bundle D.R. Rose D.R. Biochem. Soc. Trans. 1993; 21: 437-441Crossref PubMed Scopus (32) Google Scholar, 10Rose D.R. Przybylska M. To R.J. Kayden C.S. Oomen R.P. Vorberg E. Young N.M. Bundle D.R. Prot. 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Biochemistry. 2002; 41: 13575-13586Crossref PubMed Scopus (88) Google Scholar, 17Calarese D.A. Scanlan C.N. Zwick M.B. Deechongkit S. Mimura Y. Kunert R. Zhu P. Wormald M.R. Stanfield R.L. Roux K.H. Kelly J.W. Rudd P.M. Dwek R.A. Katinger H. Burton D.R. Wilson I.A. Science. 2003; 300: 2065-2071Crossref PubMed Scopus (673) Google Scholar). This is remarkable in the view of the more than 250 entries containing Fab structures that have been deposited in the Protein Data Bank (18Berman H.M. Westbrook J. Feng Z. Gilliland G. Bhat T.N. Weissig H. Shindyalov I.N. Bourne P.E. Nucleic Acids Res. 2000; 28: 235-242Crossref PubMed Scopus (27319) Google Scholar). One of the possible reasons for this unbalance could be that carbohydrates in general are not very immunogenic, producing mostly low affinity antibody responses of the IgM isotype, as mentioned above. 14F7 is thus an exception to the rule, thanks to a successful immunization procedure (5Carr A. Mullet A. Mazorra Z. Vázquez A.M. Alfonso M. Mesa C. Rengifo E. Pérez R. Fernández L.E. Hybridoma. 2000; 19: 241-247Crossref PubMed Scopus (100) Google Scholar). Here, we report the crystal structure of the unliganded Fab fragment of 14F7 at 2.5 Å resolution. This structure provided the starting point for computer docking studies with the saccharide moiety of N- glycolyl GM3, NeuGcα3Galβ4Glcβ. The resulting molecular model will provide a basis for further genetic manipulation of the binding site to improve tumor targeting. Fab 14F7 was prepared from purified mAb using papain digestion. Digestion time, temperature, and papain: mAb ratio were optimized following the progressive degradation of the 50 kDa heavy chain band on a 10% SDS-PAGE under reducing conditions. A 3 mg/ml mAb solution in 100 mm potassium phosphate buffer, pH 7.0, 10 mm cysteine, 10 mm EDTA was incubated with papain (1:100 papain:mAb ratio) at 37 °C for 4h. Enzymatic digestion was stopped with 20 mm iodoacetamide (Sigma) and loaded on a protein A-Sepharose column (Amersham Biosciences) to retain the Fc and undigested mAb, while the flow-through contained the Fab. The Fab fraction was then dialyzed extensively against 25 mm Tris-HCl, pH 8.8. For further purification, the Fab solution was loaded on a Mono Q 5/5 ion exchange column (Amersham Biosciences) and the isoforms (presumably produced by cleavage on secondary proteolytic sites during papain digestion) were separated with a shallow linear gradient obtained by mixing 25 mm Tris-HCl, pH 8.8, and 25 mm Tris-HCl, pH 7.5, over a period of 100 min at a flow rate of 0.5 ml/min. The main peak was collected and concentrated to 10 mg/ml using a 10 kDa cut-off disposable ultrafiltration device (Millipore). Initial crystallization experiments from a mixture of Fab 14F7 isoforms were unsuccessful, but once the purification protocol was significantly improved, crystals could be obtained readily within 20 min to a few days, provided that the protein solution was freshly prepared. Crystals were grown by vapor diffusion using the hanging drop technique at room temperature. The crystallization conditions were rather broad, with the precipitant of choice being PEG (ranging from PEG 400 to PEG 8000), buffered at pH 7.0 to 9.0, with or without the addition of various salts as additives. The crystal shapes varied from plates to rods and needles. Crystals from many conditions were tested for diffraction and all of them were isomorphous. The space group was P212121 with unit cell dimensions a = 52.5 Å, b = 78.9 Å, c = 121.9 Å, corresponding to a Matthews coefficient, VM, of 2.5 Å3/dalton (40Matthews B.W. J. Mol. Biol. 1968; 33: 491-497Crossref PubMed Scopus (7917) Google Scholar) with one molecule per asymmetric unit. Two crystals were used for final data collection. Both of them grew in droplets formed by a 1:1 mixture of the protein solution (10 mg/ml in 25 mm Tris, pH 8.8) and the reservoir solution, containing 15% PEG 400 and 100 mm Hepes buffer at pH 7.0. Data collection was performed at BL711 at the MAX-lab II synchrotron in Lund, Sweden, on a MAR CCD detector. Since shock-freezing of the crystals in a nitrogen stream resulted in strongly anisotropic diffraction, we decided to collect data at room temperature, exposing five different spots on the same crystal, a long needle. To achieve higher completeness and redundancy, a second crystal was used (data collection at 10 °C). All available data to 2.5 Å resolution were processed and scaled with XDS (19Kabsch W. J. Appl. Crystallogr. 1988; 21: 67-71Crossref Scopus (594) Google Scholar, 20Kabsch W. J. Appl. Crystallogr. 1988; 21: 916-924Crossref Scopus (1681) Google Scholar, 21Kabsch W. J. Appl. Crystallogr. 1993; 26: 795-800Crossref Scopus (3225) Google Scholar), resulting in 17,766 unique reflections. Statistics for the final data set are summarized in Table I.Table IData collection and refinement statistics Values in parenthesis refer to the highest resolution shell, 2.6–2.5 Å, for the final data set.Data Resolution (Å)2.5 Completeness (%)97.9 (97.5) Multiplicity4.2 (3.5) Rmerge (%)6.2 (36.7) 〈I/σI〉13.8 (3.5) I/σI>3 (%)81.0 (50.9) Wilson B (Å2)56.4Model No. of residues427 No. of protein atoms3348 No. of solvent molecules37Refinement Rfree (%)23.8 Rcryst (%)18.1Average B-factor Protein (Å2)51.7 Main chain (Å2)49.8 Side chain (Å2)53.8 Solvent molecules (Å2)51.1Stereochemistry R.m.s.d.aR.m.s. deviations from ideal geometry (39) in bonds (Å)0.009 R.m.s.d.aR.m.s. deviations from ideal geometry (39) in angles (°)1.60 R.m.s.d. B-factor for bonded atoms Main chain (Å2)3.7 Side chain (Å2)6.0PDB ID code1RIHa R.m.s. deviations from ideal geometry (39Engh R.A. Huber R. Acta Crystallogr. Sect. A. 1991; 47: 392-400Crossref Scopus (2543) Google Scholar) Open table in a new tab The structure was solved by Molecular Replacement with the program AMoRe (22Navaza J. Acta Crystallogr. Series A. 1994; 50: 157-163Crossref Scopus (5028) Google Scholar), using the Fab coordinates PDB 1C5C as the search model. The sequence was corrected, and the model was subjected to rigid body refinement with four separate rigid domains [light chain constant (CL)/variable (VL) domains and heavy chain constant (CH)/variable (VH) domains] followed by simulated annealing (23Adams P.D. Pannu N.S. Read R.J. Brünger A.T. Proc. Natl. Sci. U. S. A. 1997; 94: 5018-5023Crossref PubMed Scopus (383) Google Scholar). Six percent of all reflections were used for the test set (24Brünger A.T. Nature. 1992; 355: 472-475Crossref PubMed Scopus (3860) Google Scholar). Thereafter, positional and temperature factor refinement with CNS (25Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D. 1998; 54: 905-921Crossref PubMed Scopus (16957) Google Scholar) were alternated with molecular rebuilding (program O, (26Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13009) Google Scholar)) in several rounds. Water molecules were placed at peak positions of σa-weighted Fo - Fc maps and 2Fo - Fc maps greater than 3σ and 1σ, respectively, and checked at the graphics display. 37 water molecules are included in the final model. Refinement results are summarized in Table I. The structure was validated against composite annealed omit maps from CNS. In a Ramachandran plot (27Ramakrishnan C. Ramachandran G.N. Biophys. J. 1965; 5: 909-933Abstract Full Text PDF PubMed Scopus (696) Google Scholar) produced by PROCHECK (28Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar), 87.0% of the residues are located in the most favored regions of the plot and 12.2% are in the additional allowed regions. Two residues are in generously allowed regions and, finally, one residue, Ala L51, is in a disallowed region, slightly outside a generously allowed region. L51 is positioned in a γ-turn in complementarity-determining region loop 2 (CDR L2) and is found in a similar place of the Ramachandran plot in many reported Fab structures. The real space correlation coefficient (r.s.c.c.) for this residue against a composite annealed omit map is 75%. The sequences for the constant domains have not been determined, but were taken from the Kabat data base (29Johnson G. Wu T.T. Nucleic Acids Res. 2001; 29: 205-206Crossref PubMed Scopus (80) Google Scholar) (www.kabatdatabase.com/). The sequence for H187–H188 was first chosen as Pro-Arg, however, the electron density clearly indicated that H188 must be a tryptophan. An examination of the sequence data base revealed that Trp H188 is always accompanied by a threonine residue at H187. The real space correlation coefficients for these two residues are 93% (Thr H187) and 87% (Trp H188). Here, we use the Kabat numbering scheme for the variable regions, which for this particular antibody introduced insertion letters in the heavy chain sequence after positions 52 (52A), 82 (82A to 82C), and 100 (100A to 100H). Preparation of Protein and Ligand—The crystal structure of the 14F7 Fab fragment was taken as the starting geometry for dynamics and docking simulations. The CL and CH1 constant domains and all water molecules were removed, thus leaving only the Fab variable domains (Fv) for calculations. Energy minimizations and dynamics simulations were performed using the program CHARMM, version 24 (30Brooks B.R. Bruccoleri R.E. Olafson B.D. States D.J. Swaminathan S. Karplus M. J. Comp. Chem. 1983; 4: 187-217Crossref Scopus (13939) Google Scholar). Both polar and non-polar hydrogen atoms were added to the protein and, subsequently, an energy minimization was carried out in three steps, in order to refine the stereochemistry. First, harmonic constraints of 50 kcal/mol/Å2 were applied to all α carbons during 100 steps of a steepest descent geometry relaxation. Thereafter, a similar cycle was performed with the constraints reduced to 10 kcal/mol/Å2. Finally, the harmonic constraints were removed to perform 500 steps of conjugate gradient minimization. A distance dependent dielectric constant (ϵ = 8r) and a 12 Å cutoff distance were used for non-bonded interactions. The terminal disaccharide moiety of N-glycolyl GM3 (NeuGcα3Galβ) used in the docking procedure was built using the program CHARMM, based on topology files included in the Quanta2000 package (Accelrys Inc.). As for the protein receptor, all hydrogens were explicitly included. The third carbohydrate residue, 4Glcβ1, was added after docking of the disaccharide since it does not participate in any protein interactions. Glycosidic dihedral angles are defined as described in Refs. 31Imberty A. Delage M.-M. Bourne Y. Cambillau C. Pérez S. Glycoconj. J. 1991; 8: 456-483Crossref PubMed Scopus (70) Google Scholar, 32Siebert H.-C. Reuter G. Schauer R. von der Lieth C.-W. Dabrowski J. Biochemistry. 1992; 31: 6962-6971Crossref PubMed Scopus (126) Google Scholar, 33Nyholm P.-G. Pascher I. Biochemistry. 1993; 32: 1225-1234Crossref PubMed Scopus (81) Google Scholar. Conformational Sampling in the Binding Site Region using Molecular Dynamics—Molecular dynamics was employed to explore the conformational space of the receptor binding site, using the energy-minimized structure as starting geometry. Most of the amino acids in the complementarity determining regions and several other neighboring residues were allowed to move. It should be noted that conformational sampling was not restricted to the side chains, but also included the backbone atoms. The structure was initially heated from 0 to 300 K during 6 ps and equilibrated at this final temperature during another 12 ps. Thereafter followed a 1000-ps simulation with a 0.001-ps integration step. The SHAKE algorithm as implemented in CHARMM was used to constrain bonds to hydrogens. The same non-bonding parameters as for energy minimization were applied. Coordinates were collected every 500 steps (0.5 ps), so that at the end, 2000 coordinate frames were stored in a trajectory file. These coordinate frames were subsequently clustered using an r.m.s.d. cutoff of 1 Å. Docking Simulations of the 14F7-NeuGc-GM3 Complex—Docking simulations were performed employing a modified version of the program DOCK 3.5 (34Kuntz I. Science. 1992; 257: 1078-1082Crossref PubMed Scopus (891) Google Scholar, 35Gschwend D.A. Kunz I.D. J. Comput. Aided Mol. Design. 1996; 10: 123-132Crossref PubMed Scopus (77) Google Scholar) called DOCKdyna. 4E. Moreno, to be published. This program allows the screening of a large number (thousands) of receptor conformations for binding of a single ligand, by using a novel and automatic way of representing the receptor binding site (36Moreno E. León K. Proteins. 2002; 47: 1-13Crossref PubMed Scopus (20) Google Scholar) and re-calculating the force field grids used for energy evaluation for every receptor coordinate frame. Ligand flexibility was allowed. The top-ranking solutions within a scoring window of 6 kcal/mol were selected from the output and grouped into clusters, using a cutoff r.m.s.d. value of 1.5 Å, as calculated only for ring atoms of the sugar. Thereafter, the best ranking solutions, one from each cluster, were selected for visual inspection and analysis. Site-directed Mutagenesis—Modeling studies indicated that Asp H52 plays a crucial role in the binding of NeuGc-GM3. In order to probe the role of this residue for the molecular recognition of the saccharide ligand, two primers were designed, targeting the 14F7 residue Asp H52 for site-directed mutagenesis. One primer was used to mutate Asp H52 to Val (DH52V), the other one for mutation to Ile (DH52I). The substitutions were performed in the phage-displayed VL-shuffled single chain antibody fragment 3Fm (cloned in the phagemid vector pHG-1m, Heber Biotec, Cuba) derived from 14F7, which retained the original VH region and exhibits equal binding characteristics as the original 14F7 monoclonal antibody.2 The reason for using a single chain Fv with a different VL domain is that we could not obtain an Fv fragment with the original VH sequence of 14F7, probably due to expression problems in bacteria. Purified plasmid DNA from 3Fm was used as template for polymerase chain reaction (PCR) amplification reactions. The first reaction was performed using each mutation-containing forward primer and a single backward primer that anneals to the 5′-region of the phagemid vector pHG1–1m flanking the inserted antibody fragment. PCR conditions were: 50 s at 94 °C, 1 min at 55 °C, and 1 min at 72 °C (30 cycles). The mutations were thus introduced in the 5′-region of 3Fm. Amplification products were purified using PCR Preps DNA Purification System (Promega) and used to prime a second round of PCR reactions (using the same conditions) together with a single forward primer that anneals to the 3′-region of the phagemid vector flanking the inserted antibody fragment. Again, 3Fm plasmid DNA was used as template. Full-length antibody fragment DNA sequences containing the mutations were obtained. Purified PCR products were sequentially digested with ApaL I and NotI (New England Biolabs) and cloned into the same phagemid vector. Ligation reactions were electroporated separately (DH52V and DH52I) in TG1 electro-competent cells. Cells were grown overnight in 2× TY-agar plates containing 100 μg/ml ampicillin and 2% glucose. Sequencing of Selected Clones—DNA from selected clones was purified using Midi Preps™ kit (Promega). Sequencing was performed with an automated DNA sequencer ALF Express II (Amersham Biosciences) using primers corresponding both to the 5′ and 3′ vector sequences flanking the antibody fragment. Screening of Phage-displayed Antibody Fragments—Phage particles exposing antibody fragments were rescued from the mutant clones with helper phage M13 K07 on a 50-ml scale and purified from the bacterial supernatant through precipitation with 20% polyethylene glycol 6000 and 2.5 m NaCl (37Marks J.D. Hoogenboom H.R. Bonnert T.P. McCafferty J. Griffiths A.D. Winter G. J. Mol. Biol. 1991; 222: 581-597Crossref PubMed Scopus (1431) Google Scholar). Immunoreactivity of phage-displayed antibody fragments was determined by enzyme-linked immunosorbent assay (ELISA). Polyvinyl chloride microtiter plates (Costar, USA) were dried with N-glycolyl GM3 (1 μg/well) in methanol. Plates were washed with PBS and blocked with 2% skimmed milk powder in PBS (M-PBS) during 1 h at room temperature. Phage displaying antibody fragments were diluted in M-PBS (1010 particles/ml) and incubated on the plates for 1.5 h at room temperature. After washing the plates with PBS plus Tween 20 0.1% (PBS-T), an anti-M13 mAb coupled to horseradish peroxidase (Amersham Biosciences), appropriately diluted in M-PBS, was added to the wells. The plates were washed again and the substrate solution (500 μg/ml orthophenylendiamine and 0.015% hydrogen peroxide in 100 mm citrate buffer, pH 5.5) was added. The reaction was stopped after 15 min with 2.0 m sulfuric acid. The absorbance (492 nm) was determined with a microplate reader (Bio-Rad). Each sample was tested twice for the binding of N-glycolyl GM3. The 3Fm clone was included as a positive control for N-glycolyl GM3 recognition. Figures and Tables—Fig. 1 was prepared using ChemDraw from CambridgeSoft and Fig. 2 using Quanta2000/CHARMm27 modeling package from Accelrys Inc. Crystal Structure of Fab 14F7—The crystal structure of the 14F7 Fab fragment was determined at 2.5 Å resolution and refined to crystallographic R-factors of 18.1% (Rcryst) and 23.8% (Rfree), respectively, with good stereochemistry. The electron density is very well defined for a 2.5 Å structure (average r.s.c.c. for composite annealed omit map is 86%), due to the good data quality. Only a few regions in the structure are less well defined. In particular, the C-terminal residues L213-L214 and H216-H219 from the light chain and the heavy chain, respectively, could not be located in the electron density maps. For the heavy chain loop H128-H133, there is some discontinuous density, but not good enough for this loop to be correctly traced. Many Fab structures deposited in the protein data base lack this particular loop. Further, due to weak electron density for their side chains, Lys H13 and Lys H64 were modeled as alanines and Cys H215 was substituted by a glycine residue. Overall, the crystal structure of Fab 14F7 is highly similar to other Fab structures, with one noticeable variation. The complementarity determining region CDR H3 of 14F7 (residues 95–102 with eight letter insertions at 100), is among the longest found both in the Kabat data base and in the PDB, with 16 amino acid residues (see Table II). This long loop protrudes from the middle of the antibody-combining region, dividing the binding site into two well-separated zones that we will refer to as the VL and VH subsites (Fig. 2A).Table IISequence alignment of the CDR H3 loop of 14F7 compared to all other carbohydrate-binding antibodies present in the Protein Data Bank The Kabat numbering scheme is used. The lettered insertions of the CDR H3 hypervariable loop are indicated between arrows.PDB entryAntibody nameCDR H3 sequence100A 100H↓ ↓1RIH14F7ESPRLRRGIYYYAMDY1OM3, 1OP3, 1OP52G12KGSDRLSDNDP--FDA1UCB, 1CLY, 1CLZbr96GLDDGAW------FAY1MFA-ESe155-4GGHGYY-------GDY1M71, 1M7D, 1M7ISya/J6GGAVGA-------MDY1F4W, 14FX, 14FYS-20-4HFYAV--------LDY1PSKME36.1KS-----------FDY Open table in a new tab In the crystal structure of 14F7, the long CDR H3 loop is well defined by electron density (r.s.c.c. = 91%) and has rather low temperature factors (the average B-factor for this region is 41.2 Å2, compared with
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