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The Variable Domain Glycosylation in a Monoclonal Antibody Specific to GnRH Modulates Antigen Binding
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
234, 465– 469 (1997)
RC975929
The Variable Domain Glycosylation in a Monoclonal Antibody Specific to GnRH Modulates Antigen Binding Sumit Khurana, Vidya Raghunathan, and Dinakar M. Salunke1 National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India
Received November 25, 1996
Functionally important glycosylation has been identified in the antigen binding domain of an anti-GnRH monoclonal antibody. Presence of mannose and sialic acid residues is revealed from con A immunoblots and positive staining with a sialic acid detection kit, respectively. Desialylation of the antibody reduces GnRH binding, suggesting the role of terminal sialic acid residues in modulating antigen binding. The crystal structure of the Fab fragment shows electron density adjacent to the antigen binding site which may be attributed to the covalently attached carbohydrate moiety. Thus, the presence of sialic acid containing mannose-rich carbohydrate moiety near the antigen binding site of a monoclonal antibody Fab fragment is relevant for defining antibody specificity. q 1997 Academic Press
The antibody molecule is designed to achieve millions of antigenic specificities on a relatively conserved framework. It may appear that virtually any kind of binding site can be generated simply by differential spatial arrangement and chemical composition of a set of six hypervariable loops connecting b-strands. However, the analysis of a large number of antibody structures presently available indicates that the generation of diverse antibody repertoire may not be solely determined by the somatic mutations in the hypervariable loops. It has been suggested that a finite number of 1 Corresponding author. Fax: 91-11-616 2125; e-mail: dinakar@ nii.ernet.in. Abbreviations: GnRH, Gonadotrophin Releasing Hormone; Fab, Antigen Binding Fragment; HPLC, High Performance Liquid Chromatography; ELISA, Enzyme Linked Immunosorbent Assay; SDSPAGE, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis; Con A, Concanavalin A; PEG, Polyethylene Glycol; MoAb, Monoclonal Antibody; VH , Variable Region Heavy Chain; CH1, Constant Region 1 Heavy Chain; VL , Variable Region Light Chain; CL , Constant Region Light Chain; HRP, Horseradish Peroxidase; OPD, oPhenylenediamine; PDB, Brookhaven Protein Data Bank; TFA, Trifloroacetic Acid; EDTA, Ethylenediamine tetraacetic acid.
main chain conformations for five of the six hypervariable loops exists and, further, only a small fraction of these hypervariable loop conformations are preferentially utilized for recognizing a variety of antigens [1, 2]. Probably these limited topological options in the combining site are complemented by additional mechanisms [3, 4] for broadening and fine tuning the immune response. The anti-GnRH monoclonal antibody, P7 278 , is a high affinity bioneutralizing antibody against the decapeptide hormone, gonadotrophin releasing hormone (GnRH). It causes blockage of estrus cycle and termination of pregnancy in BALB/c mice possibly by competing with the GnRH receptor for binding to the hormone [5]. It has been suggested that the hormone binds to the receptor through the side chains of Trp3 and Arg8 and the N- and C-terminal residues by adopting a type II b-turn conformation [6]. GnRH plays an important role in the regulation of fertility in mammalian and sub-mammalian species. It controls spermatogenesis in males and ovulation in females through the release of gonadotrophins from anterior pituitary [7]. This paper presents evidence to suggest that the anti-GnRH antibody, P7278 , recognizes the hormone through mannose and sialic acid rich glycosylation in the antigen binding region of the Fab domain. The glycosylation appears to be involved in modulating antigen binding. EXPERIMENTAL PROCEDURES Antibody preparation and purification. Generation of the monoclonal antibody against GnRH has been described previously [8]. The mouse hybrid cell clone (P7278 ) was grown and developed as ascites tumors in the pristane primed BALB/c mice. The ascites was tapped from the intraperitoneal cavity and subjected to ammonium sulfate (40% saturation) precipitation at 47C. It was further purified by gel permeation chromatography on TSK G3000 column on Waters Prep LC3000 system. The Fab fragment was produced by incubating IgG with papain (Sigma Chemical Company, USA), typically for 7 h at 377C in 0.15M NaCl, 0.10M Tris-HCl, pH7.3, 5mM 2-mercaptoethanol and 2mM EDTA. The reaction was stopped by adding 7.5mM iodoacetamide. The cleaved Fab fragment was purified using anion exchange column (Waters DEAE 5PW). Desialylated antibody was prepared from native IgG using immo-
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bilized neuraminidase purchased from Sigma Chemical Co., USA. 0.1 units of enzyme was used per mg of protein for the digestions. After the digestion, neuraminidase was removed by centrifugation and the protein subsequently dialysed and lyophilized. The sialic acid estimation of IgG and Fab fragment was done using a commercial sialic acid kit available from Boehringer Manheim (Germany) according to manufacturers instructions. ELISA. The GnRH binding experiments of IgG and desialylated IgG were carried out by competitive enzyme linked immunosorbent assay (ELISA). The assay was carried out following the procedures described previously [9]. 1mg/well GnRH was adsorbed on microtiter plates(Nunc, Denmark) at 47C, and plates were blocked using 1.0% BSA. Serially diluted GnRH was dispensed into each well prior to adding fixed amount of IgG (or desialylated IgG). Horseradish peroxidase (HRP) conjugated anti-mouse antibody (NII reagent bank) was added and color was developed using o-Phenylenediamine (OPD) and H2O2 . The concentrations of IgG and desialylated IgG were estimated before each experiment using a micro BCA assay (Pierce Chemical Co., USA) and were confirmed to be within 2 % error. Immunoblotting and carbohydrate estimation. Concanavalin A (Con A) immunoblots were done as described previously [10]. Briefly, about 6mg of Fab was subjected to 12% SDS-PAGE and electroblotted on a nitrocellulose membrane in tris-glycine buffer at a constant voltage of 50V. The membrane was then incubated with Con A (Boehringer Mannheim, Germany) and the increasing concentrations of methyl a-D- glucopyranoside in 7ml volume each. After washing in 0.1% Tween the membrane was incubated with anti-Con A HRP conjugate (DAKOPATTS, Denmark) for 2 hours at room temperature. The bound conjugate was detected with color reaction using ABC staining kit (Vector laboratories, USA). IgG and Fab fragment were subjected to carbohydrate analysis by phenol-sulphuric acid method [11] using mannose as a standard. The final absorbance values were calculated per micromole of sugar or protein. For negative controls solution that lacked only one component that is sugar or protein were used, and all absorbance values were corrected against the background absorbance of these solutions. Crystallization and structure determination. Crystals of Fab were grown by microvapour diffusion method in hanging drops [12] from a solution of 12-15% PEG (8kD) at pH 8.5. The X-ray intensity data were collected using Nicolet Area Detector at IISc, Bangalore installed on a rotating anode X-ray source (Marconi Avionics, UK) with CuKa radiation. Intensity data were processed using XENGEN [13] suite of programs. All molecular replacement calculations were carried out using programs MERLOT [14] and AMoRe [15]. Fab models were selected from PDB [16] and used as probes for rotation/ translation function calculations. Crystallographic refinement of the structure was carried out using X-PLOR [17]. Electron Density maps were displayed with the help of program TOM FRODO [18].
RESULTS Carbohydrate Analysis Con A immunoblotting assay showed that the antiGnRH Fab fragment is glycosylated. Con A binding to Fab (Fig. 1A, lane a) suggests the presence of covalently attached high mannose carbohydrate moieties. This binding was concentration dependent (data not shown) and competitively inhibited by methyl a-D-glucopyranoside, a specific ligand of Con A (Fig. 1A, lanes a, b and c) implying that anti-GnRH Fab binds to Con A through a specific glycan moiety on Fab. The glycosylation of the Fab fragment was also analysed by phenolsulphuric acid reaction. The Fab showed positive reac-
FIG. 1. (A) The lectin immunoblot showing specific binding of Con A to the Fab fragment. Lanes a– c correspond to the binding of Con A to Fab in the presence of 0.0, 0.3 and 1.0M concentrations respectively of methyl a-D-glucopyranoside. Molecular weight markers are indicated by arrows. (B) The comparison of dose dependent binding of GnRH to desialylated IgG and to the native anti-GnRH MoAb (P7278).
tion in this assay. As expected, the glycosylation of Fab was comparatively less (about one fifth) than that in case of IgG. Sialic acid was another carbohydrate moiety present in the Fab fragment. When subjected to analysis by a
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE I
Estimation of Sialic Acid in anti-GnRH MoAb P7 278 Protein
%sialic acid
IgG Fab
4.5 (1.2) 2.4 (0.8)
commercial sialic acid detection kit (see Experimental Procedures) both Fab as well as IgG gave a positive colour reaction due to the release of glycosidically bound sialic acid. The estimation of sialic acid in IgG and Fab is shown in Table I. The sialic acid estimates are given with respect to the free sialic acid concentration in the standard serum provided in the kit. The numbers in brackets (Table I) indicate the sialic acid content in samples which were desialylated by neuraminidase treatment prior to analysis. Very low estimates of sialic acid for the pre- treated IgG and Fab suggest that the sialic acid contents in native IgG and Fab is significantly above background. The degree of sialylation of Fab is quantitatively lower than that of IgG. This is expected due to the presence of sialic acid in the Fc fragment as well [19]. Effect of Deglycosylation on Antigen Binding Desialylation altered the antigen binding properties of IgG. A competitive assay where free GnRH competes with the hormone immobilized on the microtitre plate for binding to IgG or desialylated IgG is shown in Fig. 1B. The GnRH binding ability of desialylated IgG was clearly weaker than that of the native IgG. The SDSPAGE profiles of desialylated IgG under non-reducing and reducing buffer conditions were comparable to those of native IgG. Further, the parallel binding curves of IgG and desialylated IgG indicate that both are identical in all aspects except in their antigen binding ability. The 50% inhibition of binding (IC50) for IgG reduces by half upon desialylation (Fig. 1B). The weakened affinity of desialylated IgG for GnRH suggests that sialic acid residue is close to or within the antigen binding site. Crystallography of Anti-GnRH Fab Crystals of anti-GnRH Fab belong to the orthorhombic space group P21 2121 with unit cell dimensions of ˚ . With the MataÅ202.66, bÅ156.67 and cÅ40.36 A 3 thews constant (Vm) [20] of 3.20 A /dalton and the solvent content of 62%, there appear to be two independent Fab molecules in the asymmetric unit. The selfrotation function calculations showed that these two molecules were related by the Eulerian angles, a Å57, bÅ727and gÅ3557. Structure was solved by molecular replacement using a search model, B13I2, the Fab against C-helix
peptide of myohaemerythrin [21], which has crystal packing apparently similar to that of P 7278 crystals. The crystal parameters of B13I2 have a direct relationship with those of P7 278 . Also, B13I2 has two molecules in the asymmetric unit related by approximately the same relationship as in the case of P7278 . Cross-rotation function calculated using only one molecule from the asymmetric unit of B13I2 Fab crystals as the probe, gave four distinct peaks (Table II). Peaks, i & ii arise from the superimposition of the constant (CH1:CL) domains of the probe with the corresponding domains and the peaks iii & iv arise from the superposition of the complete probe (CH1:CL / VH:VL) with the two molecules in the asymmetric unit of P7278 Fab crystals. The internal relationships between these two pairs of peaks are consistent with the results of the self-rotation function analysis confirming that the orientational interrelationship between the two molecules in the aymmetric unit of anti-GnRH Fab crystals is similar to that between the two molecules in the probe crystals (B13I2 Fab). Cross-rotation function calculations using the two Fab (B13I2) molecules in the asymmetric unit together as search model gave a single distinct peak same as peak iii. The translation solution could be unambiguously defined corresponding to the translations by ˚ , 3.5A ˚ and 5.1A ˚ along x, y and z axes respectively 8.6A (Table II) with an R value at the end of 20 cycles of 48.7%. Molecular replacement calculations carried out using AMoRe gave rotational & translational solutions, almost identical to that obtained above using MERLOT, with a correlation coefficient of 21% and a Rfactor of 49%. A projection of the refined 2Fo-Fc Fourier map is shown in Fig. 2A. The map shows electron density corresponding to the two Fab molecules in the asymmetric unit and the neighboring molecules. The model superimposes reasonably well with the electron density (the ˚ ). However, final R factor was 26% for all data upto 4.7A the unusual feature of the map is a distinct electron density significantly above background and not accounted for by the polypeptide chain in the solvent channel between the symmetry related molecules, blocking the antigen binding site. This density reappears even after solvent flattening [22] was carried out with a Gaussian boundary around the protein dimer. DISCUSSION Variable region glycosylation has been identified in the antibody against GnRH. Experiments carried out to determine the nature of the carbohydrate suggest that the glycan moiety is rich in mannose and sialic acid. The presence of a covalently attached carbohydrate moiety in the variable region of the antibody is not entirely unusual. A statistically significant number of antibodies possess potential glycosylation sites near the hypervariable loops in light or heavy chains. Analy-
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE II
Molecular Replacement Structure Solution Using Merlot and AMoRe Rotation Model Merlot A
A&B A&B AMoRe A&B
Program module
Translation
Dx
Dy
a
b
g
TRNSUM RMINIM
120.0 30.0 52.5 12.5 47.5 47.5
22.0 63.0 8.0 81.0 5.0 7.0
220.0 325.0 295.0 355.0 295.0 294.0
.0425 0.015
.0225 0.00
ROTING TRAING FITTING
51.5 56.1 57.4
3.7 4.5 5.5
287.0 288.5 285.0
.0519 .0606
.0412 .0368
CROSUM
i ii iii iv
sis of Kabat’s data base of antibody sequences[23] indicated that 17.5% of the antibody sequences possess the sequence motif (Asn-X-Ser/Thr) in the heavy chain variable domain [24]. Glycosylation of such sites has been subsequently confirmed in the case of many antibodies [24-27]. The glycosylation of anti-GnRH Fab does not appear to be fortuitous. The removal of the terminal sialic acid from P7278 by neuraminidase treatment reduces the antigen binding capacity without any structural alter-
Dz
.125 0.030
.1236 .1340
s/Corr Coeff
R factor
4.77 4.66 4.28 3.96 3.54 0.487 18.2 19.8 20.7
0.505 0.494
ations suggesting the functional relevance of the glycosylation. Influence of the Fab variable domain glycosylation on the antigen binding capacity has also been demonstrated earlier in the case of a dextran specific antibody where glycosylation of a VH residue increases its affinity significantly [25]. Similarly, the genetically engineered deglycosylation of the variable domain increases the affinity of the anti-CD33 monoclonal antibody towards the ligand [24]. The modulation of affinity towards the antigen by antibody glycosylation may not
FIG. 2. (A) Crystal structure of the Fab fragment. Projection along the c-axis of a section corresponding to the asymmetric unit of the ˚ . Contour level used is 1.5s. The extra electron density in the solvent 2Fo-Fc electron density map. Box dimensions are 1001100140A channel is indicated by an arrow. This density is uniformly present along the c-axis and connects the neighbouring rows of Fab molecules. (B) The Ca drawing of the antibody molecule indicating some of the potential glycosylation sites [25,27]. Positions labeled 1 and 2 correspond to light chain residues 25 and 93 and 3 corresponds to the heavy chain residue 58. 468
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be peculiar to the antibody against this peptide hormone since such antibody ligands also include proteins and carbohydrates. The glycosylation in the variable domain may account for the observed extra electron density in the anti-GnRH Fab structure. This electon density could arise from the covalently-linked oligosaccharide attached to the Fab molecule near the antigen binding site. Interactions between the juxtaposed carbohydrate moieties from the symmetry related Fab molecules (Fig. 2A) can localize the sugar residues which are otherwise expected to be disordered as in many glycoprotein structures. In fact, no such extra density is observed in the corresponding location of the second Fab in the asymmetric unit. The size and location of the extra electron density in the crystal structure of antiGnRH Fab is adequate for appropriately accomodating oligosaccharide residues of the size characterized in the monoclonal human l type light chain [27]. As shown in Fig. 2B, such covalently-linked carbohydrate residues have been detected in various antibody molecules near the antigen binding site. The residues 25 and 93 of V Lof human Bence Jones proteins of l type and 58 of VH of a monoclonal antibody against dextran have been indicated in the Ca drawing of the Fab molecule. The residue 58 of VH is ideally positioned to account for the glycosylation attributed to the observed electron density in P 7278 crystals. The affinity modulation through glycosylation in the antigen binding region may be associated with the generation of antibody diversity. The structural repertoire of immunoglobulin hypervariable loops is restricted to a finite number of conformations [2]. Many somatic mutation hot spots are identified in the framework region [28,29] suggesting that the residues outside the hypervariable loop regions are also important in the generation of the diverse antibody specificities. This can be achieved through conformational influences as suggested in the case of the antibody against phosphocholine [4]. Selective glycosylation within or close to the antigen combining site might also be playing similar role in the generation of antibody diversity.
2. 3. 4. 5. 6. 7.
8. 9. 10.
11. 12. 13.
14. 15. 16.
17. 18. 19. 20. 21. 22. 23.
24.
ACKNOWLEDGMENTS We acknowledge help from Mr. K. K. Sarin for ascites production, Dr. C. Shaha for immunoblotting assay and Dr. K. Suguna (National Area Detector Facility, IISc, Bangalore) for data collection. The antibody clone was a generous gift from Prof. G. P. Talwar. We thank Prof. M. Vijayan and Dr. S. Krishnaswamy for critically reading the manuscript.
25. 26. 27. 28.
REFERENCES 29. 1. Chothia, C., Lesk, A. M., Tramontano, A., Levitt, M., Smith-gill, S. J., Air, G., Sheriff, S., Padlan, E. A., Davies, D., Tulip, W. R.,
Colman, P. M., Spinelli, S., Alzari, P. M., and Poljak, R. J. (1989) Nature 342, 877– 883. Vargas-Madraso, E., Lara-Ochoa, F., and Almargo, J. C. (1995) J. Mol. Biol. 254, 497 –504. Amit, A. G., Mariuzza, R. A., Phillips, S. E. V., and Poljak, R. J. (1986) Science 233, 747–753. Chien, N. C., Roberts, V. A., Giusti, A. M., Scharff, M. D., and Getzoff, E. D. (1989) Proc. Natl. Acad. Sci. USA 86, 5532 –5536. Gupta, S. K., Singh, O., and Talwar, G. P. (1985) Am. J. Reprod. Immunol. Microbiol. 7, 104–108. Gupta, H. M., Talwar, G. P., and Salunke, D. M. (1993) Proteins: Structure, Function, and Genetics 16, 48 –56. Conn, P. M., Huckle, W. R., Andrews, W. V., and McArdle, C. A. (1987) in Recent Progress in Hormone Research (Clark, J. H., Ed.), Vol. 43, pp. 29– 68. Talwar, G. P., Gupta, S. K., Singh, V., Sahal, D., Iyer, K. S. N., and Singh, O. (1985) Proc. Natl. Acad. Sci. USA 82, 1228 –1231. Appel, J. R., Pinilla, C., Niman, H., and Houghten, R. (1990) J. Immunol. 144, 976– 983. Aravinda, S., Gopalakrishnan, B., Dey, C. S., Totey, S. M., Pawshe, C. H., Salunke, D., Kaur, K., and Shaha, C. (1995) J. Biol. Chem. 270, 1 –11. Dubios, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., and Smith, F. (1956) Anal. Chem. 28, 350– 356. McPherson, A. (1982) The Preparation and Analysis of Protein Crystals, Wiley Interscience, New York. Howard, A. J., Gilliland, G. L., Finzel, B. C., Poulos, T. L., Ohlendorf, D. H., and Salemme, F. R. (1987) J. Appl. Crystallogr. 20, 383–387. Fitzgerald, P. M. D. (1988) J. Appl. Crystallogr. 21, 273– 278. Navaza, J. (1994) Acta Crystallogr. A50, 157– 163. Bernstein, F. C., Koetzle, T. F., Williams, G. J. B., Meyer, E. F., Brice, M. D., Rodgers, J. R., Kennard, O., Shimanouchi, T., and Tasmui, M. (1977) J. Mol. Biol. 112, 535– 542. Brunger, A. T., Kuriyan, J., and Karplus, M. (1987) Science 235, 458–460. Jones, T. A. (1978) J. Appl. Crystallogr. 11, 268–272. Abel, C. A., Spiegelberg, H. L., and Grey, H. M. (1968) Biochemistry 7, 1271 – 1278. Matthews, B. W. (1968) J. Mol. Biol. 33, 491 –497. Stanfield, R. L., Fieser, T. M., Lerner, R. A., and Wilson, I. A. (1990) Science 248, 712–719. Wang, B. C. (1985) Methods Enzymol. 115, 90 –112. Kabat, E. A., Wu, T. T., Parry, H. M., Gottesman, K. S., and Foeller, C. (1992) Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, N.I.H., Washington, D.C. Co, M. S., Scheinberg, D. A., Avdalovic, N. M., McGraw, K., Vasquez, M., Caron, P. C., and Queen, C. (1993) Mol. Immunol. 30, 1361 –1367. Wright, A., Tao, M., Kabat, E. A., and Morrison, S. L. (1991) EMBO J. 10, 2717 – 2723. Wallick, S. C., Kabat, E. A., and Morrison, S. L. (1988) J. Exp. Med. 168, 1099 –1109. Okhura, T., Isobe, T., Yamashita, K., and Kobata, A. (1985) Biochemistry 24, 503– 508. Betz, A. Z., Rada, C., Pannell, R., Milstein, C., and Neuberger, M. S. (1993) Proc. Natl. Acad. Sci. USA 90, 2385 –2388. Panka, D. J., Mudgett-Hunter, M., Parks, D. R., Peterson, L. L., Herzenberg, L. A., Haber, E., and Margolies, M. N. (1988) Proc. Natl. Acad. Sci. USA 85, 3080 –3084.
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RC975929
The Variable Domain Glycosylation in a Monoclonal Antibody Specific to GnRH Modulates Antigen Binding Sumit Khurana, Vidya Raghunathan, and Dinakar M. Salunke1 National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India
Received November 25, 1996
Functionally important glycosylation has been identified in the antigen binding domain of an anti-GnRH monoclonal antibody. Presence of mannose and sialic acid residues is revealed from con A immunoblots and positive staining with a sialic acid detection kit, respectively. Desialylation of the antibody reduces GnRH binding, suggesting the role of terminal sialic acid residues in modulating antigen binding. The crystal structure of the Fab fragment shows electron density adjacent to the antigen binding site which may be attributed to the covalently attached carbohydrate moiety. Thus, the presence of sialic acid containing mannose-rich carbohydrate moiety near the antigen binding site of a monoclonal antibody Fab fragment is relevant for defining antibody specificity. q 1997 Academic Press
The antibody molecule is designed to achieve millions of antigenic specificities on a relatively conserved framework. It may appear that virtually any kind of binding site can be generated simply by differential spatial arrangement and chemical composition of a set of six hypervariable loops connecting b-strands. However, the analysis of a large number of antibody structures presently available indicates that the generation of diverse antibody repertoire may not be solely determined by the somatic mutations in the hypervariable loops. It has been suggested that a finite number of 1 Corresponding author. Fax: 91-11-616 2125; e-mail: dinakar@ nii.ernet.in. Abbreviations: GnRH, Gonadotrophin Releasing Hormone; Fab, Antigen Binding Fragment; HPLC, High Performance Liquid Chromatography; ELISA, Enzyme Linked Immunosorbent Assay; SDSPAGE, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis; Con A, Concanavalin A; PEG, Polyethylene Glycol; MoAb, Monoclonal Antibody; VH , Variable Region Heavy Chain; CH1, Constant Region 1 Heavy Chain; VL , Variable Region Light Chain; CL , Constant Region Light Chain; HRP, Horseradish Peroxidase; OPD, oPhenylenediamine; PDB, Brookhaven Protein Data Bank; TFA, Trifloroacetic Acid; EDTA, Ethylenediamine tetraacetic acid.
main chain conformations for five of the six hypervariable loops exists and, further, only a small fraction of these hypervariable loop conformations are preferentially utilized for recognizing a variety of antigens [1, 2]. Probably these limited topological options in the combining site are complemented by additional mechanisms [3, 4] for broadening and fine tuning the immune response. The anti-GnRH monoclonal antibody, P7 278 , is a high affinity bioneutralizing antibody against the decapeptide hormone, gonadotrophin releasing hormone (GnRH). It causes blockage of estrus cycle and termination of pregnancy in BALB/c mice possibly by competing with the GnRH receptor for binding to the hormone [5]. It has been suggested that the hormone binds to the receptor through the side chains of Trp3 and Arg8 and the N- and C-terminal residues by adopting a type II b-turn conformation [6]. GnRH plays an important role in the regulation of fertility in mammalian and sub-mammalian species. It controls spermatogenesis in males and ovulation in females through the release of gonadotrophins from anterior pituitary [7]. This paper presents evidence to suggest that the anti-GnRH antibody, P7278 , recognizes the hormone through mannose and sialic acid rich glycosylation in the antigen binding region of the Fab domain. The glycosylation appears to be involved in modulating antigen binding. EXPERIMENTAL PROCEDURES Antibody preparation and purification. Generation of the monoclonal antibody against GnRH has been described previously [8]. The mouse hybrid cell clone (P7278 ) was grown and developed as ascites tumors in the pristane primed BALB/c mice. The ascites was tapped from the intraperitoneal cavity and subjected to ammonium sulfate (40% saturation) precipitation at 47C. It was further purified by gel permeation chromatography on TSK G3000 column on Waters Prep LC3000 system. The Fab fragment was produced by incubating IgG with papain (Sigma Chemical Company, USA), typically for 7 h at 377C in 0.15M NaCl, 0.10M Tris-HCl, pH7.3, 5mM 2-mercaptoethanol and 2mM EDTA. The reaction was stopped by adding 7.5mM iodoacetamide. The cleaved Fab fragment was purified using anion exchange column (Waters DEAE 5PW). Desialylated antibody was prepared from native IgG using immo-
465
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bilized neuraminidase purchased from Sigma Chemical Co., USA. 0.1 units of enzyme was used per mg of protein for the digestions. After the digestion, neuraminidase was removed by centrifugation and the protein subsequently dialysed and lyophilized. The sialic acid estimation of IgG and Fab fragment was done using a commercial sialic acid kit available from Boehringer Manheim (Germany) according to manufacturers instructions. ELISA. The GnRH binding experiments of IgG and desialylated IgG were carried out by competitive enzyme linked immunosorbent assay (ELISA). The assay was carried out following the procedures described previously [9]. 1mg/well GnRH was adsorbed on microtiter plates(Nunc, Denmark) at 47C, and plates were blocked using 1.0% BSA. Serially diluted GnRH was dispensed into each well prior to adding fixed amount of IgG (or desialylated IgG). Horseradish peroxidase (HRP) conjugated anti-mouse antibody (NII reagent bank) was added and color was developed using o-Phenylenediamine (OPD) and H2O2 . The concentrations of IgG and desialylated IgG were estimated before each experiment using a micro BCA assay (Pierce Chemical Co., USA) and were confirmed to be within 2 % error. Immunoblotting and carbohydrate estimation. Concanavalin A (Con A) immunoblots were done as described previously [10]. Briefly, about 6mg of Fab was subjected to 12% SDS-PAGE and electroblotted on a nitrocellulose membrane in tris-glycine buffer at a constant voltage of 50V. The membrane was then incubated with Con A (Boehringer Mannheim, Germany) and the increasing concentrations of methyl a-D- glucopyranoside in 7ml volume each. After washing in 0.1% Tween the membrane was incubated with anti-Con A HRP conjugate (DAKOPATTS, Denmark) for 2 hours at room temperature. The bound conjugate was detected with color reaction using ABC staining kit (Vector laboratories, USA). IgG and Fab fragment were subjected to carbohydrate analysis by phenol-sulphuric acid method [11] using mannose as a standard. The final absorbance values were calculated per micromole of sugar or protein. For negative controls solution that lacked only one component that is sugar or protein were used, and all absorbance values were corrected against the background absorbance of these solutions. Crystallization and structure determination. Crystals of Fab were grown by microvapour diffusion method in hanging drops [12] from a solution of 12-15% PEG (8kD) at pH 8.5. The X-ray intensity data were collected using Nicolet Area Detector at IISc, Bangalore installed on a rotating anode X-ray source (Marconi Avionics, UK) with CuKa radiation. Intensity data were processed using XENGEN [13] suite of programs. All molecular replacement calculations were carried out using programs MERLOT [14] and AMoRe [15]. Fab models were selected from PDB [16] and used as probes for rotation/ translation function calculations. Crystallographic refinement of the structure was carried out using X-PLOR [17]. Electron Density maps were displayed with the help of program TOM FRODO [18].
RESULTS Carbohydrate Analysis Con A immunoblotting assay showed that the antiGnRH Fab fragment is glycosylated. Con A binding to Fab (Fig. 1A, lane a) suggests the presence of covalently attached high mannose carbohydrate moieties. This binding was concentration dependent (data not shown) and competitively inhibited by methyl a-D-glucopyranoside, a specific ligand of Con A (Fig. 1A, lanes a, b and c) implying that anti-GnRH Fab binds to Con A through a specific glycan moiety on Fab. The glycosylation of the Fab fragment was also analysed by phenolsulphuric acid reaction. The Fab showed positive reac-
FIG. 1. (A) The lectin immunoblot showing specific binding of Con A to the Fab fragment. Lanes a– c correspond to the binding of Con A to Fab in the presence of 0.0, 0.3 and 1.0M concentrations respectively of methyl a-D-glucopyranoside. Molecular weight markers are indicated by arrows. (B) The comparison of dose dependent binding of GnRH to desialylated IgG and to the native anti-GnRH MoAb (P7278).
tion in this assay. As expected, the glycosylation of Fab was comparatively less (about one fifth) than that in case of IgG. Sialic acid was another carbohydrate moiety present in the Fab fragment. When subjected to analysis by a
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Estimation of Sialic Acid in anti-GnRH MoAb P7 278 Protein
%sialic acid
IgG Fab
4.5 (1.2) 2.4 (0.8)
commercial sialic acid detection kit (see Experimental Procedures) both Fab as well as IgG gave a positive colour reaction due to the release of glycosidically bound sialic acid. The estimation of sialic acid in IgG and Fab is shown in Table I. The sialic acid estimates are given with respect to the free sialic acid concentration in the standard serum provided in the kit. The numbers in brackets (Table I) indicate the sialic acid content in samples which were desialylated by neuraminidase treatment prior to analysis. Very low estimates of sialic acid for the pre- treated IgG and Fab suggest that the sialic acid contents in native IgG and Fab is significantly above background. The degree of sialylation of Fab is quantitatively lower than that of IgG. This is expected due to the presence of sialic acid in the Fc fragment as well [19]. Effect of Deglycosylation on Antigen Binding Desialylation altered the antigen binding properties of IgG. A competitive assay where free GnRH competes with the hormone immobilized on the microtitre plate for binding to IgG or desialylated IgG is shown in Fig. 1B. The GnRH binding ability of desialylated IgG was clearly weaker than that of the native IgG. The SDSPAGE profiles of desialylated IgG under non-reducing and reducing buffer conditions were comparable to those of native IgG. Further, the parallel binding curves of IgG and desialylated IgG indicate that both are identical in all aspects except in their antigen binding ability. The 50% inhibition of binding (IC50) for IgG reduces by half upon desialylation (Fig. 1B). The weakened affinity of desialylated IgG for GnRH suggests that sialic acid residue is close to or within the antigen binding site. Crystallography of Anti-GnRH Fab Crystals of anti-GnRH Fab belong to the orthorhombic space group P21 2121 with unit cell dimensions of ˚ . With the MataÅ202.66, bÅ156.67 and cÅ40.36 A 3 thews constant (Vm) [20] of 3.20 A /dalton and the solvent content of 62%, there appear to be two independent Fab molecules in the asymmetric unit. The selfrotation function calculations showed that these two molecules were related by the Eulerian angles, a Å57, bÅ727and gÅ3557. Structure was solved by molecular replacement using a search model, B13I2, the Fab against C-helix
peptide of myohaemerythrin [21], which has crystal packing apparently similar to that of P 7278 crystals. The crystal parameters of B13I2 have a direct relationship with those of P7 278 . Also, B13I2 has two molecules in the asymmetric unit related by approximately the same relationship as in the case of P7278 . Cross-rotation function calculated using only one molecule from the asymmetric unit of B13I2 Fab crystals as the probe, gave four distinct peaks (Table II). Peaks, i & ii arise from the superimposition of the constant (CH1:CL) domains of the probe with the corresponding domains and the peaks iii & iv arise from the superposition of the complete probe (CH1:CL / VH:VL) with the two molecules in the asymmetric unit of P7278 Fab crystals. The internal relationships between these two pairs of peaks are consistent with the results of the self-rotation function analysis confirming that the orientational interrelationship between the two molecules in the aymmetric unit of anti-GnRH Fab crystals is similar to that between the two molecules in the probe crystals (B13I2 Fab). Cross-rotation function calculations using the two Fab (B13I2) molecules in the asymmetric unit together as search model gave a single distinct peak same as peak iii. The translation solution could be unambiguously defined corresponding to the translations by ˚ , 3.5A ˚ and 5.1A ˚ along x, y and z axes respectively 8.6A (Table II) with an R value at the end of 20 cycles of 48.7%. Molecular replacement calculations carried out using AMoRe gave rotational & translational solutions, almost identical to that obtained above using MERLOT, with a correlation coefficient of 21% and a Rfactor of 49%. A projection of the refined 2Fo-Fc Fourier map is shown in Fig. 2A. The map shows electron density corresponding to the two Fab molecules in the asymmetric unit and the neighboring molecules. The model superimposes reasonably well with the electron density (the ˚ ). However, final R factor was 26% for all data upto 4.7A the unusual feature of the map is a distinct electron density significantly above background and not accounted for by the polypeptide chain in the solvent channel between the symmetry related molecules, blocking the antigen binding site. This density reappears even after solvent flattening [22] was carried out with a Gaussian boundary around the protein dimer. DISCUSSION Variable region glycosylation has been identified in the antibody against GnRH. Experiments carried out to determine the nature of the carbohydrate suggest that the glycan moiety is rich in mannose and sialic acid. The presence of a covalently attached carbohydrate moiety in the variable region of the antibody is not entirely unusual. A statistically significant number of antibodies possess potential glycosylation sites near the hypervariable loops in light or heavy chains. Analy-
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE II
Molecular Replacement Structure Solution Using Merlot and AMoRe Rotation Model Merlot A
A&B A&B AMoRe A&B
Program module
Translation
Dx
Dy
a
b
g
TRNSUM RMINIM
120.0 30.0 52.5 12.5 47.5 47.5
22.0 63.0 8.0 81.0 5.0 7.0
220.0 325.0 295.0 355.0 295.0 294.0
.0425 0.015
.0225 0.00
ROTING TRAING FITTING
51.5 56.1 57.4
3.7 4.5 5.5
287.0 288.5 285.0
.0519 .0606
.0412 .0368
CROSUM
i ii iii iv
sis of Kabat’s data base of antibody sequences[23] indicated that 17.5% of the antibody sequences possess the sequence motif (Asn-X-Ser/Thr) in the heavy chain variable domain [24]. Glycosylation of such sites has been subsequently confirmed in the case of many antibodies [24-27]. The glycosylation of anti-GnRH Fab does not appear to be fortuitous. The removal of the terminal sialic acid from P7278 by neuraminidase treatment reduces the antigen binding capacity without any structural alter-
Dz
.125 0.030
.1236 .1340
s/Corr Coeff
R factor
4.77 4.66 4.28 3.96 3.54 0.487 18.2 19.8 20.7
0.505 0.494
ations suggesting the functional relevance of the glycosylation. Influence of the Fab variable domain glycosylation on the antigen binding capacity has also been demonstrated earlier in the case of a dextran specific antibody where glycosylation of a VH residue increases its affinity significantly [25]. Similarly, the genetically engineered deglycosylation of the variable domain increases the affinity of the anti-CD33 monoclonal antibody towards the ligand [24]. The modulation of affinity towards the antigen by antibody glycosylation may not
FIG. 2. (A) Crystal structure of the Fab fragment. Projection along the c-axis of a section corresponding to the asymmetric unit of the ˚ . Contour level used is 1.5s. The extra electron density in the solvent 2Fo-Fc electron density map. Box dimensions are 1001100140A channel is indicated by an arrow. This density is uniformly present along the c-axis and connects the neighbouring rows of Fab molecules. (B) The Ca drawing of the antibody molecule indicating some of the potential glycosylation sites [25,27]. Positions labeled 1 and 2 correspond to light chain residues 25 and 93 and 3 corresponds to the heavy chain residue 58. 468
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be peculiar to the antibody against this peptide hormone since such antibody ligands also include proteins and carbohydrates. The glycosylation in the variable domain may account for the observed extra electron density in the anti-GnRH Fab structure. This electon density could arise from the covalently-linked oligosaccharide attached to the Fab molecule near the antigen binding site. Interactions between the juxtaposed carbohydrate moieties from the symmetry related Fab molecules (Fig. 2A) can localize the sugar residues which are otherwise expected to be disordered as in many glycoprotein structures. In fact, no such extra density is observed in the corresponding location of the second Fab in the asymmetric unit. The size and location of the extra electron density in the crystal structure of antiGnRH Fab is adequate for appropriately accomodating oligosaccharide residues of the size characterized in the monoclonal human l type light chain [27]. As shown in Fig. 2B, such covalently-linked carbohydrate residues have been detected in various antibody molecules near the antigen binding site. The residues 25 and 93 of V Lof human Bence Jones proteins of l type and 58 of VH of a monoclonal antibody against dextran have been indicated in the Ca drawing of the Fab molecule. The residue 58 of VH is ideally positioned to account for the glycosylation attributed to the observed electron density in P 7278 crystals. The affinity modulation through glycosylation in the antigen binding region may be associated with the generation of antibody diversity. The structural repertoire of immunoglobulin hypervariable loops is restricted to a finite number of conformations [2]. Many somatic mutation hot spots are identified in the framework region [28,29] suggesting that the residues outside the hypervariable loop regions are also important in the generation of the diverse antibody specificities. This can be achieved through conformational influences as suggested in the case of the antibody against phosphocholine [4]. Selective glycosylation within or close to the antigen combining site might also be playing similar role in the generation of antibody diversity.
2. 3. 4. 5. 6. 7.
8. 9. 10.
11. 12. 13.
14. 15. 16.
17. 18. 19. 20. 21. 22. 23.
24.
ACKNOWLEDGMENTS We acknowledge help from Mr. K. K. Sarin for ascites production, Dr. C. Shaha for immunoblotting assay and Dr. K. Suguna (National Area Detector Facility, IISc, Bangalore) for data collection. The antibody clone was a generous gift from Prof. G. P. Talwar. We thank Prof. M. Vijayan and Dr. S. Krishnaswamy for critically reading the manuscript.
25. 26. 27. 28.
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