J. clin. Path., 28, Suppl. (Ass. Clin. Path.), 6, 1-7

Antibody structureSYDNEY COHEN

From the Department of Chemical Pathology, Guy's Hospital Medical School, London

A consideration of the overall properties of anti-bodies has for some time suggested that theirstructural features must be unique among proteinmolecules. In the first place, serum antibodies withina species share a variety of physical, chemical andantigenic properties indicating that all have acommon basic structure. However, the immuno-globulins of all animal species investigated occur inseveral forms, somewhat arbitrarily designated asclasses, subclasses, or types, and distinguished onthe basis of their chemical and biological properties.In addition, the great diversity of antigens whichevoke an immune response and the narrowly definedspecificity of the serum antibodies formed indicatethat each individual can synthesize a very largenumber of antibodies-probably more than 105-and there is conclusive evidence that these differfrom one another in their covalent structure. Thefundamental problem of antibody structure there-fore concerns the nature of the molecular modifica-tions which are superimposed upon a relativelyconstant basic configuration to generate a finitenumber of immunoglobulin classes and a very largeassortment of distinct combining specificities. Theextent to which this problem has been elucidated bystudies of protein structure is outlined in this paper.It has been reviewed by Leslie and Cohen (1973).There is an additional intriguing problem which

concerns the way that antibodies mediate complexbiological reactions including, for example, theexpression of acquired protective immunity orimmediate hypersensitivity. There are very fewinstances in which the interaction of antigen withantibody leads directly to biological effects. Suchprimary reactions are limited to the inactivation ofenzymes and toxins and the neutralization of someviruses and protozoa. In the great majority of casesthe biological expression of immune reactivityrequires interaction of antibody with either thecomplement system or with specific cell surfaces.Such reactions involve specific effector sites on theantibodies which are distributed unevenly among theclasses and subclasses of immunoglobulin. Thesesites are involved in complement- or cell-mediatedlysis, opsonization by macrophages and mast cell

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degranulation resulting in immediate hypersensiti-vity. None of these reactions is mediated by antibodyalone but all can be initiated by the combination ofantibody with antigen. How do antigens trigger theseevents and can their effects be explained in terms ofa structural modification of antibody molecules?

Structure of Immunoglobulins (Ig)

The four-chain structure of Ig, consisting of twoheavy and two light chains covalently linked byinterchain disulphide bonds (fig 1), was originally

U0 (LWL+ N

H7g~~~~~~~~~HFig 1 Diagrammatic representation of the IgGmolecule made up of two heavy and two light chains.Intrachain disulphide bonds enclosing loops of60-70amino acids occur along the length ofheavy and lightchains. Heavy lines denote the N-terminal variablestretches ofheavy (VH) and light (VL) chains. Thevertical arrow shows the site ofpapain digestion whichsplits the molecule within the hinge region of the heavychain to give Fab and Fc fragments.

proposed in 1962 by Porter for rabbit IgG, and isnow known to apply to antibodies of all vertebratespecies studied including primitive elasmobranchfish. The various classes and subclasses of Ig withina single species are differentiated on the basis oftheir heavy chains (table I). High molecular weightantibodies, such as serum IgM and seromucous

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Property Immunoglobulin

IgG IgA 1gM IgD IgE

StructuralMolecular weight 150 000 160 0001 900 000 170 000 185 000

(serum)370 000(secretory)

Heavy chainsClasses y a dSubclasses V,V2 Ys Y4 a, a, MiM,Percentage in serum 77, 11, 9, 3 90, 10

Carbohydrate (%) 2-9 7.5 118 113 12-1Light chains K, A K, A , A ,A K,AJ chains 0 + + 0 0

BiologicalAntibody activity + + + + +C fixation +V1 VY VY 0 +M, 0 0Macrophage attachment + Y1V Y + 0Placental transfer +Y Ya Y 0 0 0Seromucous secretion 0 +a, a2 0 0 +Tissue sensitizationHomologous spp 0 0 0 0 +Htterologous spp +Yi Y2 ys 0 0 0 02

Combine with Staph a p:otein +Y1 Yv V4 0 0 0 0

Table I Properties ofhuman immunoglobulins'Serum IgA is predominantly in the dimerized form in species other than human.'Human IgE does sensitize monkey but not guinea-pig tissues.

IgA, consist of polymers of the four-chain unitlinked by a J-chain, which is rich in half-cysteineresidues and common to both classes. Antibodies ofall classes have light chains of two types (K or A).

Certain features of this structure are of particularsignificance in regard to the generation of antibodyspecificity and the properties of immunoglobulineffector sites.

Ig chains contain an N-terminal variable region(V) comprising about 120 amino acid residues inwhich all heterogeneity within a subclass is localized.This was demonstrated first by Hilschmann andCraig in 1965 for human K-chains, but V-regions ofsimilar extent occur on all light and heavy chains.The remaining C-terminal portions (C) of Ig

chains within a given subclass have, by contrast, aninvariant structure apart from differences attribut-able to individual genotypes. The complete sequenceof a human yl-chain was elucidated by Edelman(1971) and considerable information is available forC-regions of other y-subclasses and for the ,u-, ot-,and E-chains. Available data reveal considerablesequence differences between heavy chain classes,eg, between yl- and g-chains while subclasses showfar greater homology.

All lg chains show a periodic arrangement ofintrachain disulphide bonds (fig 1) dividing lightchains into two domains (VL and CL), y and ot-chainsinto four domains (VH1, CHI, CH2, and CH3) andg- and E-chains into five domains. VL and VH showhomology with one another but not obviously with

C regions and are associated with antigen-bindingsites. The variable regions of light chains (VKcandVA)can be unambiguously differentiated, but variableregions of heavy chains (VH) appear common to allclasses. CH regions carry the biologically active siteswhich are variously distributed on the classes andsubclasses of Ig (table I) (reviewed by Spiegelberg,1974).The heavy chain region which contains the Cys

residues involved in interchain disulphide bondingshows considerable variation between classes andsubclasses and no homology with other sections ofthe heavy or light chain. This stretch of the y- ando-chain is rich in proline and is susceptible toproteolysis. This section, on the basis of electronmicrography and other physical measurements, hasbeen designated the hinge region (fig 1) but theextent to which the Fab arms can move relative toone another remains uncertain (Metzger, 1974).

Antibody-combining Sites

Physicochemical studies of antibody saturated withantigen have indicated that there are two discretecombining sites per 7S molecule and the subsequentlyestablished monovalency of Fab fragments andsymmetrical chain structure are consistent with this.The valency of pentameric IgM (HioLio) has provedvariable, but it now seems that IgM has 10 sitesand that binding of larger antigens may reduce theeffective valency to five sites per antibody molecule.

2 Sydney Cohen

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Antibody structure

Similarly, polymeric IgA appears to have fourcombining sites.The size of the antibody-combining sites, assessed

by determining the dimensions of antigenic deter-minants which give optimum binding, has beenestimated as 3.4 nm x 1-7 nm x 0.6 nm. Thisindicates that 10 to 20 amino acids comprise thecombining site and make direct contact with theantigenic determinant. Electron microscopy ofantibodies complexed with a divalent haptenindicates that the binding site is situated at theextreme ends of the Fab regions and has a maximumdepth of about 4.0 nm.

In general, antibodies react most strongly withthe antigen which elicited their synthesis and showgraded affinities for structurally related com-pounds. Stereo-complementarity of the antigenicdeterminant and the antibody-combining site isthought to provide the major contribution to thenon-covalent associations which determine speci-ficity. It is therefore surprising that high affinityantibodies may show strong cross reactions withhaptens of quite distinct structure. This suggests thatwhereas weak hydrophobic interactions betweenantigen and a closely fitting antibody site may bepredominant in low affinity antibodies, additionalstronger forces with less stereospecificity mayoperate in high affinity antibodies; their nature wouldpresumably vary with the chemical identity offunctional groups in the antigenic determinant.Another explanation of unexpected cross specificitiesis that the antibody cavity may contain more thanone binding site and that distinct small haptensmight adhere to separate sites on opposite walls ofthe cavity, and that steric hindrance accountsfor competitive inhibition of one hapten by theother.

Direct evidence that antibody specificity isdetermined by amino acid sequence arose from astudy in which complete reduction and unfoldingof Fab fragments produced concomitant loss ofantibody activity, while removal of the denaturantwas associated with significant recovery ofthe originalactivity. This indicates that the chain folding whichgenerates combining specificity is dependent on theprimary sequence of the Fab fragments. The pre-sence of variable sequences confined to the N-terminal portions of Fab fragments again suggestedthat primary structure generates combining speci-ficities. Analysis of the variable sequences in anti-bodies of defined specificity has been hampered bythe heterogeneity of most purified antibodies raisedagainst even the simplest hapten and also by therarity of myeloma proteins having specific bindingaffinities within the range for elicited antibodies.Comparative sequence studies on monoclonal

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proteins have revealed, within the V-regions of bothheavy and light chains, three short stretches withhigh sequence variability (Wu and Kabat, 1970).Hypervariable regions in both H and L chains arebrought into close proximity by chain folding andby the N-terminal intrachain disulphide bridge. Thelocation of combining sites has been sought byemploying hapten determinants attached to achemical group which will interact covalently withresidues in or near to the active site. This techniqueof affinity labelling has provided results consistentwith involvement of hypervariable regions from bothlight and heavy chains in forming the combiningcavity.

Conclusive evidence on the structure of the activesite is being obtained by x-ray crystallography ofpurified, homogeneous Fab fragments. The 0-28 nmresolution of such a fragment from a myelomaprotein has provided a three-dimensional model ofFab. This contains four globular subunits corre-sponding with the variable (VH and VL)and constant(CHI and CL) domains of Fab. The hypervariableregions of light and heavy chains occur in closeproximity at one end of the molecule and enclose acavity which probably represents the combiningsite (Poljak, Amzel, Avey, Chen, Phizackerley, andSaul, 1973). Further studies have revealed that ahapten having specific affinity for this Fab fragmentis indeed bound in the shallow groove betweenheavy and light chains and appears to be in contactwith segments of the Ig sequence which contain thehypervariable regions (Amzel, Poljak, Saul, Varga,and Richards, 1974).

lg Effector Sites

The IgG Fc fragment (molecular weight 50 000)prepared by papain hydrolysis retains many activesites of the original molecule, including the capacityto fix complement, attach to mast cells and macro-phages, combine with staphylococcal protein A,regulate IgG catabolic rate, and cross the placentalmembrane and gut wall of the newborn (table II).The sites responsible for these properties are there-fore contained within the two C-terminal intrachaindisulphide loops of the y-chain (CH2 and CH3, figs1 and 2). Smaller fragments of Fc, referred to asFc' or pFc', which comprise only the C-terminaldomain (CH3) can be prepared by hydrolysis withpepsin or papain (fig 2). These fragments carry someisotypic and allotypic determinants, combine withrheumatoid factors of certain specificities, and mayretain activity for macrophage and B cell attachment(table II). Evidence has been obtained for comple-ment-fixing activity in the CH2 domain.

Certain Ig effector sites appear to be localized in

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4Sydney Cohen

IPAPAN FII~~ ~ ~~~,I

223 3\ O4344161226 229 261 321 367 425

-Cz-s2;-z--:s;-;H 6 COOK

C2 ICR3

PEPSIN

Fig 2 Diagrammatic representation of the Fcfragment ofhuman IgG, containing the CH2 andCH5, domains (fig 1) and showing sites ofcleavage by papain andpepsin to yield Fc' andpEc' pieces containing the CH, domain.Residues are numberedfrom the N-terminus;numbers in the centre refer to cystine residues.

Fp C1

the (Fab')21 fragment, possibly within the CH1domain (fig 1). Guinea pig IgG12 fails to fix comple-ment in the presence of antigen or lyse antigen-coated cells in the presence ofcomplement. However,washed specific precipitates containing IgGi anti-bodies do fix complement by an alternate pathwaybeginning with C3, the third component of comple-ment. The sites which fix Clq3 for conventionalcomplement activation are present on the Fc

Activity Fc Fc'

(CH2, CH3) (CHi3) (CH2)

Binding of:Macrophages + +,-B-lymphocytes + +K-cells + -Complement + +Rheumatoid factor +Staph a protein +

Membrane transmission +Catabolism control +

Table II Biological activities ofFc fragment

fragment of IgG2 but absent from IgGi, whereasthose which initiate the C3 shunt mechanism are

present on (Fab')2 fragments from guinea pig IgGiand IgG2 and also on human IgA. Localization ofbiologically active sites in other classes of Ig hasnot been thoroughly studied. However, the Fcfragment of IgE contains the sites responsible forthe attachment to isologous tissues while its C-

'(Fab') is a fragment produced by pepsin which splits the moleculeon the C-terminal side of the interheavy chain disulphide bridge (seefig 1). When the latter is also split two Fab' fragments result.'The subclass of IgG containing y, heavy chains (see table I).'One of the three subunits of the first component of complement, Cl.

terminal Fc' fragment is inactive in this regard. TheFc fragment of human IgA has not been isolated,but mouse and rabbit IgA Fc can be prepared and,from the latter species, binds the secretory compo-nent characteristically associated with seromucousIgA.The failure to isolate active fragments from the

Fc portions of Ig molecules has meant that thestructure of the effector sites remains obscure. TheIgG sites which react with Clq and bind to macro-phages and the IgE sites which attach to mast cellsare all inactivated by mild reduction suggesting thatintegrity of interheavy chain disulphide bonds in thehinge region is essential.

Available evidence suggests that where the samebiological activity is mediated by different Ig classes,the sites involved are structurally distinct. Forexample, mouse 7S and 19S antibodies both bindto macrophages but not competitively, and only the19S binding is dependent on the presence of Ca++ions. In other instances, different Ig classes may bindthe same receptor, but with different affinities,indicating that their reactive sites are structurallydistinct. For example, the affinity of Clq is greatestfor IgG3 and progressively less for IgG, and IgG2.

Triggering of Ig-mediated Biological Reactions

Certain biological activities are mediated by singleIg molecules, eg, the control of lg catabolic rate,transfer across placental and gut membranes, andcombination with staphylococcal protein A (fig3). However, other reactions, including immediatehypersensitivity, complement activation and opso-nization by macrophages are not initiated by

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Antibody structure

Placental transferA MM-

Gastro-intestinal transferA 1 Control catabolic rate

Combination staph aureus protein A

B w* IgE miiidiated hypersensitivity

c Complement activationC 7 ?Macrophage endocytosis

Fig 3 Ig-mediated effector mechanisms. A Sites withbiological activity on single antibody molecules. B andC High- and low-affinity sites on free antibodies whichcan combine with acceptor sites but require crosslinkage to an adjacent antibody molecule to becomebiologically active.

individual Ig molecules, but only by more than oneantibody linked by polyvalent antigen (fig 3,B and C). These reactions therefore require anantigen-dependent interaction between Fab and Fcsections of Ig molecules (reviewed by Metzger, 1974).

Antigen as a Trigger for Antibody-mediatedReactions

IgM and IgG antibodies in free solution combinereversibly with the first component of complement(Cl), but this does not initiate the subsequent stepsof complement activation. In the case of antigenattached to cell surfaces, the interaction with Clby two adjacent IgG molecules fixed to the cellantigens is sufficient to initiate the full cycle oflysis. Antigen density on the cell surface is thereforea critical factor in determining the occurrence ofcomplement-mediated cytotoxic reactions (fig 4).Thus, the non-lytic nature of anti-Rh antibodies isattributed to the comparative paucity of D antigensites on the erythrocyte surface. Similarly, theadsorption of host components onto cell surfacesmay alter the antigen density of established tumoursand parasites and permit evasion of complement-mediated cytolysis, which nevertheless remainseffective against homologous cells newly introduced

Fig 4A Wide separation of the antigenic determinantsand the reactive IgG molecules on a cell surface withconsequent failure to initiate complement activation.Fig 4B Cross-linked IgG molecules on a cell surfaceinitiate complement-mediated lysis.

into the host-a phenomenon referred to as concomi-tant immunity.Mast cells have homocytotrophic antibodies (IgE)

bound to their surface membranes (fig 5A) but aretriggered for histamine release only after the boundmolecules have become cross linked, usually bypolyvalent antigen (fig SB). By contrast, the majorityofmonovalent haptens are totally ineffective (fig SD).The requirement for cross linking implies that thedensity of cell-bound IgE of given specificity is acritical factor determining antigen-induced hyper-

Fig 5 Diagram illustrating the requirements for IgE-mediated hypersensitivity reactions (see text).

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Sydney Cohen

sensitivity (fig 5C). The nature of the cross linkagebetween IgE molecules appears unimportant sincemultivalent antigen and divalent anti-IgE antibodiesare both potent initiators, whereas monovalentantigens or Fab fragments from anti-IgE antibodiesare ineffective. Similarly, chemically aggregated IgEdirectly induces immediate hypersensitivity reactionsin human subjects and guinea pigs, though thedosage required is greater than that needed with theother cross-linking mechanisms.

Studies of cytophilic antibody have shown thatnative Ig molecules of appropriate class bind to thesurface of macrophages; in addition, the attachmentof antigen-antibody complexes to macrophages canbe inhibited by excess free IgG. These observationsindicate that cytophilic sites, at least on IgG, areavailable on the surface of native molecules. Theamount of antibody bound to macrophages and theaffinity of binding are increased by cross linking withpolyvalent antigen at equivalence, but are unaffectedby monovalent or excess polyvalent antigen. Particlessuch as red blood cells, after reaction with cytophilicantibody, become attached to macrophages andremain bound to the surface if left at room tempera-ture or 4°C. Incubation of such preparations at 370causes phagocytosis of all attached red cells. Phago-cytosis occurs without complement when the opso-nizing antibody is IgG but complement componentsare required for ingestion of membrane-boundparticles coated with IgM. Whether cross linking ofIgG by antigen is a required trigger for phagocytosisis unknown, since the influence of antigenic valencyupon endocytosis has not been evaluated.

There appear to be two ways in which cross linkingof antibody could initiate biological reactions whichinvolve sites on Fc sections of Ig molecules.

IG CONFORMATIONAL CHANGECombination with antigen could expose or generatebiologically active sites whose affinity is not fullyexpressed in the native molecule; combination withappropriate receptors would then trigger thebiological event. This sequence would seem likelyif native Ig did not bind receptors and did not inhibitbinding by antigen-antibody complexes. In fact, asoutlined above, the sites for Cl fixation, mast cellattachment and macrophage binding are all activein free Ig of the appropriate class; it remainspossible that the binding affinity of Ig sites isincreased or new sites are exposed by an antigen-dependent conformational change and that thistriggers the receptor to initiate the biologicalsequence. However, physico-chemical studies ofimmunoglobulins in solution provide little evidencefor antigen-induced conformational changes (Metz-ger, 1974; Leslie and Cohen, 1973).

LATTICE FORMATIONThe reactivity of effector sites on native Ig moleculesand the failure to demonstrate convincingly antigen-mediated conformational changes in antibodymolecules leave open the possibility that biologicaleffects are induced by lattice formation which followsthe interaction of Ig with multivalent antigen. Thiscould occur where the Fc regions of free antibodieshave low affinity binding sites, eg, for Clq or formacrophage receptors, so that antigen-antibodycomplexes have increased functional affinity byvirtue of the number of sites in close proximity. In amulticomponent system (such as the complementsystem) the increased functional affinity of thecomplex could cause retention of the first componentfor sufficient time to allow for activation and inter-action with further components, thereby initiatingthe full sequence of the response. A similar mechan-ism seems possible for antibody-induced phagocyto-sis by macrophages.The influence of multivalency upon overall

affinity is illustrated by the fact that free IgG and the7S subunit of IgM bind similar amounts of Clqwhile pentameric IgM, which presumably containsfive complement-fixation sites per molecule, is 15times more active. Larger aggregates of IgG andIgM bind up to 100 times more Clq on a weightbasis. These data correlate with the observation thattwo or more IgG molecules in close proximity onthe cell surface are required to produce a lyticlesion whereas a single IgM molecule is sufficient toproduce the same effect.The mechanism of mast cell degranulation medi-

ated by IgE is of interest since the native antibody hasa high affinity for cell surface receptors and yetstimulates histamine release only after cross-linking.The trigger may involve conformational changeswithin the antibody which generate either additionalbinding activity or conformational changes withinreceptors at the cell surface. However, it is simpler topropose that lattice formation between bound IgEmolecules leads to aggregation of cell surfacereceptors and this provides the trigger for mast celldegranulation (fig 5).

Conclusions

Antibody molecules show a high degree of individualantigen-binding specificity but may share certainbiological properties, such as the capacity to fixcomplement, which are characteristic of the class ofIg to which they belong. This functional dualism isgenerated by peptide chains which are unique inhaving variable N-terminal (V) regions and constantC-terminal (C) regions.The two antigen-binding sites of each four-chain

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Antibody structure 7

Ig molecule are contained within the V-regions ofboth heavy and light chains. Specificity is determinedby primary structure. X-ray diffraction studies showthat combining sites are generated by the shorthypervariable stretches in heavy and light chains.The stereochemical configuration of the antibodysite is regarded as the prime determinant of specifi-city.

Biological effector sites occur predominantly inthe Fc portion of the molecule which comprises thetwo C-terminal intrachain disulphide loops of theheavy chains. Little information is available aboutthe location, chemical structure, specificity, oraffinity of these sites. All appear accessible in nativeIg and some, including those responsible for placen-tal and gastrointestinal transfer of Ig molecules andfor the concentration-dependent control of lgGcatabolic rate, are active in free molecules. Othereffector sites are present on free Ig molecules andinteract with their relevant receptors, but the fullbiological sequence is initiated only after the boundantibody has become cross linked, usually by poly-valent antigen. This is true for complement activa-tion, IgE-mediated hypersensitivity and possibly alsofor opsonin-induced phagocytosis.The cross linkage of Ig leads to lattice formation

and this may trigger the biological sequence byproducing aggregation or conformational change inthe receptor. An Ig lattice signal is consistent with

the known presence of effector sites on native Igmolecules, the failure of monovalent or excess poly-valent antigen to induce reactions, and the trig-gering which occurs with diverse forms of crosslinking. Ig conformational changes may have a rolein initiating biological reactions, but their occurrencedoes not seem mandatory.

References

Amzel, L. M., Poljak, R. J., Saul, F., Varga, J. M., and Richards, F. F.(1974). The three-dimensional structure of a combining region-ligand complex of immunoglobulins NEW at 3-5 A resolution.Proc. nat. Acad. Sci. (Wash.), 71, 1427.

Edelman, G. M. (1971). Antibody structure and molecular immuno-logy. Ann. N. Y. Acad. Sci., 190, 5.

Hilschmann, N., and Craig, L. C. (1965). Amino acid sequencestudies with Bence Jones proteins. Proc. nat. Acad. Sci. (Wash.),53, 1403.

Leslie, R. G. Q., and Cohen, S. (1973). The active sites of immuno-globulin molecules. In Essays in Fundamental Immunology, I,edited by I. Roitt, p. 1. Blackwell, Oxford.

Metzger, H. (1974). Effect of antigen binding in the properties ofantibody. Advanc. Immunol., 18, 169.

Poljak, R. J., Amzel, L. M., Avey, H. P., Chen, B. L., Phizackerley,R. P., and Saul, F. (1973). Three-dimensional structure of theFab fragment of a human immunoglobulin at 2-8 A resolution.Proc. nat. Acad. Sci. (Wash.), 70, 3305.

Porter, R. R. (1962). In Basic Problems of Neoplastic Disease, editedby A. Gellhorn and E. Hirschberg, p. 177. Columbia UniversityPress, New York.

Spiegelberg, H. L. (1974). Biological activities of immunoglobulins ofdifferent classes and subclasses. Advanc. Imnmunol., 19, 259.

Wu, T. T., and Kabat, E. A. (1970). An analysis of the sequences ofthe variable regions of Bence Jones proteins and myelomalight chains and their implications for antibody complemen-tarity. J exp. Med., 132, 211.

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