ANTIBODY STRUCTURE AND THE GENERATION OF B-CELL DIVERSITY

WHAT ARE ANTIBODIES? Antigen specific proteins produced by plasma cells Belong to immunoglobulin superfamily Located in blood and extravascular tissues, secretions and excretions Bind pathogenic microorganism and their toxins in extracellular compartments Secreted form of immunoglobulins

WHAT ARE IMMUNOGLOBULINS? Antigen specific proteins produced by B lymphocytes Belong to immunoglobulin superfamily Bound to surface of B lymphocytes Function as binding (receptor) sites for specific antigens Antigen receptor sites on mature B lymphocytes IgM IgD Membrane-bound form of immunoglobulins

WHAT IS THE IMMUNOGLOBULIN SUPERFAMILY Proteins with structural feature first defined in immunoglobulins Characteristic structural feature Sequence of Domains providing stable conformation Domain Polypeptide (100 to 110 amino acids) chain folded into sandwich (2 slices of bread) held together by disulfide bond IG superfamily members Antibodies, B cell receptors, T cell receptors, MHC molecules and others

STRUCTURE OF ANTIBODIES Antibodies are glycoproteins composed of Polypeptide chains and carbohydrate Monomeric structure Polypeptide chains 2 identical heavy chains 2 identical light chains Polypeptide chains joined by disulfide bonds Carbohydrate

STRUCTURE OF ANTIBODIES Polypeptide chains have variable and constant regions Variable N (amino)-terminal of polypeptide chain Antigen binding site Constant C (carboxyl)-terminal of polypeptide chain Binding sites for cell surface receptors and complement Structure represented by the letter “Y” Y shaped molecule cleaved by protease papain Fragment antigen binding (Fab) Fragment crystallizable (Fc)

CLASSES (ISOTYPES) OF IMMUNOGLOBULINS Classes based on constant region of heavy chains Immunoglobulin A (IgA) Immunoglobulin D (IgD) Immunoglobulin E (IgE) Immunoglobulin G (IgG) Immunoglobulin M (IgM) Differentiation of heavy chains Length of C region, location of disulfide bonds, hinge region, distribution of carbohydrate Classes have different effector functions

CLASSES (ISOTYPES) OF IMMUNOGLOBULINS Additional classification based on light chains Kappa Lambda Each IG has either kappa or lambda, not both IgG kappa IgG lambda No functional differences between light chains

IT’S GREEK TO ME Heavy chains, light chains and other molecules of the immune system identified using letters of the Greek alphabet

THREE DIMENSIONAL STRUCTURE OF ANTIBODIES Antibodies function in setting of infectious process Proteolytic enzymes, salt and pH differences Antibodies remain stable based on Sequence of domains Single domain consists of 100 – 110 amino acids folded into compact and stable conformation Domains Variable (V) Single V domain in H and L chains Constant (C) Single C domain in L chains Three to four (C) domains in H chains

ANTIGEN BINDING SITES OF IMMUNOGLOBULINS Antigen binding sites formed from hypervariable regions Heavy chain V domain Light chain V domain Hypervariable regions of V domains Amino acid sequence differences concentrated Flanked by less variable framework regions Three hypervariable regions in each V domain Hypervariable regions also called Complementarity-determining regions (CDR)

ANTIGEN BINDING SITES OF IMMUNOGLOBULINS Antigen binding sites vary with size and shape of antigen Part of antigen to which antibody binds Antigenic determinant (Epitope) Antigen-Antibody binding based on non-covalent forces Hydrogen bonds Affinity Strength of binding of one molecule to another by a single binding site Avidity Overall strength of binding between two molecules

ANTIBODIES AS DIAGNOSTIC AND THERAPEUTICS AGENTS Based on specificity and affinity of antibodies Both applications require large quantities of identical antibodies Monoclonal antibodies Monoclonal antibodies are produced using hybridoma cell line Hybridoma cell line Derived from single antibody producing cell fused with myeloma cell (neoplastic plasma cell)

IMMUNOGLOBULIN DIVERSITY IN B-CELLS BEFORE ENCOUNTER WITH ANTIGEN Immune system capable of producing a limitless number of different immunoglobulins/antibodies Mechanism Genes for IG organized differently Genes exist as nonfunctional segments Variable (V), Joining (J), Diversity (D), Constant (C) Genes are inherited in this form Germline form (germline configuration)

IMMUNOGLOBULIN DIVERSITY IN B-CELLS BEFORE ENCOUNTER WITH ANTIGEN Expression Gene segments must be rearranged into functional gene Gene Rearrangement Takes place during development of B-cells Mechanism of somatic recombination Genes for IG located at 3 chromosomal locations Heavy chain locus on chromosome 14 Kappa light chain locus on chromosome 2 Lambda light chain locus on chromosome 22

GERMLINE ORGANIZATION OF HUMAN IG HEAVY CHAIN AND LIGHT CHAIN LOCI Lambda light chain locus Gene segments 30 (V), 4 (J) and 4 (C) Kappa light chain locus 40 (V), 5 (J) and 1 (C) Heavy chain locus 65 (V), 27 (D), 6 (J) and 9 (C)

CONSTRUCTION OF LIGHT CHAIN AND HEAVY CHAIN VARIABLE REGIONS Constructed from 2 segments 1 (V) segment 1 (J) segment Heavy chain Constructed from 3 segments 1 (D) segment

SOMATIC RECOMBINATION Performed by enzymes with cut and rejoin DNA Directed by Recombination signal sequences (RSS) Recombination signal sequences Recognition sites for enzymes Recombination occurs between different types 9 / 12 / 7 9 / 23 / 7 Mechanism follows the 12/23 rule Ensures segments joined in correct order

MECHANISMS OF GENETIC DIVERSITY IN V-REGION OF IMMUNOGLOBULINS Random combination of V and J segments in light chain genes V, D and J segments in heavy chain genes Addition of P (palindromic) and N (non-templated) nucleotides at junctions of gene segments during recombination Junctional diversity Association of H and L chains in different combinations

CONSTRUCTION OF B-CELL SURFACE IMMUNOGLOBULINS Following rearrangement of VH gene segments, two CH loci are transcribed IgM IgD M and D constant segments Located nearest variable segments M and D transcript processed by Cleavage, polyadenylation and splicing IgM and IgD enter endoplasmic reticulum

SURFACE IMMUNOGLOBULINS ASSOCIATED WITH PROTEINS TO COMPLETE ANTIGEN RECEPTOR In ER, IgM and IgD associated with transmembrane proteins Ig-alpha Ig-beta Transmembrane proteins Transport M and D to B cell surface Communication of antigen binding to inside of B cell Tails interact with intracellular signaling molecules Complex of IgM and IgD with Ig-alpha and Ig-beta forms B-cell receptor

DIVERSIFICATION OF ANTIBODIES AFTER B-CELLS ENCOUNTER ANTIGEN Mature, naïve B cell has membrane bound IgM and IgD antigen receptors Binding of antigen initiates proliferation and differentiation of B-cells into plasma cells During differentiation, B cells switch from making immunoglobulin to antibody M and D isotypes IgM Produced in large amounts Provides protective immunity IgD Produced in small amounts No known function

MECHANISM OF SWITCHING FROM IMMUNOGLOBULIN TO ANTIBODY Surface and secreted forms derived from same heavy chain gene by alternative RNA processing Each heavy chain C gene has Membrane coding (MC) region Secretion coding (SC) region Mechanism involves a switch in cleavage, polyadenylation and splicing From pAm region to pAs region

DIVERSIFICATION OF ANTIBODIES AFTER B-CELLS ENCOUNTER ANTIGEN Following antigen activation of B-cells, additional diversification occurs in V domain by Somatic hypermutation Introduction of random single nucleotide substitutions (point mutations) throughout V regions of H and L chains Mechanism poorly understood More common in hypervariable regions (CDRs)

OUTCOME OF SOMATIC HYPERMUTATION Gives rise to some antibodies with higher Affinity for antigen Affinity Strength of binding of one molecule to another by a single binding site Higher affinity antibodies are produced as immune response proceeds Affinity maturation

THE PRIMARY HUMORAL IMMUNE RESPONSE Immune response initially produces IgM antibodies then switches to IgG antibodies Question Why switch from IgM to IgG? Answer Limited effector mechanisms for IgM Range of effector mechanisms for IgG Mechanism Isotype or class switching

ISOTYPE OR CLASS SWITCHING Process by which B cell changes class of IG produced while preserving antigenic specificity Involves somatic recombination which attaches different heavy chain constant region to variable region Occurs only during active immune response Mechanisms involves recombination between Switch sequences (regions)

CLASSES, SUBCLASSES AND PHYSICAL PROPERTIES OF IMMUNOGLOBULINS Subclasses are numbered according to plasma concentration

FUNCTIONS AND PROPERTIES OF ANTIBODY Neutralization Direct inactivation of pathogen or toxin thereby preventing its interaction with human cells Opsonization Coating of pathogens for more efficient phagocytosis Activation of complement More efficient phagocytosis Direct killing

IgM ANTIBODY OF THE IMMUNE RESPONSE First isotype produced in primary response May or may not be produced in secondary response Produced before B cells undergo somatic hypermutation Occurs as pentamer with J chain Found primarily in blood and lymph Multiple binding sites confers high avidity and compensates for low affinity of monomers Highly effective in complement activation Functions as rheumatoid factor

IgG ANTIBODY OF THE IMMUNE RESPONSE Second isotype produced in primary response Primary isotype of Secondary immune response Memory immune response Represents approximately 75% of total serum IG Four subclassses (1-4) Different effector functions Transported across placenta Functions as rheumatoid factor

IgA ANTIBODY OF THE IMMUNE RESPONSE Two subclasses (IgA1 and IgA2) and two forms (monomeric and dimeric) Monomeric Located in blood and extracellular spaces Predominately IgA1 Ratio of IgA1 to IgA2 is 10:1 Functions as rheumatoid factor Dimeric Located in mucous membranes and secretions Predominately IgA2 Ratio of IgA2 to IgA1 is 3:2 J chain like IgM

IgE AND IgD ANTIBODIES OF THE IMMUNE RESPONSE Binds with high affinity to receptors on mast cells, basophils and activated eosinophils Longer half-life when cell bound Initiates a strong inflammatory reaction to parasites Involved in allergic reactions IgD Antigen receptor on mature B-cells No other known function