Antibody Struc | Immunoglobulin G | Antibody

Antibody structure

Overview
1. Antibodies belong to a class of proteins called immunoglobulins 2. Antibody molecules belong to one of five classes i.e. IgG, IgM, IgA, IgD & IgE 3. Immunoglobulins are “Y” shaped proteins. The “arms” of the “Y” bind antigens. The tail of the “Y” is responsible for biological activity eg. C’ activity or binding to cells 4. Ability of immunoglobulins to bind antigen determ. by AA sequence in variable region

General Characteristics of antibody
Antigen-specific products of B cells First of the molecules participating in immune response to be characterized, best understood Basic building block, immunoglobulin domain, is used in molecules of both immune system & other biological recognition systems Antibody molecule has 2 separable functions: 1) specific binding to antigen eliciting response 2) biological activity - recruit cells & components designed to destroy agent to which ab developed

Serum and plasma
Serum

Blood Plasma Clot

Blood + anticoagulant

Cells

Chromatography Molecular sieving Ion exhange Affinity Isolation & characterization Salting out/ Dehydration Ammonium sulfate Alcohol precipitation Ultracentrifugation Immunochemical .

Chromatography Molecular sieving - + + + + + + + Ion exchange Affinity .

Salting out Ammonium sulfate (half saturated) Ethanol (90%) .

Density gradient ultracentrifugation Displacement = Svedberg 60 Ce .0 on 0 X G .0 ntr 0 0 if .1 ug 00 ati .

Kabat & Tiselius (1939) + Albumin α β Globulins -Discovered that hyperimmunizing rabbits resulted in increased γ -Purification revealed antibody activity resided in this serum portion γ .

Electrophoretic analysis of serum Sample application Separation by charge + Cathode + - Anode + Albumin α β Globulins γ .

Protein concentration Characterizing chains Albumin Broad peak of γ α 1α 2 β γ = heterogenous proteins + normal - Need pure Ig to chemically analyze Narrow peak of γ = homogenous proteins Protein concentration + multiple myeloma - Bence-Jones proteins in urine of multiple myeloma pts. κ or λ . = dimers of immunoglobulin light chains.

papain. identical to each other . univalent (can’t ppt) one binding site each.Porter (England) Treatment with proteolytic enzyme.crystallizable) Fab SS SS Fc SS Fc crystallizable (thus homogenous) can’t bind ag responsible for biological activity of molecule after ag bound to Fab portions Fab Fab specifically bind antigen. resulted in three approximately equal sized fragments: 2 capable of ag rx (fragment antigen binding) 1 could be crystallized (fragment.

000 daltons All immunoglobulins basic unit consisting of 4 polypeptides i.e.Eddleman (USA) Treatment with mercaptoethanol = 4 chains: (mercaptoethanol breaks S-S bonds) SH HS SH HS SH HS 2 chains = 53. 2 L . 2 H.000 daltons 2 chains = 22.

& C-terminal Fab Papain digestion results in cleavage @ “N” terminus in proline hinge region @ disulfide bridge N-terminus Fab SS SS SS Fc Fab2 Pepsin digestion results in cleavage @ “C” terminal portion. resulting in a divalent fragment (Fab2) joins by S-S & several Fc fragments SS SS SS C-terminus .Proteolytic enzyme digestion reveals N.

Cleavage of Ig SH HS w ced u Red ME SH HS SH HS 4 polypeptide chains Fab Fab SS SS SS Papain 2 Fragment ag binding 1 Fragment crystallizable Pe ps in 1 Fab2 SS SS SS Several small pieces Fc .

immune Myeloid myeloma .Myeloma cells Plasma cell becomes tumorous = myeloma produces homogenous (monoclonal) Ig Myeloma Ig = myeloma “proteins” Myeloma proteins may be purified & structurally analyzed AA sequence in L & H chains Normal. non immune Normal.

domain SS C terminus C terminus Variable (VL) Constant (CL) . domain Identical sequences in C-termin.Light chain sequences N-terminus Fab N-terminus SS SS Fab 214 AA in two domains Analysis from several myelomas Different sequences in N-termin.

Heavy-chain sequence N-terminal domain varies (HV) Other 3 domains constant (HC) Flexible Hinge Region N-terminus SS SS Heavy chain consists of 445 AA in 4 domains Fab Fab SS C terminus .

34 CDR 2 = 50 .56 CDR 3 = 89 -97 Framework regions = regions where AA seq.determining regions (CDR) CDR = 6-10 AA CDR 1 = 24 .HV3) Location of antigen binding site AA sequence det.% var Sequence variability not distributed evenly in variable region CDR 1 CDR 2 CDR 3 Variation restricted to 3 regions Hypervariable regions (HV1. shape of ag binding site (paratope) Determine epitopes to which Ig binds = complementary.HV2. is relatively constant (FR) Heavy chain CDR CDR 1 = 31-35 CDR 2 = 50-65 CDR 3 = 95-102 % var Wu & Kabat plot .

linked by disulfide bonds CDR Disulfide bonds CDR 1 Each layer formed several stretches 3 conformation = β strand Layers = β sheet Order of β strands is characteristic for each sheet 3 D structure = Ig fold CDR 2 C terminus Β strands Β strands .Structure of variable & constant domains (X-ray crystallography) Light chain C domain Light chain V domain N terminus Two layers.

.Opening to reveal β strands comprising each β sheet Principal difference between C & V domains V domain has 2 more β strains forming extra loop unique strands C domain V domain Order & orientation characteristic for each domain β strands lettered sequentially according to occurrence in AA sequence in dom.

Location of CDR & FR in Ig L & H chains CDR 1 FR1 FR2 2 FR3 3 FR4 CL FR1 1 FR2 2 FR3 3 FR4 CH1 H CDR .

L & H chain folding to yield 3 CDR in each chain to form walls of ag binding groove .

500 Light chain Heavy chain .Immunoglobulin consists of 4 polypeptide chains N terminus Ag binding site Variable regions VH CH1 Ag binding site VL CL Constant region Disulfide CH2 bonds CH3 C terminus EM Rabbit Ig X 2.042.

g.λ individual of species produces both types. both L chains = either κ or λ . mouse 95%κ .Immunologic analysis of immunoglobulins Ig (like most other proteins) stimulate ab in other animal species All species have two major classes of L chains i. human 60% κ ) in any Ig molecule. CHO content. κ . size & biological function . never one of each Ig of all species consist of 5 classes (isotypes) differ in structure of H chains H chains among isotypes differ serologically.e. ratio of κ : λ varies by species (e.

enzyme-.g.) .e. 2ε .H chains H chain confers unique biologic properties of molecule e. but ab molecule H chains are identical ( i. receptor binding .. C’ activation with ag Immunoglobulin isotype IgM IgG IgA IgD IgE Heavy chain µ γ α δ ε Individual of species produces all H chains. 1/2 life. 2δ etc. in proportion characteristic for species.

but not κ 1λ 1γ 1α 1 IgE isotype = κ 2ε 2 or λ 2ε 2 ….. .Isotypic (Class) structure of Ig L H L H L H L H IgG isotype = κ 2γ 2 or λ 2γ 2..etc….

in proportion characteristic for species. 2δ etc. but ab molecule H chains are identical ( i. 1/2 life. 2ε . enzyme-..H chains H chain confers unique biologic properties of molecule e. C’ activation with ag Immunoglobulin isotype IgM IgG IgA IgD IgE Heavy chain µ γ α δ ε Individual of species produces all H chains.g.) .e. receptor binding .

042.Immunoglobulin consists of 4 polypeptide chains N terminus Ag binding site Variable regions VH CH1 Ag binding site VL CL Constant region Disulfide CH2 bonds CH3 C terminus EM Rabbit Ig X 2.500 Light chain Heavy chain .

etc…... .Isotypic (Class) structure of Ig L H L H L H L H IgG isotype = κ 2γ 2 or λ 2γ 2. but not κ 1λ 1γ 1α 1 IgE isotype = κ 2ε 2 or λ 2ε 2 ….

HingeAA between C 1 & C 2 region Consists of approx 12 H H No homology between hinge & other Heavy chain domains Angle = 60 0 AA sequence unique for each class & subclass Angle = 900 Angle = 0o .

AA sequence in hinge region Hinge region Light Chain Cys -Arg-Val-Glu-Pro-Lys-Ser-Cys-Asp-Lys-Thr-His-Thr-Cys-Pro-Pro-Cys -Pro-Ala-Pro-Glu-Arg-Val-Glu-Pro-Lys-Ser-Cys-Asp-Lys-Thr-His-Thr-Cys-Pro-Pro-Cys -Pro-Ala-Pro-GluCys Light Chain Papain Fab region Fc region .

allowing flexibility between two Fab arms of the Y-shaped antibody. papain) to generate the Fab & Fc fragments . allows open & close to accommodate binding to two epitopes.Characteristics of hinge region Immunoglobulins (with possible exception of IgM & IgE) contain hinge between CH1 & CH2 No homology between AA sequence of hinge & heavy chains AA sequence differs with different classes Comprised of many cysteine and proline residues Cysteine involved in formation of interchain disulfide bonds Proline prevents folding in a globular structure. because it is open. it can be cleaved by proteases (e.g.

γ 2. highest # S-S IgG4 = monoval. 150 kDa Small size ppt in surfaces (e. MW approx..g. autoab to clot fac Autoab to DNA = IgG1. no aggl ppt rx. & ppt ag Only activates classical C’ p’way Subclasses show close overall relation Heavy chains = γ 1. cross placental barrier) Opsinize. aggl. highest catabolic rate.IgG3 IgG3 IgG4 .IgG Highest concentration in serum Plays major role in immune dfns.γ IgG1 IgG2 All normal indiv. have all IgG1>IgG2>IgG3>IgG4 IgG3 has shortest 1/2 life.γ 3.

Two L chains (either κ or λ but not both) Each H chain = 50 kD Each L chain = 25 kD 150 kD.Structural features of IgG Two γ H chains. “γ globulin” HV Variable Least anodic of all serum proteins Constant + Anode Cathode . 7S.

19S SS SS J chain S S S SS SS SS Doesn’t have hinge region has additional H domain Has a J chain (one of 2 Ig isotypes) 15 kD S SS . Macroglobulin (M) SS 900 kD.Structural features of IgM SS SS SS SS SS SS Pentamer (5) First Ig produced following immun.

saliva. 7S. mucus. bind to basal membranes of epithelia via secretory piece In serum.Structural features of IgA Secretory piece SS SS J chain Principal Ig in external secretions e.g. no secretory piece Secretory component protects IgA from proteolytic digestion SS SS SS SS .sweat. gastric fluid & tears Major Ig of colostrum & milk. provides neonate with major source of intestinal protection against pathogens 165 kD. primarily a monomer. migrates as fast γ Plasma cell forms basic IgA molecule with J chain which form dimers (second Ig to contain J chain) When released from plasma cell.

migrates as fast γ No interchain S-S bridges in H chains Readily denatured .Structural features of IgD Primarily a B cell antigen receptor Long exposed hinge region 170kD. 7S.

cross links IgE on mast cell surface. migrates as fast γ Contains an extra domain (CH4) which binds to mast cells & basophils May remain attached for long time when ag reappears.Structural features of IgE Sometimes called reaginic ag Mediates allergies (Type I hypersensitivies) 190 kDa. release mast-cell granules & signs of anaphylaxis . 8S.

involve changes of 1-3 AA in constant region. known allotypes IgG: Gm (Ig Markers) Km (markers on κ chain Idiotype = one of several thousand Ig. depends on existence of allelic forms of the Ig . all people have Allotype = genetic difference.Immunoglobulin variations Isotype = class of Ig. result of different forms of the same gene. each of which directed toward specific epitope .

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