ImmunoglobulinsImmunoglobulins

StructureStructure

DefinitionsDefinitionsImmunoglobulin is a generic term that refers to a Immunoglobulin is a generic term that refers to a diverse group of molecules found in the blood and diverse group of molecules found in the blood and tissue fluidstissue fluids

They are soluble globulin molecules and they They are soluble globulin molecules and they generally migrate in an electrophoretic field at or near generally migrate in an electrophoretic field at or near the gamma globulin fractionthe gamma globulin fraction

An antibody is an immunoglobulin molecule capable of An antibody is an immunoglobulin molecule capable of binding specifically with a known substance (antigen)binding specifically with a known substance (antigen)

Immunoglobulins are synthesized by B lymphocytes Immunoglobulins are synthesized by B lymphocytes and terminally differentiated B lymphocytes or plasma and terminally differentiated B lymphocytes or plasma cellscells

Introduction Introduction The first to show that antibodies reside in the gamma The first to show that antibodies reside in the gamma globulin fraction were Kabat and Tiselius in 1939 when globulin fraction were Kabat and Tiselius in 1939 when they performed serum electrophoresis and they called they performed serum electrophoresis and they called them immunoglobulinsthem immunoglobulins

From the broad electrophoretic peak, it is clear that a From the broad electrophoretic peak, it is clear that a heterogeneous collection of immunoglobulin heterogeneous collection of immunoglobulin molecules with slightly different charges is presentmolecules with slightly different charges is present

This heterogeneity was one of the early obstacles in This heterogeneity was one of the early obstacles in attempts to determine the structure of antibodies, attempts to determine the structure of antibodies, since analytical chemistry requires homogeneous since analytical chemistry requires homogeneous crystalizable compounds as starting proteinscrystalizable compounds as starting proteins

Certain diseases (multiple myeloma and light chain Certain diseases (multiple myeloma and light chain disease) provided the solutiondisease) provided the solution

Recently the hybridoma technology has made Recently the hybridoma technology has made available antibodies in pure form available antibodies in pure form

Porter in 1959 treated antibody with papain and Porter in 1959 treated antibody with papain and produced three fragments of equal sizeproduced three fragments of equal size

Two of the fragments were found to retain the Two of the fragments were found to retain the antibody’s ability to bind antigen specifically but could antibody’s ability to bind antigen specifically but could no longer precipitate the antigen from solutionno longer precipitate the antigen from solution

These two fragments were referred to as FabThese two fragments were referred to as Fab

The third fragment could be crystallized out of solution The third fragment could be crystallized out of solution and called FCand called FC

ENZYME DIGESTION OF IMMUNOGLOBULINS

Fragment “antigen-binding”

Fragment crystallisable

Papain

Pepsin

At about the same time, Edelman discovered At about the same time, Edelman discovered that when the immunoglobulin molecule was that when the immunoglobulin molecule was extensively reduced of by mercaptoethanol, it extensively reduced of by mercaptoethanol, it fell apart into four chains, two identical heavy fell apart into four chains, two identical heavy chains (53000 Daltons) and two identical light chains (53000 Daltons) and two identical light chains (22000 Daltons)chains (22000 Daltons)

Noble prize was awarded for Porter and Noble prize was awarded for Porter and Edelman for revealing antibody structure Edelman for revealing antibody structure

Antibody structure

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LIGHT HEAVY H+L Antibody

dimer

StructureStructureAn immunoglobulin is composed of four polypeptide An immunoglobulin is composed of four polypeptide (glycoprotein) chains; two light and two heavy chains (glycoprotein) chains; two light and two heavy chains linked by disulfide bondslinked by disulfide bonds

Five different types of heavy chains; Five different types of heavy chains; αα, , , µ, , µ, δδ, and , and εε..

Two types of light chains; kappa (k) and lambda(Two types of light chains; kappa (k) and lambda(λλ) ) with a ratio of 3:2with a ratio of 3:2

An individual B cell or immunoglobulin monomer An individual B cell or immunoglobulin monomer expresses either k or expresses either k or λλ but not both but not both

Within an immunoglobulin molecule, the two heavy Within an immunoglobulin molecule, the two heavy chains and light chains are identical chains and light chains are identical

Schematic diagram of structure of Ig

Some immunoglobulin molecules are basic Some immunoglobulin molecules are basic monomers and others are composed of multiple monomers and others are composed of multiple copies (dimers or pentamers) of identical copies (dimers or pentamers) of identical immunoglobulin monomersimmunoglobulin monomers

Both heavy and light chains can be divided into Both heavy and light chains can be divided into regions or domains, homologous portions of an regions or domains, homologous portions of an immunoglobulin chain, each composed of immunoglobulin chain, each composed of approximately 110 amino acids and contains an approximately 110 amino acids and contains an intradomain disulfide bridgeintradomain disulfide bridge

(a) Heavy and light chains arefolded into domains, each containingabout 110 a.a. & an intrachain disulfide bond that forms a loop of 60 amino acids.

(b) The μ and Є heavy chainscontain an additional domain that replaces the hinge region

Light chains contain two regions, a variable (VLight chains contain two regions, a variable (VLL) and a ) and a constant ( Cconstant ( CLL) domain) domain

Heavy chains contain a single variable (VHeavy chains contain a single variable (VHH) and ) and multiple constant domains(Cmultiple constant domains(CHH1, C1, CHH2, C2, CHH3, C3, CHH4)4)

Variable regions in both heavy and light chains are so Variable regions in both heavy and light chains are so named because of the extensive variation in the amino named because of the extensive variation in the amino acid sequence found in immunoglobulin molecules acid sequence found in immunoglobulin molecules made by different cellsmade by different cells

The amino acid sequence determines the The amino acid sequence determines the conformational structure of Vconformational structure of VHH and V and VLL

The combination of a light variable and a heavy The combination of a light variable and a heavy variable region forms a pocket that constitute the variable region forms a pocket that constitute the antigen-binding region of the immunoglobulin moleculeantigen-binding region of the immunoglobulin molecule

Hinge RegionHinge RegionMade up predominantly of cysteine and proline Made up predominantly of cysteine and proline residuesresidues

It permits flexibility between the two Fab arms of It permits flexibility between the two Fab arms of the Y shaped moleculethe Y shaped molecule

It allows Fab to open and close to accommodate It allows Fab to open and close to accommodate binding to two epitopes separated by a fixed binding to two epitopes separated by a fixed distancedistance

Variable DomainsVariable DomainsThree areas of hypervariability occur between Three areas of hypervariability occur between less variable stretches called less variable stretches called Framework Framework regionsregions

Because they bind specifically to epitopes, the Because they bind specifically to epitopes, the hypervariable regions are termed hypervariable regions are termed complementarity -determining regionscomplementarity -determining regions (CDRs) (CDRs) of the L and H chains; CDR1, CDR2, and of the L and H chains; CDR1, CDR2, and CDR3CDR3

V D JCDR1 CDR2 CDR3

V JCDR1 CDR2 CDR3

Immunoglobulin FragmentsImmunoglobulin FragmentsFabFab: produced by papain cleavage (2 Fab): produced by papain cleavage (2 Fab)

FCFC: produced by papain cleavage: produced by papain cleavage

FdFd: it is the heavy chain portion of an Fab fragment : it is the heavy chain portion of an Fab fragment cleaved by papaincleaved by papain

F(ab)2F(ab)2: it is the dimeric molecule produced by pepsin : it is the dimeric molecule produced by pepsin cleavage which fragments the FCcleavage which fragments the FC

FdFd── : it is the heavy chain portion of an Fab fragment : it is the heavy chain portion of an Fab fragment cleaved by pepsin cleaved by pepsin

Immunoglobulin ClassesImmunoglobulin ClassesFive different classes or isotypes depending Five different classes or isotypes depending on heavy chain antigenicityon heavy chain antigenicity

Monomers, dimers or pentamersMonomers, dimers or pentamers

Monomers are divalent having two identical Monomers are divalent having two identical antigen- binding sitesantigen- binding sites

General structures of the five major classes of antibody

IgMIgMFirst to be produced in an immune response. M for First to be produced in an immune response. M for macroglobulin, M. Wt. about 10macroglobulin, M. Wt. about 106 6 Daltons, Daltons, sedimentation coefficient 19S, Has an extra Csedimentation coefficient 19S, Has an extra CHH domaindomain

Cell surface bound monomer or secreted pentamer. Cell surface bound monomer or secreted pentamer. Five basic units (pentamer) joined by a J chainFive basic units (pentamer) joined by a J chain

(150.000 Daltons) synthesized by B cells or plasma (150.000 Daltons) synthesized by B cells or plasma cellscells

Five antigen-binding sites instead of the expected Five antigen-binding sites instead of the expected valence of 10 due to conformational constraints valence of 10 due to conformational constraints imposed by polymerizationimposed by polymerization

6-8% of serum immunoglobulins 6-8% of serum immunoglobulins

IgGIgGMonomer, M. Wt 150.000, sedimentation Monomer, M. Wt 150.000, sedimentation coefficient 7S. Least anodic of all serum coefficient 7S. Least anodic of all serum proteinsproteins

Four subclasses; IgGFour subclasses; IgG11, IgG, IgG22, IgG, IgG33, and IgG, and IgG44

Predominant immunoglobulin in blood, lymph, Predominant immunoglobulin in blood, lymph, CSF, and peritoneal fluidCSF, and peritoneal fluid

72-80% of serum immunoglobulins72-80% of serum immunoglobulins

IgAIgAThe major immunoglobulin in external secretions such The major immunoglobulin in external secretions such as saliva, mucous, sweat, gastric fluid, and tearsas saliva, mucous, sweat, gastric fluid, and tears

Monomer (serum) or dimer (secretions), M.Wt 165000, Monomer (serum) or dimer (secretions), M.Wt 165000, sedimentation coefficient 7S sedimentation coefficient 7S

Two subclasses; IgATwo subclasses; IgA1 1 andand IgAIgA22

13-19% of serum immunoglobulins13-19% of serum immunoglobulins

Serum IgA is predominantly monomeric and Serum IgA is predominantly monomeric and monomeric IgAmonomeric IgA1 1 accounts for about 90% of serum IgAaccounts for about 90% of serum IgA

Secretory Ig ASecretory Ig ASecretory Ig A has a J chain and a secretory Secretory Ig A has a J chain and a secretory piece (70.000 Daltons) which is synthesized by piece (70.000 Daltons) which is synthesized by epithelial cells to facilitate passage of secretory epithelial cells to facilitate passage of secretory IgA into mucous secretions and to protect it IgA into mucous secretions and to protect it from cleavage (secretory IgA is more resistant from cleavage (secretory IgA is more resistant than serum IgA)than serum IgA)

Secretory IgA: - IgASecretory IgA: - IgA1 1 90% in secretions above 90% in secretions above

the diaphragmthe diaphragm - IgA- IgA2 2 10% in lower GI10% in lower GI

Structure and formation of secretory lgA

IgDIgDMonomer, M. Wt. 180.000, sedimentation Monomer, M. Wt. 180.000, sedimentation coefficient 7Scoefficient 7S

A major surface component of many B cells but A major surface component of many B cells but present in very low concentrations (< 1% of present in very low concentrations (< 1% of serum immunoglobulins) in serum where it has serum immunoglobulins) in serum where it has no function.no function.

It is not secreted by plasma cells and it is It is not secreted by plasma cells and it is uniquely susceptible to proteolytic cleavageuniquely susceptible to proteolytic cleavage

IgEIgEMonomer, M. Wt 200.000, sedimentation Monomer, M. Wt 200.000, sedimentation coefficient 8Scoefficient 8S

It has an extra CIt has an extra CHH domain domain

Less than 0.001% of serum immunoglobulinsLess than 0.001% of serum immunoglobulins

Binds with high affinity to mast cells and Binds with high affinity to mast cells and basophils (Homocytophilic)basophils (Homocytophilic)

Antigenic determinants of immunoglobulins

AllotypesAllotypesAllelic forms of the same protein as a result of the Allelic forms of the same protein as a result of the presence of different forms of the same gene at a presence of different forms of the same gene at a given locusgiven locus

Allotypic differences at known loci usually involve Allotypic differences at known loci usually involve changes in only one or two amino acids in the changes in only one or two amino acids in the constant region of a chainconstant region of a chain

Important genetic markers inherited as dominant traits; Important genetic markers inherited as dominant traits; Gm on Gm on chain, Km (previously Inv) on kappa chains, chain, Km (previously Inv) on kappa chains, and Am on and Am on αα chain chain

The genes encoding the markers are expressed The genes encoding the markers are expressed codominantly, so that an individual may be codominantly, so that an individual may be homozygous or heterozygous for a given marker homozygous or heterozygous for a given marker

IdeotypesIdeotypesThe antigenicity of the variable region of FabThe antigenicity of the variable region of Fab

May or may not block binding of the antibody to the May or may not block binding of the antibody to the antigen depending whether produced against CDRs or antigen depending whether produced against CDRs or Framework sequence. An ideotype represents an Framework sequence. An ideotype represents an “internal immage” of the epitope“internal immage” of the epitope

Public or cross reacting epitopes are ideotypes on Public or cross reacting epitopes are ideotypes on different antibodies produced against the same different antibodies produced against the same epitope. Private epitopes react with only a particular epitope. Private epitopes react with only a particular antibody moleculeantibody molecule

Regulation of an Immune response (Jerne’s Theory)Regulation of an Immune response (Jerne’s Theory)