Transcript Antibody Structure and B Cell Diversity

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
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*
*
*
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 BCELLS 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
* Gene segments
* 40 (V), 5 (J) and 1 (C)
* Heavy chain locus
* Gene segments
* 65 (V), 27 (D), 6 (J) and 9 (C)
CONSTRUCTION OF LIGHT CHAIN AND
HEAVY CHAIN VARIABLE REGIONS
* Light chain
* Constructed from 2 segments
* 1 (V) segment
* 1 (J) segment
* Heavy chain
* Constructed from 3 segments
* 1 (V) segment
* 1 (D) segment
* 1 (J) 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 Bcells 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
* 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
Classes
Subclasses
IgG
IgG1, IgG2, IgG3, IgG4
IgA
IgA1, IgA2
IgM
IgD
IgE
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
* IgE
* 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