BLOCK 3: IMM: (3.4) ANTIBODIES - PART 3

B-cell Receptor Structure

• The B-cell receptor (BCR) is a transmembrane protein composed of an immunoglobulin molecule and associated signaling proteins.
• The immunoglobulin molecule consists of a heavy chain and a light chain, both with variable and constant regions.
• The heavy chain has a secretion coding (SC) region followed by a membrane coding (MC) region.
• The SC region is responsible for the secreted form of the BCR, while the MC region is responsible for the membrane-bound form.
• Alternative mRNA processing determines whether the BCR is secreted or membrane-bound.
• The polyadenylation site after the MC region (PA mu M) results in a transmembrane BCR.
• The polyadenylation site before the MC region (PA mu S) results in a secreted BCR.

B-cell Receptor Complex

• The BCR complex is composed of the BCR itself (immunoglobulin molecule), Igα and Igβ signaling proteins.
• The BCR complex is responsible for intracellular signaling upon antigen binding.
• The cytoplasmic tails of Igα and Igβ contain immunoreceptor tyrosine-based activation motifs (ITAMs).
• ITAMs are phosphorylated upon antigen binding, triggering downstream signaling pathways.

Secretion of Antibodies

• Secretion of antibodies is another example of alternative mRNA processing.
• Alternative splicing of the heavy chain mRNA results in a secreted form of the antibody.
• The textbook provides an example of IgM, which can be either membrane-bound or secreted.
• The secreted form of IgM lacks the MC region, allowing it to be released from the cell.

Immunoglobulin Isotypes

• Different immunoglobulin isotypes are defined by their heavy chain constant region, indicated by Greek letters.
• Isotypes also differ in their N-linked carbohydrate groups.
• All immunoglobulins are monomers in their membrane-bound form.
• IgD, IgE and IgG are monomers in their soluble, secreted form.
• IgA exists as monomers and dimers, while IgM exists mainly as pentamers.

Immunoglobulin Isotype Functions

• Each isotype has specialized functions:
  • Major functions are represented by dark red shading
  • Lesser functions are represented by dark pink
  • Minor functions are represented by pale pink.
  • Opsonization refers to the ability of the antibody to facilitate phagocytosis.
  • Antibodies that activate the complement system indirectly cause opsonization through complement activation.

Isotype Switching

• Involves somatic recombination events that attach different heavy-chain constant region genes to the existing variable region exon, also known as class switching.
• This occurs through recombination between switch regions (S) with deletion of DNA between them.
• Switch regions are targeted by Activation-induced cytidine deaminase (AID), causing nicks in DNA strands.
• AID and the repair process of damaged DNA are essential for both somatic hypermutation and isotype switching.

Isotype Switching Mechanism

• The nicks in DNA facilitate recombination between the switch regions leading to the excision of intervening DNA and the juxtaposition of the rearranged variable region with a different constant region gene.
• The first switch in a clone of B cells is from the μ isotype to another isotype.
• Subsequent switching to other isotypes can occur later.

Isotype Switching in Memory B Cells

• Antibodies produced by memory B cells that have experienced prolonged or repeated exposure to the same epitope might undergo isotype switching, induced by T cell cytokines.
• The availability of multiple isotypes with the same specificity allows the humoral response to activate various mechanisms against the same epitope.
• Serial reactivation of memory B cells allows the isotype switch to occur during each restimulation.
• IgM is the predominant isotype in primary responses, while secondary responses are mostly IgG with significant amounts of IgA and IgE.

Immunoglobulin Gene Clusters

• Gene clusters encoding κ (kappa) light chains are located on chromosome 2.
• Gene clusters encoding λ (lambda) light chains are located on chromosome 22.
• The heavy chain gene cluster is located on chromosome 14.
• The potential antigen-binding combinations are greater than 26 million.

Immunoglobulin Basic Structure

• Light chains, termed κ (kappa) or λ (lambda) are on chromosomes 2 & 22, respectively.
• Five types of heavy chains are encoded on chromosome 14:
  • Mu (μ)
  • Delta (δ)
  • Gamma (γ)
  • Epsilon (ε)
  • Alpha (α)
• Isotypes are genetically different forms of light chains (κ & λ) and of heavy chains (μ, δ, γ, ε, & α).
• Immunoglobulin class or subclass is determined by the heavy chain isotype.

Immunoglobulin Class Structure

• IgM is secreted as a pentamer of immunoglobulin monomers.
• The IgM pentamer is held together by a polypeptide called the J chain for "joining chain".
• Monomers are cross-linked by disulfide bonds to each other and to the J chain.
• The lack of a hinge region in IgM monomer makes the molecule less flexible than others, but this is compensated by the pentamer having five times as many antigen-binding sites as IgG.
• With pathogens possessing multiple identical epitopes on their surface, IgM can attach with several binding sites simultaneously.

IgA Dimer Formation

• IgA is synthesized as a dimer in mucosal lymphoid tissue, associated with the same J chain found in pentameric IgM.
• In dimeric IgA, monomers are disulfide bonded to the J chain but not to each other.

IgG Flexibility

• The most flexible part of the IgG molecule is its hinge.
• The hinge allows the Fab arms to wave and rotate, accommodating the antibody to the orientation of epitopes on pathogen surfaces.
• The “elbow” within the Fab adds more flexibility, allowing variable domains to bend with respect to constant domains.
• The “wagging” of the Fc tail allows IgG molecules bound to antigen to accommodate the binding of C1q and other effector molecules.

IgG Subclasses

• The four IgG subclasses have different and complementary functions.
• Differences in hinge structure, number of disulfide bonds, and amino acid sequences, particularly glycine and proline residues, influence hinge flexibility.

IgG4 Functional Monovalency

• IgG4 is synthesized in a form that has two heavy chains, two light chains, and two identical antigen-binding sites.
• Molecules of IgG4 can interact and exchange one heavy chain and its associated light chain in circulation.
• This property results in most IgG4 molecules in circulation having TWO different binding sites for antigen.
• Therefore, they interact with a pathogen or a protein antigen through only one binding site.

Immunoglobulin Gene Rearrangement

• Before immunoglobulin light-chain (κ or λ) and heavy-chain genes can be expressed, rearrangements of gene segments are needed to produce exons encoding V regions.
• After rearrangement, the genes are transcribed into primary transcripts containing both exons and introns.
• The introns are spliced out, producing mRNAs that translate into kappa or lambda light chains and mu heavy chains.
• These chains assemble inside the cell and are expressed as membrane-bound IgM at the cell surface.