HKMU Faculty of Medicine Department of biochemistry & Molecular Biology First semester January 2016

BASIC IMMUNOGLOBULIN STRUCTURE Immunoglobulins generally assume one of two roles: immunoglobulins may act as plasma membrane bound antigen receptors on the surface of a B-cell or ii) as antibodies free in cellular fluids functioning to intercept and eliminate antigenic determinants. In either role, antibody function is intimately related to its structure

Immunoglobulins: Are composed of four polypeptide chains: two "light" chains (lambda or kappa), and two "heavy" chains 1.alpha 2.delta 3. gamma 4.epsilon 5.mu The type of heavy chain determines the immunoglobulin isotype (IgA, IgD, IgG, IgE, IgM, respectively).

Light chains are composed of 220 amino acid residues while heavy chains are composed of amino acids. -Each chain has "constant" and "variable" regions. Variable regions are contained within the amino (NH 2 ) terminal end of the polypeptide chain (amino acids 1-110).

When comparing one antibody to another, these amino acid sequences are quite distinct. Constant regions, comprising amino acids (or ), are rather uniform, in comparison, from one antibody to another, within the same isotype. "Hypervariable" regions, or "Complementarity Determining Regions" (CDRs) are found within the variable regions of both the heavy and light chains.

These regions serve to recognize and bind specifically to antigen. The four polypeptide chains are held together by covalent disulfide (-S-S-) bonds

- Structural differences between immunoglobulins are used for their classification -. As stated above, the type of heavy chain an immunoglobulin possesses determines the immunoglobulin "ISOTYPE ".

More specifically, an isotype is determined by the primary sequence of amino acids in the constant region of the heavy chain, which in turn determines the three-dimensional structure of the molecule. Since immunoglobulins are proteins, they can act as an antigen, eliciting an immune response that generates anti-immunoglobulin antibodies.

However, the structural (three-dimensional) features that define isotypes are not immunogenic in an animal of the same species, since they are not seen as "foreign". For example, the five human isotypes, IgA, IgD, IgG, IgE and IgM are found in all humans and a result, injection of human IgG into another human would not generate antibodies directed against the structural features (determinants) that define the IgG isotype

. However, injection of human IgG into a rabbit would generate antibodies directed against those same structural features. Another means of classifying immunoglobulins is defined by the term "allotype". Like isotypes, allotypes are determined by the amino acid sequence and corresponding three-dimensional structure of the constant region of the immunoglobulin molecule

Unlike isotypes, allotypes reflect genetic differences between members of the same species. This means that not all members of the species will possess any particular allotype. Therefore, injection of any specific human allotype into another human could possibly generate antibodies directed against the structural features that define that particular allotypic variation

A third means of classifying immunoglobulins is defined by the term "idiotype". Unlike isotypes and allotypes, idiotypes are determined by the amino acid sequence and corresponding three-dimensional structure of the variable region of the immunoglobulin molecule. In this regard, idiotypes reflect the antigen binding specificity of any particular antibody molecule

BASIC IMMUNOGLOBULIN FUNCTION Antibodies function in a variety of ways designed to eliminate the antigen that elicited their production. Some of these functions are independent of the particular class (isotype) of immunoglobulin

These functions reflect the antigen binding capacity of the molecule as defined by the variable and hypervariable (idiotypic) regions. For example, an antibody might bind to a toxin and prevent that toxin from entering host cells where its biological effects would be activated

Similarly, a different antibody might bind to the surface of a virus and prevent that virus from entering its host cell. In contrast, other antibody functions are dependent upon the immunoglobulin class (isotype). These functions are contained within the constant regions of the molecule.

For example, only IgG and IgM antibodies have the ability to interact with and initiate the complement cascade. Likewise, only IgG molecules can bind to the surface of macrophages via Fc receptors to promote and enhance phagocytosis.

GENERATION OF ANTIBODY DIVERSITY The immune system has the capacity to recognize and respond to about (Ten million) 10 7 different antigens. This extreme diversity can be generated in at least three possible ways: 1.Multiple genes in the germ line DNA. 2.Variable recombination during the differentiation of germ line cells into B-cells. 3.Mutation during the differentiation of germ line cells into B-cells.

The figure shows the genetic makeup of a germ line cell and a mature B-cell at the loci controlling heavy chain production. Germ line DNA has many (up to 200) different variable (V) region genes, in addition to 12 diversity (D) region genes and four joining (J) region genes.

During differentiation of this cell into the B- cell, rearrangement of the DNA occurs. This rearrangement aligns one of the many V genes with one of the D genes and one of the J genes, producing a functional VDJ recombinant gene. Since any of the genes may recombine with any others, this rearrangement has the potential to generate 200(v) x 12(D) x 4(J) = 9600 different possible combinations

1.The same type of event occurs in the genes encoding the immmunoglobulin light chains where about 200 different V regions may recombine with about 5 different J regions giving rise to 200 x 5 = 1000 possible light chains. 1.Since in any particular B-cell, any light chain combination can occur along with any heavy chain combination, the total possible immunoglobulin combinations approaches 10 7 (9600 x 1000).

Antibodies: Are glycoproteins are built of subunits containing –two identical light chains (L chains), each containing about 200 amino acids –two identical heavy chains (H chains), which are at least twice as long as L chains The first 100 or so amino acids at the N-terminus of both H and L chains vary greatly from antibody to antibody – they are termed variable (V) regions –unless members of the same clone (often not even then!), no two B cells are likely to secrete antibodies with the same V region –the amino acid sequence variability in the V regions is especially pronounced in 3 hypervariable regions also called CDR’s –the tertiary and quaternary structure of the antibodies brings the 3 CDR’s of both the H and L chains together to form the antigen binding site which binds the antigen epitope

Only a few different amino acids sequences are found in the C- terminus of the H and L chains and these are called constant (C) regions –two different kinds of C regions for L chains -- kappa (κ) and lambda (λ) –five different kinds of C regions for H chains mu (µ) chains, IgM gamma (γ) chains, IgG alpha (α) chains, IgA epsilon (ε) chains, IgE delta (δ) chains, IgD –each of these 5 kinds of H chains can pair with either lambda or kappa L chains

Constant-Region Domains The carboxyl-terminal domains of immunoglobulins display considerably less sequence variability within a given isotype than observed for V-region domains. These domains are referred to as constant (C) regions. H-chain C regions are numbered (C H 1, C H 2, C H 3, and C H 4) beginning with the most V-region proximal domain. The C region domains of the H-chain have been shown to be responsible for many aspects of antibody function, including interaction with Fc receptors, complement fixation, transplacental transfer, the ability to form multimers, and the capacity to be secreted across mucosal surfaces. Because different H-chain isotypes have different C region domains, these capabilities vary with the class of the particular antibody. Five major classes of H-chain C regions exist: (γ, α, µ, δ, ε). As a direct consequence of the correlation between the H-chain class of an antibody and its resultant effector functions, immunoglobulins have been named according to there heavy chain, i.e., IgG, IgA, IgM, IgD and IgE.

Hinge Region Immunoglobulins have a hinge region located C-terminal of the the C H 1 domain of their H-chains. In the case of the H-chains of the µ and ε isotypes the hinge is so elongated that it is actually an extra immunoglobulin domain explaining the presence of a fourth C domain in these isotypes. Other heavy chain isotypes (γ, α, and δ) use shorter stretches of polypeptide, which are thought nonetheless to have evolved from the C µ/ε 2 domain. Hinge regions are encoded by separate exons. The hinge region permits a considerable degree of flexibility between the antigen-binding and effector-interacting components of the immunoglobulin molecule.

Concept: All classes of antibody have the same basic structure. Two identical heavy chains and two identical light chains: each of which contributes to antigen binding. The Structural Organization of the Human Immunoglobulin Isotypes.

Structure of the 5 Major Classes of Antibody

Immunoglobulin G (IgG) The most abundant class in serum ~80% of the total serum immunoglobulins. IgG is found also in the interstitial spaces. The IgG molecule consists of two λ H-chains and two κ or two λ L-chains. In man there are four subclasses of IgG. IgG fixes complement IgG is the only immunoglobulin to cross the placenta. IgG reacts with FcR’s on phagocytic cells to promote opsonization.

IgD and IgE The principal role of IgD: Located on the surface of B-lymphocytes and along with IgM serves the function of antigen recognition by the B cell. IgE binds to mast cells and basophils through a high affinity FcR and is involved in mediating hypersensitivity reactions.

Study questions 1.Structures of all immunoglobulins 2. Molecular basis of ab diversity 3.Terminologies ie allotypes, idiotypes etc