Structure of an immunoglobulin molecule

Antibodies

  • Antibodies are produced by B cells and represent their primary contribution to the adaptive immune response.They were the first molecules of the specific immune response to be characterised and remain the best-studied immune proteins.Collectively, antibodies belong to a family of plasma proteins known as immunoglobulins.

Overview Of The Structure and Function of Immunoglobulins (Antibodies)

An immunoglobulin molecule has two distinct functions:

    1. Specific antigen binding: It recognizes and binds molecules from the pathogen that triggered the immune response.
    2. Effector recruitment: It recruits cells or molecules capable of destroying the targeted pathogen.
  • These functions are structurally separated within the antibody molecule:
    • Variable region: Responsible for antigen binding, this region is highly diverse among antibodies, allowing each immunoglobulin to recognize a unique antigen. The complete antibody repertoire of an individual is sufficiently diverse to recognize virtually any antigen.
    • Constant region: Responsible for effector functions, this region is less variable and exists in five forms, defining the antibody isotypes. Each isotype is specialized in activating specific effector mechanisms.
  • Diversity generation: The remarkable diversity of antibodies arises from genetic recombination of immunoglobulin-coding genes.
  • Activation of B cells: A B cell does not produce antibodies until stimulated by a specific antigen. Antigens are recognised by surface immunoglobulins on B lymphocytes. Binding of an antigen to these surface receptors is essential for B cell activation and their differentiation into antibody-producing plasma cells.

General Structure of an Immunoglobulin Molecule

A – Historical Overview

  • The first structural model of an immunoglobulin was proposed by Pauling in 1940, depicting a molecule with two antigen-binding sites connected by a rigid bridge.

  • In the 1960s, Porter and Nisonoff demonstrated that enzymatic digestion of IgG produced fragments with distinct functional properties, defining the Fab (antigen-binding) and Fc (effector function) fragments.

  • Further studies using reduction and acid dissociation revealed that immunoglobulins are composed of heavy and light chains connected by disulfide bonds.

  • Building on these findings, Fleischman (1964) proposed a model of the immunoglobulin as two identical heavy chains, each linked to an identical light chain by disulfide bridges.

  • Studies on myelomas and plasmacytomas greatly advanced our understanding of antibody structure. While antibodies were known to be a heterogeneous pool of serum proteins, myeloma patients produced large amounts of a single, easily purified immunoglobulin.

  • Purified immunoglobulins enabled the generation of antisera, classification into isotypic, allotypic, and idiotypic groups, and crystallographic studies that provided a three-dimensional representation of the molecule.

  • These combined insights laid the foundation for the current structural model of immunoglobulins.

B – General Structure of Immunoglobulin Molecules

  • Immunoglobulins have a Y-shaped structure, composed of three segments of roughly equal size connected by a flexible hinge region.

  • All immunoglobulins share this basic architecture: two pairs of heavy and light chains linked by disulfide bonds.

  • Within this family, there are five immunoglobulin classes (IgM, IgD, IgG, IgA, and IgE) which differ biochemically and functionally.

B-1 Basic Structure of an IgG1 Molecule

  • The overall structure of all antibodies is sufficiently conserved that IgG can serve as the prototypical example of a basic antibody structure.

  • IgG molecules are symmetric multichain proteins with a molecular mass of approximately 150 kDa.

  • They consist of four polypeptide chains in a 2:2 ratio:

    • Heavy chains (H): ~50 kDa each

    • Light chains (L): ~25 kDa each

  • Each IgG molecule contains two identical heavy chains and two identical light chains in equimolar proportion.

B-1-1 Heavy Chains

  • There are five types of heavy chains, designated by the Greek letters γ, α, μ, δ, ε, corresponding to the five immunoglobulin classes: IgG, IgA, IgM, IgD, and IgE.

  • Some classes are further divided into subclasses, as in IgG and IgA.

  • The C-terminal region of each heavy chain determines the functional activity of the antibody.

B-1-2 Light Chains

  • There are two types of light chains, κ (kappa) and λ (lambda).

  • Each light chain type can combine with any heavy chain type.

  • Each antibody molecule contains two identical light chains.

  • The κ/λ ratio varies between species; in humans, it is approximately 2:1. Deviations from this ratio can indicate tumorigenic B cells.

  • Light chains do not determine the functional properties of the antibody.

B-2 Domain Organization of the Immunoglobulin Molecule

  • Heavy and light chains exhibit two fundamental structural features:

    • Each chain consists of globular sequences (~110 amino acids each), called domains, stabilized by intra-chain disulfide bonds.

      • Light chains: 2 domains (VL and CL)

      • IgG heavy chains: 4 domains (VH, CH1, CH2, CH3)

    • The amino-terminal (variable) domains of both heavy and light chains vary greatly between antibodies, while the remaining domains are constant.

  • Y-shaped structure:

    • Each arm of the Y is formed by a light chain paired with the N-terminal half of a heavy chain.

    • Pairings: VH with VL and CH1 with CL, forming the antigen-binding site.

    • The stem of the Y consists of the C-terminal regions of the heavy chains.

      • CH3 domains interact with each other.

      • CH2 domains, containing carbohydrate moieties, do not interact directly, contributing to flexibility and effector function.

B-3 Enzymatic Sensitivity of the Immunoglobulin Molecule

  • Proteolytic enzymes targeting specific peptide sequences have been used to dissect immunoglobulin structure and determine which regions are responsible for distinct antibody functions.

  • Papain digestion: Partial digestion with papain cleaves the antibody into three fragments:

    • Two identical fragments (Fab  “fragment antigen binding”) correspond to the two arms of the antibody and retain antigen-binding ability. Each Fab consists of a complete light chain and the VH and CH1 domains of a heavy chain.

    • One fragment (Fc “crystallizable fragment”) does not bind antigen but is easily crystallizable at low ionic strength. It comprises the CH2 and CH3 domains of both heavy chains and mediates interactions with effector cells and molecules.

  • The exact profile of fragments depends on the location of disulfide bonds in the hinge region between CH1 and CH2 domains. Papain cleaves amino-terminal to the disulfide bonds.

  • Pepsin digestion: Pepsin cleaves carboxy-terminal to the disulfide bonds, generating a F(ab’)₂ fragment, which contains the two Fab fragments linked by disulfide bonds. The remaining portion is further degraded into small fragments.

    • The F(ab’)₂ fragment retains antigen-binding capability but cannot interact with effector cells or molecules.

B-4 Flexibility of the Immunoglobulin Molecule

  • The hinge region connecting Fc and Fab regions is highly flexible, allowing independent movement of the two arms.

  • Some flexibility is also present between the variable (V) and constant (C) regions, enabling the Fab arms to bind antigens spaced at variable distances.

B-5 Domains of the Immunoglobulin Molecule

  • Constant and variable domains share a similar overall structure but are not identical.

  • Each domain is composed of a polypeptide folded into two antiparallel β-sheets, with hydrophobic residues buried between the layers:

    • One sheet contains four segments, the other three segments, connected by a single disulfide bond.

  • Variable domains are larger and contain an additional fold, forming three closely spaced hypervariable loops that constitute the antigen-binding site.

  • This hypervariable region is directly responsible for specific antigen recognition.

C – Structure of Different Immunoglobulin Classes and Subclasses

Most mammals have five immunoglobulin classes: IgM, IgG, IgA, IgD, and IgE. These classes differ in molecular weight, charge, amino acid composition, and glycosylation. Within each class, antibodies show additional heterogeneity. Following electrophoresis, immunoglobulins appear heterogeneous and separate into gamma (γ) and alpha (α) fractions in normal serum. All immunoglobulins are glycosylated, with sugar content varying from 2–3% in IgG to 12–14% in IgM, IgD, and IgE.

C-1 IgG

  • IgG is the predominant immunoglobulin in normal serum, representing approximately 75% of total Ig (8–18 g/L).
  • IgG is divided into four subclasses (IgG1–4).
  • Molecular weight: ~146 kDa for monomeric 7S IgG; IgG3 is slightly heavier due to a longer heavy chain (γ3).
  • IgG distributes evenly in intravascular and extravascular compartments.
  • Primary function: neutralization of bacterial toxins; IgG is the major antibody in secondary immune responses.
  • Each IgG subclass has a unique number and distribution of disulfide bonds:
  • Heavy-light chain linkage (hinge region) varies: IgG2, IgG3, and IgG4 differ.
  • Heavy-heavy chain bonds: 2 for IgG1 and IgG4, 4 for IgG2, 15 for IgG3.

C-2 IgM

  • Represents ~10% of total Ig (1–2 g/L).
  • IgM is a pentamer, each heavy chain ~65 kDa, total ~970 kDa.
  • Confined mainly to the intravascular compartment, IgM forms the bulk of “natural antibodies” and dominates the primary anti-infectious response.
  • Structural features:
    • Heavy (μ) chains differ from γ chains in amino acid sequence and contain an additional constant domain.
    • The five subunits are linked by disulfide bonds.
    • μ chains are highly glycosylated.
    • Contains a J-chain (137 amino acids, cysteine-rich), which mediates pentamer formation.

C-3 IgA

  • Accounts for 15–20% of serum Ig (3.5–4.5 g/L).

  • 80% of human IgA is monomeric in serum.

  • Major antibody in mucosal secretions (saliva, colostrum, milk, bronchial, and urogenital secretions).
  • Secretory IgA (sIgA):
    • Exists in two subclasses: IgA1 and IgA2, primarily dimeric (~385 kDa).
    • Includes a secretory component synthesized by epithelial cells and a J-chain for dimerization.
    • Secretory component facilitates mucosal transport and protects IgA from proteolysis.
    • IgA1 dominates serum; IgA2 dominates mucosal secretions.
    • Heavy chain structure: 1 variable domain + 3 constant domains; CH1 and CH2 domains have an additional intrachain disulfide bond.

C-4 IgD

  • <1% of plasma Ig.
  • Found in high amounts on the surface of circulating B lymphocytes.
  • Biological function is not fully defined, but IgD is critical in B cell activation and differentiation upon antigen recognition.
  • Contains one disulfide bond between heavy chains; δ chains are highly glycosylated.

 

C-5 IgE

  • Present in trace amounts in serum.
  • Found on mast cells and basophils via high-affinity FcεRI receptor.
  • Functions in anti-parasitic immunity (helminths) and immediate hypersensitivity reactions.
  • Heavy chain (~550 amino acids) with one variable and four constant domains, explaining its higher molecular weight.