2

Comparison of BCR and TCR

BCR & TCR: Receptors bind to molecular surfaces (usually proteins) Receptors exist in trillions of varieties Receptors share structural & sequence homology but are not identical.

Antibody

BCR Surface Ig/Ab 1984 960s

Experiment Immunized rabbit with Ovaalbumin (Ag) Serum collected Two portions P1 & P2 P1 on gel electrophoresis P2 treated with OVA ppt removed and then sample on electrophoresis gel Mobility vs amount of protein plotted on graph

1939, Tiselius & Kabat: Demonstration of the presence of Ab in a particular serum protein fraction
albumin

Amount of protein

globulins

E1

E2

Mobility on electrophoresis
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albumin

Amount of protein

globulins

E1

E2

Immune serum Ag adsorbed serum

Mobility

Biochemical characterization of Ab
Gerald Edelman, in 1959

Reducing agent Ab 2 x 50 kDa + 2 x 25 kDa 150 kd (Beta-mercaptoethanol/BME )

S-S BME SH + HS

BME + + +

Papain

By Rodney Porter 1959

Rodney Porter built a model Ab molecule Y shaped Stem and two branches Branch consist of Lc + Hc (half) Stem consists of two Hc (remaining halves) The structure of free tips of the branches bind specifically to Ag Separately Lc and Hc are inactive

Gerald M. Edelman Born 1929, New York Rockefeller University

Rodney R. Porter 1917 1985, England University of Oxford Oxford, United Kingdom
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Challenge?
Ab purified from serum N-terminal sequencing of Lc/Hc Frustrating!!!!! Data made no sense? Multiple amino acids at each position Sample heterogeneous Homogeneous source required Myeloma proteins produced by B cell tumors Hilschmann and Craig (1965): Lc Sequenced The first half varies variable region The second half identical constant region The antigen binding site must be at the tip of the branch.
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12

Roberto Poljack

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Structural Features of antibody: crystal structure

Antibody molecule composed of structurally similar subunits called Immunoglobulin (Ig)- like domains. These are a feature of many, many proteins in the immune (and neural!) systems (the Ig superfamily of proteins). The V-region is one such domain and each C-region another. Each of these domains contains an internal disulfide bond Structured as a barrel of F-pleated sheets (2 sets of anti-parallel strands). It is important to remember that the structure of the BCR is the same as the structure of the antibody - the only difference is that it is membrane bound. The membrane bound and secreted forms of immunoglobulin are splice variants.

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15

Folding pattern of CL and VL domains

 Each domain is a barrel- shaped structure  strands running in opposite directions (antiparallel) pack together to form two sheets (shown in yellow and green in the diagram of the C domain) Sheets held together by a disulfide bond The strands are lettered sequentially with respect to the order of their occurrence in the amino acid sequence of the domains; the order in each sheet is characteristic of immunoglobulin domains The strands C' and C'' that are found in the V domains but not in the C domains are indicated by a blue shaded background The characteristic four-strand plus three-strand (C-region type domain) or fourstrand plus five- strand (V-region type domain) arrangements are typical immunoglobulin superfamily domain building blocks, found in a whole range of other proteins as well as antibodies and T-cell receptors
A barrel : hollow cylindrical container

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Antibodies
Class of glycoproteins called immunoglobulins Y shaped Arms of Y recognize and bind antigen Tail of Y responsible for biological activity Present in body fluids (saliva, tears, urine and milk) Blood serum has highest concentration Found floating free in blood serum Also act as B-cell receptor (antigen binding receptor)

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BASIC STRUCTURE OF IMMUNOGLOBULINS

Light Chain Heavy Chain
VH CL VL

Heavy Chain

Disulfide bonds
CH1

Hinge Region
CH2

Carbohydrates
CH3

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BASIC STRUCTURE OF IMMUNOGLOBULINS  Four chain (polypeptides) structure  Identical light chains (23kD) = 2  Identical heavy chains (50-70kD) = 2  Inter-chain disulfide bonds  Intra-chain disulfide bonds  Both chains are divided into variable(V) and constant(C) regions based on variability in the amino acid sequences  Light Chain - VL and CL / Heavy Chain - VH and CH  Hinge Region is flexible/mobile/swing  Carbohydrates (glycoproteins) attached to the CH2 domain

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STRUCTURE OF THE VARIABLE REGION

Hypervariable (HVR) or complementarity determining regions (CDR)

 Region that bind to epitopes  Antigen binding site (ABS)  Determines the binding of Ig to a specific epitope/antigen  Also called complementarity determining regions (CDR)  CDR: short, 6 10 aa
Framework regions

 Less variability in aa sequence as compared to HVR

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Differences in the aa sequences in the constant region of the heavy chains

Five different classes 1. IgG - Gamma heavy chains 2. IgM - Mu heavy chains 3. IgA - Alpha heavy chains 4. IgD - Delta heavy chains 5. IgE - Epsilon heavy chains
Specific structural and functional properties

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Chain composition of the five immunoglobulin classes in humans

25

26

1. 2. 3. 4.

Most abundant class in serum 80% of total serum Ig Contains two K-heavy chains The subclasses differ in the number of disulfide bonds and length of the hinge region
27

 Numbered according to their decreasing average serum concentrations Subclasses differ in the size of the hinge region and also in the number and position of the interchain disulfide bonds (thick black lines) linking the heavy chains. Notable feature of human lgG3 is its 11 inter-chain disulfide bonds

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IgG Biological Function

IgG (1, 3, 4) is the only class of Ig that crosses the placenta and protects the developing fetus IgG (3>1>2) Effective complement activator (Classical pathway) IgG (1,3>4>2) bind with high affinity Fc receptors on phagocytic cells and mediate opsonization.

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Constitute 5 - 10% of total serum Igs B-cell membrane bound IgG is monomeric Free form of Ab is pentameric Pentamers held by S-S bonds between C-terminus of heavy chain domains (C4+ C4) & (C3+ C3) Fc in center and 10 Ag binding region (Fab) on periphery Pentamer contains J (joining)chain (linked by S-S to 2 of 10 chains)
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Functions: 1. Secretory IgA protects mucous membranes from bacterial colonization and viral infection (entry site for pathogens) 2. Important line of defense against bacteria such as Salmonella, Vibrio cholerae etc. 3. Complex of sIgA and pathogen removed by phagocytosis/peristalsis of gut 4. Mothers milk = sIgA = protects new born 5. IgA is the major class of Ig in secretions - tears, saliva, mucus

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Structure and formation of secretory lgA,

Only 10-15 % of the total Ig in serum, but predominant in external secretions

Most serum IgA exists as a monomer, although dimers, trimers, & tetramers are sometimes present.  In contrast, secretory IgA is formed during transport through mucous membrane epithelial cells ( dimer or tetramer )
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One Disulfide bond  0.2% of total serum Ig  Its role in serum is uncertain  IgD along with IgM is major membrane bound Ig expressed on B-cell/ BCR  No biological effector function has been identified for IgD
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 IgE is the least common serum Ig  It binds very tightly to Fc receptors on basophils and mast cells even before interacting with Ag  Involved in allergic reactions
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Allergen cross-linkage of receptor-bound lgE on mast cells

Despite its extremely low serum conc. ( 0.3ug/ml) IgE mediates H.S ( hay fever, asthma, hives, anaphylactic shock )

FIGURE 4-16
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Antigenic determinants/Epitopes on immunoglobulins

Ab (glycoproteins) can themselves function as potent immunogens to induce Ab responses.
+ : powerful tools for the study of B-cell development : Ab responses/ Immunotechniques - : obstacles to passive Ab therapy

Three major catagories of epitopes on Ig molecules: Isotype, Allotype, & Idiotype Each located in characteristic portion of the molecule

36

ISOTYPE  Isotypic determinats are constant region determinants  Collectively defines H-chain class and subclass & L-chain type and subtype (alpha, delta, mu, gamma, epsilon, kappa, lambda)  Each isotype in encoded by a separate constant region gene.  All members of a species carry the same constant region gene (different/multiple alleles possible)  Different species inherit different constant-region genes The isotypic determinants on an Ab from one species will be recognized as foreign in another species

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Allotype
 Although all members of a species inherit the same set of isotypic genes, multiple alleles exist for some of the genes. [ subtle (1~4 a.a.) differences in some, but not all, members of a species] Ab to allotypic determinants can arise from a blood transfusion or be produced by a pregnant mother in response to paternal allotypic determinants on the fetal Ig.

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IDIOTYPE
 VH and VL domains have unique a.a seq. = able to recognize different Ag  Each unique region can function as antigenic determinants called Idiotope  Region outside Ag binding site in V - region can also act as Idiotype The sum of the individual idiotopes (each individual antigenic determinant) is called idiotype of the antibody  An idiotope can be the actual antigen-binding site or not  injection of a homogeneous Ab into a recipient who is genetically unidentical to the donor can produce anti-idiotype Ab

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Antigenic determinants on Ig

Antigenic determinants of immunoglobulins

( Among different inbred strains )

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FIGURE 4-17

How mIg/BCR mediate activating signal after contact with an Ag? Cytoplasmic tail very short General 3 aa mIgM mIgD =structure mIgA = 14 aaof = mIg + Ig the=B-cell a/Ig b mIgG mIgE 28 aa Transmembrane protein complexes

receptor (BCR)

Too short to associate with signaling molecules (PTKs, GPs)

mIg does not constitute the entire Ag binding receptor

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B-Cell Receptor

General structure of the B-cell receptor (BCR)

BCR is a transmembrane protein complex Composed of mIg + S-S linked Heterodimer + Ig a/Ig b

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 Membrane glycoproteins  Affinity for Fc portion of Ab  Important for biological functions

43

Four distinct roles of Fc binding proteins

 The movement of Ab across cell membranes : poly IgR for sIgA & to some extent, pentameric IgM  The transfer of lgG from mother to fetus across the placenta : FcRN (neonatal Fc receptor)  Trigger effector functions : Opsonization or ADCC Cross-linking of FcR generates immuno-regulatory signals that affect cell activation, differentiation, etc

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B-Cell Receptor

The structure of a number of human Fc-receptors

Fc-binding polypeptide

Accessory signal-transducing polypeptide

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FIGURE 4-19

Ig Superfamily

Some members of the Ig superfamily

@ They have the characteristic Ig-fold structure >>> common evolutionary ancestry ( gene duplication & later divergence ) : facilitating interactions between membrane proteins occurring between the faces of b pleated sheets as well as Ag binding
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FIGURE 4-20

Polyclonal Antibody
 Derived from different B-cell lines  Natural mixture of antibodies secreted against a specific antigen  Each Ab recognizes a different epitope  Produced by immunizing an animal with the appropriate antigen  The immunized animals serum is collected  Antibodies can then be purified from the serum  Less expensive

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Monoclonal Antibodies

The conventional polyclonal antiserum contains a mixture of monoclonal antibodies

mAb is specific for one epitope on a complex Ag 48

FIGURE 4-21

Monoclonal Antibody (mAb or moAb) :

Derived from a single B-cell line Monoclonal antibodies recognize one epitope only. Process begins by immunizing an animal with an antigen. The animals spleen is removed. B-cells are fused with myeloma cells resulting in hybridomas. Hybridomas are screened to find those producing antibodies to the antigen with which they were immunized. Each hybridoma cell is derived from one B-cell so the antibodies that a clonal population of hybridoma cells produce are monoclonal antibodies. Expensive to produce
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Monoclonal antibody and Hybridoma

50

Monoclonal Antibodies

mAb have important clinical uses in diagnostic, imaging, & therapeutic agents

( Toxins used to prepare immuno-toxins include ricin, Shigella toxin, & diphtheria toxin )

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FIGURE 4-22 (a)

Monoclonal Antibodies

Diphtheria toxin binds to a cell-membrane receptor(L.) & a diphtheria-immunotoxin to a tumor-associated antigen(R.)

mAb or Fab specific for

FIGURE 4-22 (b) 52

PrePre-clinical and Clinical development programs (200 major companies in 2004)

Chemicals vs Biologicals Biologicals vs Antibody

Gene therapy 14 18 Others rProteins 38

978 Chemicals

399 Biological
91 Vaccine 238 Antibody

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