Basic Antibody Structure

Chapter 4. Immunoglobulin Structureand Function12*- Light chains consist of 2domains (C and V).*- Heavy chains have 4-5domains (depending onthe class of antibody)*Heavy chain 446 aa1. Functional Regions2. Types of chains3. Constant &Variable regions4. Glycoprotein1- Each heavy and light chainis made up of a number ofdomains ( Ig folding or Igdomains).Light chain 214aa2- Each domain is about 110amino acids in length andcontains an intrachaindisulfide bond betweentwo cysteines about 60amino acids apart.Basic Antibody Structure-150,000 molecular weight Multiple myeloma cancerous plasma cells Monomer 150,0001234What is the difference?- Constant (C) andVariable (V) regions1

RECAP:Pepsin100,000 MW(Fab)231Papain2 (45,000)1 (50,000)2- The Fc region plays NO role in antigen binding.Mercaptoethanol2 (50,0000)2 (25,000) 2 H 2 L1- Papain breaks antigen molecules into 2 Fab fragmentsand an Fc fragment.- Pepsin breaks antibody molecules into an F(ab’)2fragment and a VERY SMALL pFc’ fragment.- Mercaptoethanol treatment results in 2 heavy and 2light chains- Complexes of antibodies cross-linked by antigen arecalled “immune complexes”.2 Fab Fc21. Constant region - aminoacid sequence in the Cterminal regions of the H andL chains is the same.1132. Variable region - aminoacid sequence in the Nterminal regions of the H andL chains is different. Thisregion provides antibodieswith unique specificity.3. Hyper-variable regions areregions within the variableregions (greaterspecificities).Summary Molecule consists of Constant and Variable regionsfor both Light and Heavy chains (CH, VH, CL, VL) Ig molecule made of domains Domains 110 aa Each antigen-binding site is made up of the Nterminal domain of the heavy and the light chains IgM and IgE possess 4 CH domains (CH1-CH4)while IgG, IgA and IgD have 3 CH domains (CH1CH3). Hinge region is missing. Hypervariable regions in the Variable regions ofboth H and L chains.Figure 3.3-Within the variabledomains are threeregions of extremevariability.Complementarity-Determining Regions, or CDRs.These are referred to asthe hypervariableregions.These regions of thevariable domainsactually contact theantigen.They therefore make upthe antigen-bindingsite.These regions are alsocalled thecomplementaritydetermining regions, orCDRs.Heavy ChainLight Chain2

H chain CDRs- A simulated antigenbinding site showinghow the CDRs formpoints of contact withthe antigen.RECAP:- Antibodies are comprised of repeating 110 aa units referred toas domains or Ig folds.- The C-terminal domains are constant from antibody toantibody (within a class).- The constant region domains are responsible for all functionsof antibody other than antigen binding (opsonization, ADCC,complement activation) Biological Function!L chain CDRs- The N-terminal domains are variable from antibody to antibodyand are referred to as “variable domains”.- The variable domains contain 3 hypervariable regions - theCDRs.- The CDRs of the V domains in both H and L chains make upthe antigen-binding site.Antibody-Mediated EffectorFunctions Binding to Antigen OPSONIZATION: FcR in Macrophages andneutrophils COMPLEMENT ACTIVATION: IgG and IgM ADCC – NK cells trough FcR CROSSING EPITHELIAL LAYERS – IgA (butalso IgM) CROSSING PLACENTA- IgGFcγ receptors enhance phagocytosis of foreign cells/particlescoated with IgGAntibody made in response to foreign cells (cells/viralparticles/bacteria etc) will bind to those cells.Macrophages (and neutrophils) possess receptors for the Fcregion of IgG.Binding of macrophage Fc receptors to antibody bound tocells/particles facilitates and increases phagocytosis ofcells/particles.ADCC - Antibody-dependent cellular cytotoxicity - mediated by IgGAntibody made in response toforeign cells (cells/viralparticles/bacteria etc) willbind to those cells.Cells of the innate immunesystem (neutrophils,eosinophils, macrophages,NK cells) possess receptorsfor the Fc region of IgG.These cells bind to antibodyon the surface of foreign cellsand release lytic compounds lysis.Monomer,Dimer,andPentamerKuby Figure 14-123

Structural Variants of the Basic Immunoglobulin MoleculeRelative abundance in normal serum:Different heavy chains can be usedThere are five major types of heavy chain -- five major classes(isotypes) of antibody- gamma -- IgG (in humans 4 subclasses: IgG1, IgG2, IgG3, IgG4)- mu -- IgM- alpha -- IgA (in humans, 2 subclasses: IgA1, IgA2)- delta -- IgD- epsilon -- IgEIgG8 - 16 mg/mlIgA1.4 - 4 mg/mlIgM0.5 - 2 mg/mlIgD0.003 - 0.04 mg/mlIgE17 - 450 ng/ml ( 0.0005 mg/ml)IgDThe function of antibody varies depending on which heavy chain Most abundant insecondary responses-Crosses placenta (FcRn)-Complement activation-Binds to FcR inphagocytesFigure 3.15aCrosses placentaComplement ActivatorFc bindingCrosses placentaComplement ActivatorFc bindingCrosses placentaComplement Activator- Best Complement activation- First Ab produced inneonate- First antibodyproduced after challenge- Mucosal transport(to some degree)- Monomer on B cells- J chain: polymeric4

12- Dimer in mucosal secretions- Mucosal transport- Monomer in circulation- J chain (polymeric) and Secretory componentsSecretory ComponentRole of IgE in allergic reactionsIgE antibodies mediate the immediatehypersensitivity (allergic) reactions thatare responsible for symptoms of hayfever, asthma, hives and anaphylacticshock.IgE binds to Fc receptors on themembranes of blood basophils andtissue mast cells.IgD- Role unknown- Present on thesurface ofMATUREB cells Marker!!Cross-linkage of receptor-bound IgEmolecules by antigen (allergen)induces degranulation of basophilsand mast cells.A variety of pharmacologically activemediators present in the granulesare released, giving rise to allergicmanifestationsSUMMARY- IgA and IgM are secreted across epithelial surfaces- IgG, IgD and IgE can be found only within thebody - in serum or lymph.- IgA and IgM are also found in serum and lymph BUTIN ADDITION can also be found in secretions such asmucous secretions, saliva and tears.- The IgA and IgM found in external secretions differsfrom that found in serum by the presence of anadditional component referred to as the "secretorycomponent".Antigenic Determinants onImmunoglobulins Abs are glycoproteins and themselves veryimmunogenic Epitopes on immunoglobulins are dividedinto:– ISOTYPIC– ALLOTYPIC– IDIOTYPIC- This component is acquired as the IgA or IgM istransported across the epithelial cell barrier.5

**Constant region determinants that define each antibodyclass and subclassThe function of antibody varies depending on which heavy chainis used.Allelic variation (Allotypes): IgG of a particular class may beslightly different between individuals (e.g. variation in theIgG amino acid sequence)Note: This type of variation has no effect on antibodyfunction.RECAP - Sequence variation in antibodies:1. Different light changes - no significantfunctional effect2. Different heavy chains - very significantfunctional effect - isotypic variation3. Allelic variation between individuals - no largefunctional effect - allotypic variationGenerated by variation in amino acid sequence in the VH andVL. Most exactly, in the CDRs in the V regionsVariation in the antigen binding site (Idiotypes)4. Variation in the antigen-binding site - idiotypicvariationRemember: Idiotype Ag binding siteB Cell Receptor (BCR):- Short cytoplasmic tail (328 aa) .signaling?- Signaling through ahomodimer, Ig-α and Ig-β- Ig molecule Ig-α/Ig-β isthe BCR- The homodimer moleculeis member of the Igsuperfamily groupIg Superfamily Divergence from a common gene ancestor codingfor 110 aa. A member MUST have a “typical” Ig domain orfold 110 aa with an intra chain disulfide bond50-70 aa apart. Most members do not bind Ag!! Then, they mustfacilitate interaction with surface proteins You must know members with roles in: a) immunefunction, b) Receptor/Signal transduction, and c)Adhesion6

Immune FunctionReceptorsNeonatalMonoclonal Antibodies Kohler & Milstein 1975 Fusion of normal, activated B cell andplasmacytoma (cancerous plasma cell) Hybrid: immortal, secrete Ab, hypoxanthineRESULTS:Plasmacytoma VS B cell Plasmacytoma:– Cancerous plasma cell (Immortal)– Does not secrete Abs– Lacks HGPRTSpleen B cellHybrid**Plasmacytoma Normal spleen B cell– Limited life span– Secretes Abs– Possess HGPRTDie in cultureImmortal, SecretesLacks HGPRTAb, Possesshypoxanthine (HGPRT)7

12Applications?345 DiagnosisResearchTreatmentAffinity VS AvidityAffinity (polyclonal Ab) high becauseof multiple epitopesAvidity (monoclonal Ab) low affinity buthigh avidity because of strong epitopeAb interactionIgG - Most abundant Ig of internal body fluids (serum, extracellular fluids) - combats microorganisms andtoxins within the body tissues.The EndIgA - Most abundant Ig in mucous secretions - protects external surfaces of the bodyIgM - The first class of antibody produced during an immune response. Present both in internal bodyfluids and in secretions.IgD - Functions not well defined. Found mostly on the B cell plasma membraneIgE - Increases during parasitic infections. Causes symptoms of allergy.Complementfixation byclassicalpathwayAbility to crossthe placentaBinds to mastcells andbasophilsBinds tomacrophagesand polymorphsIgGIgAIgMIgDIgE - -- -------- -- 8

Kuby Figure 14-12 ADCC - Antibody-dependent cellular cytotoxicity - mediated by IgG Antibody made in response to foreign cells (cells/viral particles/bacteria etc) will bind to those cells. Cells of the innate immune system (neutrophils, eosinophils, macrophages, NK cells) possess receptors for the Fc region of IgG. These cells bind to antibody