160 lmmundogy Today, vo[. 3, No. 6, I082 The three-dimensional structure of antibodies Markus Marquart and Johann Deisenhofer Max-Planck lnstitut l'{ir' Biochemie, Abteilung Strukturforschung II, D-8033 Martinsried, F.R.G. Antibody molecules are glycoproteins which occur in vertebrate species. They recognize and bind an enor- mous variety of tbreign substances (antigens) and sub- sequently trigger further defense mechanisms at the molecular or cellular level. Specific recognition requires surface structures complementary to the antigen and hence a huge variety of antibody molecules. In contrast the effector functions need identical interaction sites in all antibody molecules. The determination of the primary structure of immunoglobulins a-3 and the X-ray crystallographic studies of several antibody molecules and frag- ments4,S,Tjo,12 i5 led to an advanced understanding of the way in which antibodies meet these opposing requirements. FAB Fc IGG Fig. 1 Schematic representation of an IgGl immunoglobulin molecule. The arms of the Y-shaped molecule arc lorrncd by the Fab parts, the stem is made up by the Fc part. The light chains are linked to the heavy chains by a disulphide bridge close to the C-tcrminus. The two heavy chains are connected via two disulphide linkages in the hinge region. Fig. / is a schematic drawing of an antibody molecule of class IgG]. It is composed of two identical heavy chains and two identical light chains with mol. wts of 50,000 and 25,000, respectively. Both types of polypeptidc chain are folded into domains: the four domains of the heavy chain are VH, GHI, CH2, and CH3; the light chain consists of the two domains VL and GL. All domains except CH2 are arranged in pairs which arc held together by non-covalent forces. Inter-chain disulfide bridges provide further stability. Among antibody molecules of a given class and species, the V-domains differ considerably in amino acid sequence, whereas the G-dol'nains have identical sequences. The V-domains are composcd of about 110 amino acid residues at the N-terminal end of heavy and light chains. The VH-VL pair together forms the antigen binding site; different antibody specificities are the result of different amino acid sequences of the V-domains. The sequence variability in V-domains is most pronounced in a few hypervariable regions. On the other hand the framework residues are well con- served. The constant domains CtI2 and GH3 are involved in effector functions such as complement activation and binding to receptors on certain cell types. There is significant homology between the amino acid sequences of all G-domains, and of the framework residues of V-domains. Proteolytic cleavage at the hinge region yields stable and functional fi'agments: the antigen-binding frag- ment Fab, and the Fc fragment (Fc was the first anti- body fi'agment obtained in crystalline form) ¢'. A B F E H 0 C D X ARRANGEMENT OF SFRANDS IN IMMUNOGLOBULIN DOMAINS X N-TERMINUS UP, • C-TERMINUS UP Fig. 2 Schematic drawing of tile strand topology in a V- domain viewed parallel to the strands. (x) and (0) indicate N- and C-terminal ends of" the strands point- ing towards the observer. Elsevier Biomedical Pless 1982 01674919/82/0000~)00D/$2 75
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`Immunol(Jgy To@, vol. 3, No. 6, /982 161 Besides IgG1, several other classes (IgM, IgA, IgD, IgE) and subclasses of immunoglobulins have been identified; the differences between these are located in the constant region of the heavy chain. The two types of light chain (kappa, lambda) can combine with heavy chains of any class. Domain folding The general folding pattern in all immunoglobulin domains is very similar. It is shown schematically in Fig. 2 for a V-domain. The folding is characterized by two pleated sheets connected by an internal di- sulphide bridge linking strands B and C. The two sheets cover a large number of hydrophobic amino acid side chains. Despite that gross similarity there exist substantial differences when one compares V- and C-domains: C-domains lack strand X, strand D is very short (2-3 amino acids) and connected to strand E. In addition the length of the loop regions in C-domains is different from V-domains, thus changing the overall shape con- siderably. VH and VL, on the other hand, show only minor differences when compared with each other (except in the hypervariable regions) as do CL, CH1 and CH3. CH2 represents yet a third type of domain, differentiated from the other C-domains mainly by the branched carbohydrate chain linked to it. It will be discussed in more detail below. Domain-domain interaction Two kinds of domain interactions occur in immuno- globulins: lateral (or trans) interactions and longi- tudinal (or cis) interactions. In lateral interactions immunoglobulin domains other than CH2 strongly assnciate to form modules VL-VH, CL-CHI, CH3-CH3. In V modules Vii may be replaced by VL to tbrm light chain V dimers as seen in the Bence-Jones protein fragments Rei or' Au 7 9 In Bence-Jones proteins, which are light chain dimers, one of the light chains simulates the Fab [)arts of the heavy chain, as described for Mcg I(~. V modules associate in a different way than C modules do. In V modules HGCI) faces (see Fig. 2) of the domains get into contact, in C modules the ABFE faces are involved. A considerable loss of accessible surface area 11 is connected with contact formation of the immuno- globulin domains. It amounts to 1760 h a, 1923 A~- and 2180 A 2 for VL-Vtt, CL-CH1 modules of IgG KoP 2,13 and the CH3-CH3 module of an human Fc fragment H,~s respectively. In VL VH association both framework residues and amino acids fi'om hyper- variable segments are involved. A comparison of V- domain amino acid sequences of different animal species shows that the contacting framework residues are highly conserved. Also the constant domain residues participating in lateral contact are either invariant or replaced by homologous residues in dilltrent imnmnoglobulin chains. This low degree of sequence variability for the residues important for lateral contact formation provides an explanation for the fact that different L-chains can associate with different IF-chains to give intact immunoglobulins. In addition to the extensive Van der Waals contacts, there exist a few trans hydrogen bonds, in which mainly polar side chain groups are involved. There are two salt linkages in Kol CL-CH1 contact: Glu 125 light chain - Lys 214 heavy chain, Glu 126 light chain - Lys 148 heavy chain, which have their analgon in CH3 - CH3 pairing: Glu 356 - Lys 439, Glu 357 - Lys 370. CH2 is an exception, as it forms a single unit without lateral domain interactions (see Fig. 3)*. Instead it interacts with bound carbohydrate, which is attached to Asn 297. The CH2 residucs that arc involved in carbohydrate contact are, with a few exceptions, structurally in the same positions as the residues that form the CH3 CH3 contact (face ABFE in Fig. 2). This demonstrates that the carbohydrate in CH2 provides a substitute for the C-C con- tact and presumably helps to stabilize the CH2- domain. The branched carbohydrate forms a few hydrogen bonds with the CH2-domain, but the dom- inant interactions are hydrophobic in nature. The carbohydrate covers a hydrophobic patch of the protein made up of Phe 241, 243, Val 262, 264, Tyr 296, Thr 260, Arg 301, which would otherwise be exposed to the solvent. The loss of accessible surface area of one CH2 domain is 522 A2, which is only about halt" as much eovered surface area as seen in CH3-CH3 contact (1080 X2). This observation could explain the apparent 'softness' of {hose parts of the CH2-domain, as seen in the crystal structurC 4.~, which are most remote from the CH3-CH2 interface. The functional relevance of carbohydrate in anti- bodies is unclear. It might be involved in intraccllular movements of the glycoproteins and in secretion 1<Is. It may well be that the origin of the altered functional properties of carbohydrate-free antibody variants is structural destabilization. In contrast to the extensive lateral interactions, nonbonded longitudinal interactions along the heavy chain or light chain are much weaker or do not exist at all. However, they are interesting because con- formational changes in antibodies affect those inter- actions. Fig. 3, which represents the Fc part of an IgG1 molecule shows the CH2-CH3 interaction. With a loss in accessible surface area of 778 ~2 this contacl has roughly one third of the size of CH3-CH3 contact. The residues that participate in CH2-CH3 contact are highly conserved in all lg classes, suggesting thal this contact is likely to be found in IgG and lgA and as CH3-CH4 contact in IgE and IgM. * Most readers will need a stereo viewer (commercially available) to see in three dimensions the structures shown in the paired diagrams on pages 162, 163 and 166.
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`Fig. 3 Stereo drawing of a space filling model of human Fc-frag-. merit. The molecule is buih from two identical polypeptide chains (chain l, chain 2), and identical carbohydrate groups. Both halves are related by approximate diads. Fig. 4 IgG1 molecule Kol. The Fab parts and the hinge segment are well ordered in the Kol crystals, the Fc part is disordered and not visible. Fig. 5 Amino acid comparison of residues 98-119 (Eu num- bering) of M603, New, Kol and Eu heavy chains. The underlined residues were left out in Fig. 6e. PLEASE NOTE We regret that for technical reasons it has not been possible to reproduce Figs 3, 4, 6, 7 and 8 with the colour coding that allows different parts of the molec- ules to be distinguished. The full-colour diagrams, with explanatory legends, can be found in the personal monthly edition of bnmun- ology Today datedJune 1982. End of VH 98 M603: Cys Ala Arg Asn Tyr New : Cys Ala Arg Asn Leu Kol : Cys Ala Arg Asp Gly Eu : Cys Ala Gly Gly Tyr Tyr Gly Ser Thr lie Ala Gly Cys Gly His Gly Phe Gly lie Tyr Ser D segment lie Cys Ser Ser Ala Ser Cys Phe
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`; 2 ., 53 53 Fig. 6 Antigen binding region of IgG1 Kol. (a) The extended third hyper- variable loop of the heavy chain folds into the putative antigen binding pocket. (b) C ~ backbone and sidechains of Kol antigen binding pocket. (c) Artificial deletion of nine residues in the third hyper- variable segment of Kol, which makes it of equal length with IgG1 Eu 28, reveals a deep curved cleft. q ) m m Gly Pro J segment 110 119 Try Tyr Phe Asp Val Try Gly Ala Gly Thr Thr Val Thr Val Ser Ser Asp Val Try Gly Gin Gly Ser Leu Val Thr Val Ser Ser Asp Tyr Try Gly Gin Gly Thr Pro Vat Thr Val Ser Ser Pro Glu Glu Tyr Asn Gly Gly Leu Val Thr Vat Ser Ser
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`164 Immunology Today, vo/. .3, No. G, 1982 The CH2-CH3 orientation is found to be somewhat variable and influenced by external forces. In the Fc fragment crystals the two chemically identical chains are in a different environment. As a consequence the CH2-CH3 orientation varies by about 6 ° . In Fc-Protein A complex crystals this arrangement differs slightly from that of Fc crystals .5. More drastic changes are observed in VH-CH1 and VL-CL longitudinal contacts, when chemically different Fab fragments are compared. These differences in longitudinal arrangement are most con- veniently described by an elbow angle, which is enclosed by the pseudo diads relating VL to VH and CH1 to CL respectively. The elbow angle may vary from more than 170 ° to 135 ° when we compare Kol Fab with McPc Fab 12,13J9,2°. In two cases the elbow angles of the same molecule in two different crystal lattices were compared and found to differ by 8 ° and 17 ° respectively 19,2~. In Fab New, with an elbow angle of approximately 137 ° , there exist a few longitudinal contacts between VL and CL and VH and CH12223, whereas there are no non-bonded longitudinal contacts in intact Kol and Fab Kol (see Fig. 4), which are characterized by an open elbow angle. We interpret these observations to mean that in Fab Kol the V-C arrangement is flexible in solution. In the crystal the molecule is stablized by packing interactions; these will be discussed from a different point of view later. The antigen-binding area Comparison of amino acid sequences of variable parts has demonstrated the hypervariability of some segments. These were considered to be involved in antigen binding z4. Indeed, crystal structure analyses of Ig fragment-hapten complexes show that haptens bind in a cleft or depression formed by the hyper- variable segments. The VL dimer of Rei 7,9 may serve as an illustrative example. The symmetrically arranged hypervariable regions form a deep slit-like pocket around the diad relating the two VL monomers. The walls of the slit are lined by tyr,osines 49, 91, 96, Asn 34 and Gin 89; the bottom of the pocket is formed by Ty.r 36 and Gin 89. A trinitrophenyl group binds to the Rei fragment and fills the binding pocket completely. Another example of an IgG fragrnent-hapten complex is Fab New, which is known to bind among other ligands a hydroxy derivative of vitamin K125. The hypervariable segments of New form a shallow groove with approximate dimensions of 16 x 7 A and a depth of 6 A. McPc 603, a mouse IgA (~) Fab fragment 2° binds phosphorylcholine. The site of hapten binding is a large wedge shaped cavity, with dimensions 15 x 20 and a depth of 12 A. Only five of the six hypervariable regions contribute to the formation of the cavity: L- chain hypervariable regions one and three, and all three H-chain hypervariable regions. The second hypervariable region of L-chain is screened from the cavity by the first hypervariable loop of [.-chain and the third hypervariable loop of H-chain. The deeper cavity in McPc603, as compared to Fab New, is due to longer hypervariable loops. The lirst hypervariable region of L-chain and the third hypervariable region of H-chain is three residues and the second hyper- variable loop of the H-chain is two residues kruger in McPc603 than in New. Phosphorylcholine occupies only a small part of the cavity and interacts via Van der Waals fbrces, electro- static: interactions, and hydrogen bonds with the protein. In contrast to the above examples IgG Kol shows no cleft or depression in the antigen-binding region. In IgG Kol the heavy chain has a rather long third hyper- variable loop, which contains six residues more than M603 and eight more residues than Fab New. The amino acid sequences of the third hypervariable regions of M6032(', New 23, Kol iv and E,u 28 are com- pared in Fig. 5. The sequence alignment and classifi- cation in VH, D and J segment 2<> is somewhat arbitrary, especially for the beginning of theJ segment as a nucleotide sequence has been determined only for M6032('. The additional residues in Kol with the nearly palindromic amino acid sequence -GIy-Phe- Cys-Ser-Ser-Ala-Ser-Cys-Phe-Gly fold into the puta- tive antigen binding site and fill it completely (see Fig. 6a,b). The two cysteins are disulphide bridged and form the start and endpoints of a short antiparallel 13- sheet, comprising residues -Cys-Ser-Ser-Ala-Ser-Cys-. If in a model building experiment nine residues are cut from the third hypervariable region of the Kol heavy chain, thus making it of equal length with IgG1 Eu 2s, a deep curved cleft appears (Fig. 6c), which easily could accommodate haptens. With respect to the anti- gen binding area lgG Kol thus looks as if it carried its own hapten in form of an extended third hyper- variable loop. Another peculiarity of lgG Kol might be of interest in that context. In the Kol crystal lattice the hypervariable parts of one molecule touch the hinge and spatially adjacent segments of a symmetrically related molecule. This contact consists of three salt linkages (Arg 49 light chain-COOH light chain, Asp 50 light chain-Arg 215 heavy ehain, Asp 53 heavy chain-I.ys 134 heavy chain), a few hydrogen bonds and extensive Van der Waals interactions. Thus, the lattiee contact found in Kol crystals might give an instructive model for antibody-antigen interaction, as antigens are usually macromolecules which cover a much larger part of the antibody than haptens do. The hinge segment The hinge segment which covalently links Fab and Fc parts, has a unique primary and spatial structure. Its central region consists of two parallel disulphide- linked poly L-proline helices with an amino acid sequence-Cys-Pro-Pro-Cys-12-% In the IgG1 subclass represented by the Kol molecule the poly-proline double helix is short (Fig. 7). However, in IgG3 the hinge sequence is quadruplicated ~ and model build-
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`Immunology Today, voL 3, No. 6, 1982 165 ing suggests that the poly-pro!ine segment of this molecule may be more than 100 A long. The poly-proline segment, a relatively rigid struc- ture, is flanked on both sides by flexible segments: The segment on the N-terminal side is well defined in the crystal lattice of Kol due to crystal packing inter- actions, but it lacks internal interactions, that would provide stability in solution. The C terminal segment is disordered and flexible in Kol crystals and in the Fc crystal structure .3,~5. The rigid hinge segment allows independent movement of the Fab arms and the Fc part. There is direct evidence for flexibility in the crystal lattice of Kol ~3.~') and Zie ~j. This is in contrast to the abnormal IgG protein Dob, which lacks a hinge region 32. The significance of the hinge for Fab-Fc flexibility is obvious. Complement binding The binding of the Clq component of the CI complex to antigen-antibody complexes is the first step in the classical pathway of complement activation~3.3L The Clq head pieces bind to the CH2 domains of antibodies >,3c'. Protein A, a constituent of the cell wall of Slaphylococcus aureus, binds to the Fc- part of antibody molecules of certain classes and sub- classes, but does not interfere with complement binding. The determination of the crystal structure of the complex between FB (one of the four Fc-binding domains of protein A 37) and Fc-fragment showed that protein A binds at the CH2-CH3 contact ~5,3s. Fig. 8 shows a space-filling model of the FB-Fc complex. The area of CH2 not covered by FB must contain the Clq binding site. In view of the size of the Clq head pieces (mol. wt 50,000) it appears unlikely that they can bind at the inner sides of CH2, i.e. near the carbo- hydrate. The most plausible binding site is therefore near the tip of CH2 on the outer side of the domain. It is worth mentioning that this region is disordered in crystals of the FB-Fc complex which indicates that this part of the CH2 domain is flexible. Possibly, flexibility is required for antibody CIq interaction. Summary and perspectives Investigations of the three-dimensional architecture of" antibodies have elucidated the folding of the polypcptide chains into domains, and the spatial arrangement of the domains. The structural basis for understanding antibody specificity and antibody flexibility was obtained. Segmental flexibility is an important property of antibodies: Flexible segments of the polypeptide chains at the switch and hinge regions allow the Fab fragments to change their shape and their relative orientation. Conformational changes of this kind are necessary to meet the geometric require- ments which arise on binding of antibodies to multi- valent antigens. The understanding of the cffector functions of anti- body molecules is much less complete. One of the central problems is the explanation of the strong enhancement of Clq binding to antigen-antibody complexes as compared to free antibody molecules. Two mechanisms have bc'en considered (for a review see Ref. 39): since Clq is multimeric with at least six antibody binding sites, binding may be enhanced by the formation of antigen-antibody aggregates through crosslinking. Alternatively, antigen binding might induce a contbrmational change in the Fc-part which enhances affinity for CIq. There is strong evidence for the importance of aggregation, but a mixed mechanism which involves aggregation and a conformational change cannot be ruled out. The studies described here were almost exclusively carried out with myeloma or Bence-Jones proteins because these were the only homogeneous immuno- globulins which could be obtained in sufficient quantity. However, in most cases the specificities of such molecules is unknown. Recently, large amounts of homogeneous antibodies elicited against strepto- coccal or pneumococcal polysaccharides became available from certain rabbit and mouse strains 4°,<. These sources, and the use of hybrids obtained from myeloma and spleen cells have made it possible to obtain homogeneous antibodies of defined speci- ficity ~2,43, Structural studies of" 'natural' antigen-anti- body complexes can be expected to lead to a more complete understanding of antibody function. Crystallographic work on a specific antibody and of its antigen is already in progress H. Acknowledgements We thank Prof. R. Huber for helpful discussions. References 1 Edehnan, G. M. 11970) Sci. Am. August, 81-87 2 Porter, R. R. (1976) Sci. Am. October, 81 87 3 Hilschmann, N. (1969) .Vhlurwi.~.~enschqfier~ 56, 195-205 4 Edmundson, A. B., Ely, K. R. and Abola, E. E. 11978) (]<ml. Top. Mol. bromine/. 7, 95-118 5 Amzel, L. M. and Po!iak , R..J. (1979) Ann. Rev. Bimhem. 48, 961-997 6 Porter, R. R. (1958).Valure(Lorulor*) 182, 670-67I 7 Epp, O., Colman, P. M., Fehlhammer, H., Bode, W., Schiflbr, M., Huber, R. and Palm, W. 11974) Eur..7. Biochem. 45, 513-524 8 Fehlhammer, I1., Schiller, M., Epp, O., Colman, P. M., Lattman, E. E., Schwager, P., Steigemann, W. and Schramm, H..J. 11975) lhopll),s. S/rue/. Me~haTdrm 1, 139-146 9 Epp, O., Latnnan, E. E., Schiffer, M., Hubcr, R. and Palm, W. 11975) Bio('hemi.rlr}, 14, 4943-4952 10 £dmundson, A. B., Ely, K. R., Abola, R. R., Schitler, M. and Paniagiatopoulos, N. (1975) Biochemist O, I4, 3953-3961 11 Lee, B. and Richards, F. M. (1970). 7. Mo/. Bml. 55,379-400 12 Colman, P. M., 1)eisenhofer, .J., Huber, R. and Palm, W. 11976) .7. Md. Bid. 100, 257-282 13 Marquart, M., IDeisenhofer, J., Huber, R. and Palm, W. (1980).}*. Mcd. Biol. 141,369-392 14 l)eisenhofer, J., Colman, P. M., El) p, O. and Huber, R. (1976) th2p/w-Se~,ler[~ Z. Physid. (.T/era. 357, 1421-1434 15 Deisenhofer, J. (1981 ) Biochemi.~l U 20, 2361 23711 16 Melchers, F. (1973) Biochemirt[}, 12, 1471-1476 17 Weitzman, S. and Scharft, M. 1). (1976).7. M,d. Bin/. 102, 237 252 18 Hickman, S., Kulczycki, A..Jr, Lynch, R. G. and Kornteld, S. (1977),7. Bid. (kern. 252, 4402~408
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`Fig. 7 Conformation of the hinge region as seen in IgGl Kol. Fig. 8 Space filling model of the FB (protein A) - Fc complex. 19 Matsushima, M., Marquart, M., Jones, T. A., Colman, P. M., Barrels, K., Huber, R. and Palm, W. (1978) J. Mol. Biol. 121, 441 459 20 Segal, D. M., Padlan, E. A., Cohcn, G. It., Rudikoff, S., Potter, M. and Davies, D. R. (1974) P~cJc..N}zll Acad. Sci. U.S.A. 71, 4298-4302 21 Abola, E. E., Ely, K. R. and Edmundson, A. B. (1980) Biochemislrj 19, 432-439 22 Po{jak, R. J., Amzel, L. M., Chen, B. L., Phiackerley, R. P. and Saul, F. (1974) Proc. Nail Acad. Sci. U.S.A. 71, 3440-3444 23 Saul, F., Amzel, L. M. and Poljak, R.J. (1978) J. Biol. {,'hem. 253,585-597 24 Wu, T. T. and Kabat, 1'2 A. (1970).7. Exp. Med. 132, 211-250 25 Amzel, L. M., Po!jak, R. J., Saul, F., Varga, J. M. and Richards, F. F. (1974) Proc. Nail Acad. Sci. U.S.A. 71, 1427-1430 26 Early, P., Huang, H., Davis, M., Calame, K. and Hood, L. (1980) Cell 19, 981-992 27 Schmidt, W., Jung, H. D., Palm, W. and Hilschmann, N. (1981) private communication 28 Cunningham, B. A., Rutishauser, U., Gall, W. E., Gottlieb,P. 1)., Waxdal, M. J. and Edelman, G. M. (1970) Biochemistry 9, 3161-3170 29 Sakano, H., Maki, R., Kurosawa, Y., Roeder, W. and Tonegawa, S. (1980) NaVarre (L~mdon) 286,676-683 30 Michaelson, T. E., Frangione, B. and Franklin, E. C. (1977) J. Biol. (,'hem. 252,883 889 31 Ely, K. R., Cohnan, P. M., Abola, E. E., Hess, A. C., Peabody, D. S., Parr, D. M., Conncll, G. E., Lauschinger, C. A. and Edmundson, A. B. (I 978) Biochemistry 17, 820-823 32 Silverton, E:W., Navia, M. A. and Davies, D. R. (1977) Pror. fll?~/l Acad. Sci. U.S.A. 74, 8140-5144 33 Mueller-Eberhard, H.J. (1975) Amz. Rev. Biockem. 44, 697-724 34.Porter, R. R. and Reid, K. B. M. (1979) Adv. Prof. (,'hem. 33, 1 71 35 Connell, G. E. and Porter, R. R. (1971) Biocaem. 7. 124, 53P 36 Yasmeen, D., Ellersnn, J. R., Dorrington, K.J. and Painter, R. H. (1976).7. [mmzmrd. 116, 518 526 37 Sjoedahl, J. (I977) Eur. J. Biochem. 78,471-490 38 Deisenhofer, J., Jones, T. A., Huber, R., Sjoedahl, J. and Sjoe- quist, J. (1978) Z" Physiol. C/~em. 359, 975-985 39 Metzger, H. (1978) (,'hnl. Top. Mol. Immurwl. 7, 119-148 40 Jaton, j.-c., Huser, If., Braun, D. G., Givol, D., Pecbt, J. and Schlessinger, J. C. (1975) Biochemi.~lry 14, 5312-5315 41 Braun, D. G. and Huser, H. (1977) in Progress in lmm~moLc~gy HI (Mandel, T. E., Cheers, C. H., Hosking, C. S., McKenzie,[. F. C. and Nossal, G. J. v., eds) pp. 255-264, Elsevier Nnrth- Holland, Amsterdam, New York, Oxford 42 Koehler, G. and Milstein, C. (1975) JVature (London) 256, 495-497 43 Melchers, F., Potter, B. M. and Bethesda, N. W. (eds) (1978) C~trr. Top. Microbiol. [mmlmol. 81 44 Colman, P. M., Gough, K. H., Lilley, G. G., Blagrove, R. J., Webster, R. G. and Laver, W. G. (1981)J. MoL Biol. 152, 609-614
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