Antibody Structure and Function Part I

Fundamentals of Immunoglobulins and Antibodies

Immunoglobulins versus Antibodies:
- Immunoglobulins: These are glycoproteins synthesized by B cells. They can exist in a membrane-bound form on the surface of B cells.
- Antibodies: These are glycoproteins synthesized and secreted by plasma cells.
  - Specificity: They exhibit exquisite specificity for an epitope of the molecules that comprise the antigen.
  - Circulation: They circulate as major components of the blood plasma.
The Transition from B Cell to Plasma Cell:
- A Resting B cell displays membrane-bound immunoglobulin (Ig) as a receptor.
- Upon encounter with an antigen (e.g., a bacterium), the B cell is stimulated.
- The Stimulated B cell gives rise to antibody-secreting plasma cells.
- Specificity Retention: Plasma cells secrete antibodies that possess the exact same antigen specificity as the original membrane-bound immunoglobulin found on the resting B cell.

Structural Composition of the Antibody Molecule

Basic Unit: Antibodies are glycoproteins built from a basic unit consisting of four polypeptide chains:
- Two Identical Light (L) Chains.
- Two Identical Heavy (H) Chains.
Physical Specifications:
- Molecular Weight: Approximately $\sim 150\,\text{kDa}$ .
- Termini:
  - N-termini: The amino-terminal ends, which contain the variable regions for antigen binding.
  - C-termini: The carboxy-terminal ends, which consist of constant regions.
- Disulfide Bonds: Interchain disulfide bonds link the heavy chains to each other and link each light chain to a heavy chain, stabilizing the quaternary structure.
- Carbohydrate (CHO): Sugars are attached to the constant regions of the heavy chains.
Functional Regions:
- Variable (V) Region: Located at the N-terminal end of both heavy and light chains. It contains the antigen-binding sites.
- Constant (C) Region: Forms the stem and lower parts of the Y-shape. It determines the biological activity and class (isotype) of the antibody.
- Complementary-determining regions (CDRs): Specific sections within the variable region that physically contact the antigen.
- Hinge Region: A flexible segment between the Fab arms and the Fc tail.

The Flexibility of the IgG Hinge Region

The flexible hinge of the IgG molecule allows it to bind with both arms to various arrangements of antigens on a surface. The molecule is capable of several distinct motions:
- Waving the Fab arms: Tilting the binding arms away from or toward each other.
- Rotation of the Fab arm: Rotating around the axis of the hinge.
- Wagging the Fc tail: Movement of the constant fragment relative to the binding fragments.
- Bending of the Fab elbow: Flexibility within the Fab fragment itself.

Proteolytic Cleavage of the Y-Shaped Antibody

Papain Digestion: Partial digestion with the protease papain cleaves the antibody above the hinge region, resulting in three fragments:
- Fab (Fragment Antigen Binding): Two identical fragments, each containing one antigen-binding site.
- Fc (Fragment Crystallizable): One fragment that readily crystallizes; it mediates effector functions and interacts with cell surface receptors.
Pepsin Digestion: Digestion with the protease pepsin cleaves the antibody below the hinge region, resulting in:
- $F(ab')_2$ : A single fragment where the two antigen-binding sites remain linked together. This fragment is divalent (can bind two antigens).
- pFc': The largest remaining part of the crystallizable fragment.

The Immunoglobulin Superfamily

The similar structure of Heavy and Light chains suggests a common evolutionary ancestry. This has led to the classification of the "Immunoglobulin Superfamily," characterized by the immunoglobulin-fold domain structure.
Members of the Superfamily:
- Immunoglobulins: e.g., IgM.
- T-cell Receptor (TCR): Composed of $\alpha$ and $\beta$ chains (or $\gamma$ and $\delta$ ).
- MHC Molecules:
  - Class I: Includes the $\alpha$ chain and $\beta_2$ -microglobulin.
  - Class II: Includes $\alpha$ and $\beta$ chains.
- Ig- $\alpha$ /Ig- $\beta$ Heterodimer: Signals the B-cell receptor complex.
- T-cell Accessory Proteins:
  - CD3 (complex with $\gamma, \delta, \varepsilon$ chains).
  - CD2, CD4, and CD8.
- Adhesion Molecules:
  - VCAM-1 (Vascular Cell Adhesion Molecule-1).
  - ICAM-1 and ICAM-2 (Intercellular Adhesion Molecules).
  - LFA-3 (Lymphocyte Function-associated Antigen-3).
- Poly-Ig Receptor: Involved in the transport of IgA and IgM across epithelia.

Antigenic Determinants of Immunoglobulins

Antibodies are glycoproteins that can themselves serve as immunogens (antigens) if injected into a different organism or under certain conditions. Their epitopes fall into three categories:

Isotypic Differences:
- Antigenic determinants found in the constant region.
- These distinguish different classes (e.g., IgG vs. IgA).
Allotypic Differences:
- Determinants encoded by different alleles of the same gene.
- Even within the same class (e.g., IgG), individuals may have slight amino acid differences.
Idiotypic Differences:
- Determinants located in the variable regions ( $V_H$ and $V_L$ ).
- These reflect the unique binding specificity of the antibody.

Immunoglobulin Classes (Isotypes)

The identity of the heavy chain constant region defines the five main isotypes or classes of immunoglobulins:

Class	Heavy Chain	Subclasses	Light Chain
IgG	$\gamma$ (gamma)	$\gamma 1, \gamma 2, \gamma 3, \gamma 4$	$\kappa$ or $\lambda$
IgM	$\mu$ (mu)	None	$\kappa$ or $\lambda$
IgA	$\alpha$ (alpha)	$\alpha 1, \alpha 2$	$\kappa$ or $\lambda$
IgE	$\varepsilon$ (epsilon)	None	$\kappa$ or $\lambda$
IgD	$\delta$ (delta)	None	$\kappa$ or $\lambda$

Note: Subtype differences (such as those between $\gamma 1$ and $\gamma 2$ ) are caused by amino acid substitutions at only a few positions.

The Antigen Binding Site

The binding site is formed by the Hypervariable (HV) regions (also called Complementarity-Determining Regions or CDRs) of both the heavy-chain and light-chain variable ( $V$ ) regions.
Variability Analysis:
- If you plot variability against the amino acid residue position (roughly 1 to 120), there are three distinct peaks of extreme variability.
- Framework Regions (FR): FR1, FR2, FR3, and FR4 represent the stable regions that provide the structural scaffolding for the variable domain.
- Hypervariable Regions: HV1 (CDR1), HV2 (CDR2), and HV3 (CDR3) provide the actual contact points for the antigen.
- The HV3 region often shows the highest peak of variability.
Complementarity: The CDRs create a surface that is chemically and physically complementary to the shape and charge of the antigen.

Antibody Binding to Antigen: Noncovalent Forces

The interaction between an antibody and an antigen is entirely noncovalent. Four primary forces are involved:

Electrostatic Forces:
- Origin: Attraction between opposite charges.
- Example: Interaction between an amino group ( $\text{NH}_3^+$ ) and a carboxyl group ( $\text{OOC}^-$ ).
Hydrogen Bonds:
- Origin: A hydrogen atom is shared between two electronegative atoms (usually Nitrogen $(N)$ or Oxygen $(O)$ ).
- Example: $=O \cdots H-N-$ or similar dipolar attractions ( $\delta^-, \delta^+$ ).
Van der Waals Forces:
- Origin: Fluctuations in electron clouds around molecules polarize neighboring atoms oppositely.
- Example: Transient dipoles inducing attractions ( $\delta^- \leftrightarrow \delta^+$ ).
Hydrophobic Forces:
- Origin: Hydrophobic groups (non-polar) interact unfavorably with water and tend to pack together to exclude water molecules.
- Note: This attraction also heavily involves Van der Waals forces once the molecules are packed together.

Types of Epitopes Bound by Antibodies

Linear Epitope: The antibody binds to a contiguous, linear sequence of amino acids within a protein antigen.
Discontinuous Epitope: Also known as a conformational epitope. The antibody binds to amino acids that are brought together by the three-dimensional folding of the protein, even though they may be far apart in the primary linear sequence.

References

Murphy, K., Weaver, C., & Berg, L. Immunobiology. 10th edition. Norton, NY. 2022. Chapter 4.
Parham, P. The Immune System. 4th edition. Garland Science, NY. 2015. Chapters 4 and 9.
Kindt T.J., Goldsby, R.A., Osborne, B.A. Kuby Immunology. 6th edition. W.H. Freeman and Company, NY. 2007. Chapter 4.
Goldsby, R.A., Kindt T.J., Osborne, B.A., Kuby J. Immunology. 5th edition. W.H. Freeman and Company, NY. 2003. Chapter 4.