v. Hinge region

Development Team

Paper Coordinator: Prof. Anju Srivastava

Department of Zoology, University of Delhi Principal Investigator: Prof. Neeta Sehgal

Department of Zoology, University of Delhi

Content Writer: Dr. Haren Ram Chiary

Kirori Mal College, Department of Zoology, DU Content Reviewer: Prof. Sukhmahendra Singh

Banaras Hindu University Co-Principal Investigator: Prof. D.K. Singh

Department of Zoology, University of Delhi Paper : 10 Immunology

Module : 14 Organization and expression of Ig genes: Diversity of immunoglobulins, structural variations in immunoglobulin constant regions

Description of Module

Subject Name ZOOLOGY

Paper Name Immunology Zool 010

Module Name/Title Organization and expression of Ig genes

Module ID M14, Diversity of Immunoglobulin, structure variation in Immunoglobulin constant region

Keywords Immunoglobulin, domain, Variable region, Constant Region, CDR,

Isotype, Allotype, Idiotype

Contents

1. Learning Objective 2. Introduction 3. Historical resume 4. Immunoglobulin

i. Antibody structure

ii. Immunoglobulin fine structure iii.

Immunoglobulin domains iv.

Variable region Domain and CDRs

v. Hinge region

5. Diversity of Immunoglobulin

i.

Diversity due to domain variations ii. Diversity due to hypermutation iii. Diversity due to variable domain

iv.

Diversity due to constant domain

6. Structure variation in Immunoglobulin constant region i.

Isotype determinants

a.

Immunoglobulin A

b. Immunoglobulin D c. Immunoglobulin E d. Immunoglobulin G e. Immunoglobulin M ii.

Allotype determinants iii. Idiotype determinants

8. Summary

1. Learning Outcomes

After studying this module, you shall be able to

• Learn about the basic structure of immunoglobulin molecules

• Understand the immunoglobulin fine structure

• Evaluate the different factors leading to diversity in immunoglobulin molecule.

• Learn about the structural variation in immunoglobulin constant region leading to functional variability’s.

2. Introduction

The word immunoglobulin came at around 1930’s when a class of proteins called globulins are termed as antibodies. Immunoglobulin or antibodies are the terms used interchangeably. Antibodies are Y-shaped divalent glycoprotein structure with heavy and light chain held together by disulfide bonds. On any microorganism mediated assault of host immune system, antibodies are generated against the antigen for its clearance. Antibodies are derived from mature B-lymphocytes (WBCs) of adaptive immune system which on antigen binding become plasma cells producing clone antibodies specific to that antigen. In order to cop up with the enormous diversity of pathogens, (antigens) antibodies require frequent adaptation to interact and combat against different emerging epitopes (antigens). More than 10 billion varieties of antibodies are generated in humans accounting for distinct sites on antigen for interaction. Thus, body needs to generate this much diversity of proteins against distinct and diverse antigens. The immunoglobulin has very limited number of genes encoding

However, complex mechanisms are adopted by the human body to produce spectrum of antibody differing in their specificities and functions from that small set of antibody genes.

3. Historical Resume

By 1930, we all know that immunoglobulin belongs to globulin, a distinct class of proteins. Pauling and Pressman revealed that the antibodies have antigen binding sites called “pocket” where antigen binds non-covalently. Eisen and his colleagues suggested the divalent structure of antibodies. Cambell and Bulman, 1952, showed 700ᵒ A limit of the antibody site for antigen binding. Svedberg suggested the molecular weight of antibodies to be 160 KDa.

Porter, 1950, with the help of a protolytic enzyme known as papain, cleaved antibodies at the specific sites resulting into three fragments, one with 50 KDa MW called crystallisable “Fc” fragment and other two similar fragments with 45 KDa MW called “Fab”, fragments antigen binding. Edelam and Ponlik suggested two basic sub units of immunoglobulin as Heavy (H) and light (L) chain with 50 KDa and 20 KDa of molecular weight (MW). In 1962, Edelman and Gally elucidated the nature of Bence Jones Proteins, named after Dr. Henry Bence Jones. In the patients with myeloma, they found dimmers of immunoglobulin light chains freely secreted in urine samples and named them as Bence Jones proteins.

Later on in 1960, based on the carboxyl region sequence differences immunoglobulin were classified into different classes and sub classes. With the development of X-rays crystallography, the antibody structures with its combining sites were elucidated in 1974.

4. Immunoglobulin

Among enormous and differentiated proteins found in the body, the most fascinating and critically important molecule is antibody which provides protection against antigens or foreign molecules. The molecular structure of immunoglobulin is multifunctional with an additional advantage of gene duplication and rearrangements. Vertebrates can generate specific antigen binding immunoglobulin molecules varying from 10¹⁰- 10⁴ or may be more in diversity. This unique characteristic of immunoglobulin (Ig) expressing structurally related still different molecules comes from the evolved cellular differentiation process of Ig producing cells where single immunoglobulin molecule specific for particular antigen is secreted from a single cell. The immunoglobulin is a globular structure with

three partitioning; one is Fc region and two similar Fab segments with antigen binding region. The component of complementary system as well as surface receptors on immune cells (FcR) binds specifically to the Fc segment of immunoglobulin. Additionally, the movement of immunoglobulin across the membrane, placenta and body compartment are only possible because of these interactions.

The movement of immunoglobulin molecules at the site of inflammation is also because of such interactions. The best example of immune response elicited by this type of interaction is ADCC (antibody dependent cell mediated cytotoxicity) where the Fc region of antigen bound antibody molecule is recognized by the innate immune cells (such as Natural Killer (NK) cells) and mediate a perforin dependent killing of target cells.

i. Antibody structure

Four polypeptides chains combined together to form a Y-shaped divalent structure of antibody molecules. Polypeptide of two identical heavy chain (H) of 50 KDa molecular weight comprising of nearly 450 amino acid residues and two identical light chains (L) of 25 KDa of 214 amino acids are attached together by disulfide bonds to form a four polypeptide divalent Y-shaped structure (figure 1). These disulfide bonds are critical for maintaining the divalent structure of antibody and variations in the precise position of bond formation and number of bonds among different classes and subclasses of immunoglobulin suggested their role in the immunoglobulin structure. Antibodies of different specificities vary in the sequence and composition of heavy and light chain amino acid terminals within their first 110 amino acids. Therefore, the difference in the antibody specificity can be attributed to the diversity in the amino terminal amino acid sequences of variable regions called V region. These V regions are called VH in heavy chain and VL in light chain of immunoglobulin. CDRs or complementary determining regions are highly variable regions in both heavy and light chain of immunoglobulin, forming the antigen-binding site and traced to differences in antibodies specificities.

Figure 1: Basic structure of antibody molecule

Region beyond 110 amino acids variable region have relatively constant composition and same sequence in different antibodies and termed as constant region of heavy (CH) and light (CL) chain.

Immunoglobulin with some exceptions is glycoproteins with constant region restricted binding of carbohydrate. Although the exact function of glycosylation or carbohydrate attachment is not known but immunoglobulin glycosylation enhance their solubility and via liver affects their clearance rate from the serum.

ii. Immunoglobulin fine structure

Protein organization at the primary, secondary, tertiary and quaternary levels determines the immunoglobulin structure. Amino acid sequences consitutes the primary structure of both heavy and light chains. The primary structure forms polypeptide chains which folds upon itself to form β-pleated sheets. These extended chains form a combat globular domain as a tertiary structure. The continuous polypeptide chain of β-pleated sheets connects these globular domains (figure 2 a). Finally functional domains are formed as a quaternary structure composed of heavy and light chains globular domains. It is this functional domain that act as antigen binding sites and perform effector functions.

(a)

(b) (c)

Figure 2: Immunoglobulin fine structure, (a) β pleated sheets held together with the help of disulfide bonds in variable and constant domains of immunoglobulin (b) and (c) β strand arrangement in the globular domains of constant domain and Variable (two extra β strands) domain.

iii. Immunoglobulin domains

Amino acid sequences of heavy and light chain of immunoglobulin molecules comprised of repeated homologous units of 110 amino acids forming an independent globular motif called immunoglobulin domain.

Within this globular motif called domain, a loop of 60-70 amino acids were formed by intrastrand disulfide linkage. Light chain of immunoglobulin comprised of variable domain, denoted as VL and constant domain as CL. Heavy chain of immunoglobulin structure comprised of single variable domain depending o the class of antibody and denoted as CH1, CH2, CH3 and CH4.

Immunoglobulin domains folded into characteristic protein motifs forming a compact structure called immunoglobulin fold. Variable length of loops connects three or four anti-parallel β- strand of polypeptide. These β-strands forms β-pleated sheets and are stabilised by hydrogen bonding. Two β- pleated sheets form a sandwich structure to form immunoglobulin fold, and these β-sheets within fold structure are stabilised by conserved disulfide linkages and hydrophobic interactions.

Although, variable domain show slightly longer structure with extra β strand pair and loop connecting this extra β strands present in the β sheet structure (Figure 2 b, c).

iv. Variable region Domain and CDRs

The variability among the amino acid sequences of heavy and light chain of variable domain includes exceptionally variable short polypeptide sequence termed as Hypervariable regions (HV). These short polypeptide residues are near 25-35, 50-55 and 95-100 amino acids.

About 15- 20% of variable domain structure comprised of such hypervariable regions and three such regions are present in heavy and light chains of variable domain. The remaining stretches of polypeptide segment constituting variable domain is very much conserved in nature and termed as framework regions (FR). The hypervariable forms the antigen binding site complementary to the structure of epitope present on the antigen and thus termed as CDRs or complementary determining regions. In the Fab the six loops of hypervariable regions determine the variation of antibody specificities. Framework region (FR) functions as a scaffold that supports the six loops in place within the immunoglobulin structure.

CDRs of heavy chain of variable domain (VH) are non specific and bind with high affinity to antigen than light chain CDRs due to the involvement of more amino acids residues of VH domain in antigen binding.

v. Hinge region

A proline, cysteine, lysine and aspartic acid rich extended polypeptide sequence between the first two regions of constant domain (CH1 and CH2) in heavy chain are termed as Hinge region. It is present in the γ; β and α heavy chain providing segmental flexibility as a result it allow Fc and Fab segments to move relatively thus acting as a tether to both the segments. Moreover, when antigens display its epitopes and Fc move to bind with Fc region specific receptors on other immune cell surfaces to perform biologically effector functions. Thus, hinge region provide flexibility because of which fragments of immunoglobulin can assume different angles relative to each other in order to bind with different epitopes and other immune cells. Proline at the hinge region make it more susceptible to proteolytic enzyme driven cleavage and also provide it extended polypeptide structure, this region is vulnerable to papain and pepsin cleavage. The two heavy chains are stick together due to intrastrand disulfide bonds as a result of cysteine residues at the hinge region. Among different species and between different classes of immunoglobulin, the number of disulfide bonds varies at the hinge region. Although hinge region is absent in μ and ε chain of IgM and IgE immunoglobulin, a hinge like additional domain lies between CH2/CH2 region of 110 amino acid residues. In the γ3 and δ chain 60 amino acids are there in the hinge region while α1, α2, γ1, γ2 and γ4 have 10 amino acid residues.

5. Diversity in Immunoglobulin

i. Diversity due to domain variations

The chromosomes has relatively large segment called loci or locus. Each loci for specific genes encodes for antibodies. Each domain of the antibody is encoded by different and distinct genes. In humans, chromosome number 2 and 22 represents specific locus for light chain κ (kappa) and λ (lambda) genes while chromosome number 14 represent heavy chain genes. Each antibody structure consists of heavy and light chains both chains have variable and constant domains.

Distinct B cells (not clones) generate different antibodies with much difference in variable domains.

ii. Diversity due to hypermutation

The stimulated mature B cells rapidly differentiate and the genetic material of the cell also duplicates rapidly because of which the genetic material incorporates point mutations or changes at very high

nucleotides change per division and each SHM contributes to formation of broad range of diverse antibodies.

iii. Diversity due to variable domain

Three loops of hypervariable regions namely HV1, HV2 and HV3 constitute the variable domain.

Approximately 65 different genes located on specific locus of heavy chain encode for variable domains and they differ in their hypervariable regions. This set of genes when undergo complex combination mechanisms produce a large and diverse range of antibodies. This permutation, combination and rearrangement of genes is termed as V (D) J recombination as diverse range of antibodies are produced from different variable domains. V (variable), D (diversity) and J (joining) are segments on the genes which encodes for heavy and light chain of variable domain on the antibodies. Variation in the assembly of variable domain leads to structural variations in the immunoglobulin molecules.

iv. Diversity due to constant domain

Additional to all the above suggested variations, immunoglobulin structural variations called isotypes can be generated by linking same variable region to different constant domains of heavy chain. At the heavy chain locus, the genes for constant region (CH) are located downstream to the variable genes determining the isotype of immunoglobulin and effector functions. Based on the variation of constant domain of heavy chain immunoglobulin is classified into five major isotyopes. Secreted and membrane bound forms of each isotype is produced by alternative splicing of mRNA of immunoglobulin. Isotype switching generates structural variations unique to constant domain of heavy chains (CH region).

6. Structural variations in constant region domain

Various biological effector functions are performed by immunoglobulin are determined by the amino acid sequence of constant domain. The CH1 and CH2 domains hold together the VH and VL domains of immunoglobulin via disulfide bonds and extend the Fab segments by virtue of the hinge region, thereby, facilitating the conformation rotation of two fragments and thus, contributing to epitope binding also.

CH1 and CL domain allow random association with VH and VL domains thereby leading to a diverse repertoire of antibody molecules (antibody diversity). Thus random gene rearrangements of immunoglobulin generate antibody diversity contributed by the constant region domain.

Other constant region domains

The immunoglobulin IgM and IgE comprised of four constant domains of heavy chain without any hinge region while IgA, IgG and IgD contains three constant regions with CH1 and CH2 joined by the hinge regions. CH4 and CH3 of two identical heavy and light chains contribute as the carboxyl terminal ends of IgE and IgM and IgA, IgG, and IgD respectively (Table 1).

Table 1: Distinct classes of Immunoglobulins with different heavy chain constant domain.

Class Hinge region

Constant domain heavy chain

1

Constant domain heavy chain

2

Constant domain heavy chain

3

Constant domain heavy chain

4

IgM - + + + +

IgE - + + + +

IgA + + - + -

IgG + + - + -

IgD + + - + -

The solubility of immunoglobulin in serum is virtue of the oligosachharide side chains present at the heavy chain of constant domain (CH2) of IgA, IgG, IgD while CH3 domain of IgM and IgE (figure 3).

These CH3 domains of immunoglobulin IgM and IgE are highly accessible for the stimulation of complement system thereby contributing to the biological effector function of the immunoglobulin molecules. Immunoglobulin is classified into five classes and further sub-divided into subclasses (IgG has four sub-classes, IgA with two sub-classes). The sub-classes can be either membrane bound (mIg) or secreted immunoglobulin (sTg), both differ in their structure and function at the carboxyl terminal domain. The membrane bound (mIg) contains a short cytoplasmic tail, a transmembrane sequence and

length hydrophilic sequence at the carboxyl terminal. The spacer sequence and cytoplasmic tail vary among all isotype of immunoglobulin.

(a) (b)

Figure 3: Schematic diagram of immunoglobulin with defined variations at the constant regions, (a) IgA, IgD and IgG has three constant domains while (b) IgM and IgE has four constant domains.

At different developmental stages of humoral cells or B lymphocytes, different membrane bound immunoglobulin are expressed. The membrane bound IgM is expressed by the immature pre-B cells while mature B cells co-expressed mIgM and IgD prior to any antigenic encounter. Antigenic stimulation triggers the differentiation of mature B cells into plasma cells and memory cells (figure 5).

Plasma cells express various isotypes of secreted immunoglobulin via isotype switching while memory cells express membrane bound immunoglobulin of mIgE, mIgG, mIgM or mIgE.

Immunoglobulin as Immunogens

Immunoglobulin are proteins which can induce an antibody (immune) response, thus acting as a potent immunogen, these are potential tools to study humoral immune response and B-cell development. Three major categories of antigenic determinants are classified on the immunoglobulin molecules: (A) isotype, (b) allotype and (c) idiotype determinants.

1. Isotype determinants

On the basis of differences in the constant domain of heavy chain, immunoglobulin is categorized into classes and sub-classes. Similarly light chains are categorized into its types and sub types based on the constant region of amino acids sequences differences. These amino acid differences are called isotypic determinants (figure 4). A unique and separate constant region encodes each isotype carried by all the members of a species. Different species inherit different isotypes, a reason why antibody of one species is recognized as foreign molecules by another species.

Figure 4: Isotypic determinants differentiated by comparing different immunoglobulin class of same species eg Mouse IgG1 and Mouse IgM.

On B lymphocytes activation, the heavy chain genes undergo class switching, a phenomena producing different immunoglobulin classes and also termed as CSRs or Class switching recombination (figure 5). The plasma cells produces IgG, IgE or any other class of Ig from IgM via class switching due to change in the constant domains of heavy chain (CH). The variable domain of heavy and light chain does not undergo CSR. Hence, the antigen binding specificities of isotypes antibodies has no changes.

The immunoglobulin are classified into five major isotypes based on the amino acid sequence differences at the constant regions of heavy chain contributing to the structural and functional properties which are class specific. The five isotypes are immunoglobulin A, IgD, IgE, IgG and IgM.

Figure 5: Developmental stages B-lymphoctes and Immunoglobulin expression on isotype switching.

a. Immunoglobulin A

Immunoglobulin A comprised of four polypeptide chains in its monomer form, but it may exist in dimeric, trimeric or tetrameric forms joined by the J-chains and called secretory immunoglobulin as present in the external secretions. The human immunoglobulin A has two sub-classes IgA1 and IgA2 and the heavy chain of IgA is α-type. It constitutes 10-15% of total immunoglobulin present in the serum, predominating in the secretory fluids like tears, saliva, breast milk and mucus secretions at different organs. The secretory immunoglobulin comprised of dimer or tetramer form, J chain and secratory component in virtue of transporting Ig across cell membranes. The J (joining chain) chain polymerized the immunoglobulin in its dimeric form as in case of pentameric IgM (figure 6, 8). The monomer form of IgA was 160 KDa M.W. and the secretory component is of 70 KDa molecular weight secreted from the mucus membrane lining epithelial cells.

Figure 6: A dimer form of IgA immunoglobulin, 1: Heavy chain, 2: Light chain, 3: J chain, 4: Secretory Protein

The Fc region of immunoglobulin binds with one of the five immunoglobulin-like domain of secretory component and stabilised by disulfide bonds between the Fc region of dimeric IgA and first Ig like domain of secretory component. Plasma cells are populated at the mucus membrane surface secretory IgA in abundance (5-15g) than any other immunoglobulin. The pentameric poly-Ig receptor is expressed on the glandular epithelia of lacrimal, salivary and mammary glans and on mucosal epithelia (urogenital tract, lungs, digestive tract). The plasma cells migrate to sub mucosa of mucus membrane and secrete immunoglobulin A, which tightly binds with the poly-Ig receptor and stimulate coated pit formation thereby leading to endocytosis of IgA receptor complex via receptor mediated cellular uptake pathway. These vesicles carrying Ig-A receptor complex when fuses with the luminal plasma membrane, the receptor is enzymatically cleaved from the luminal membrane and released together with IgA molecules as secretory component in the mucus secretion (figure 7). In the protease rich microenvironment of mucosal membrane the polymeric Ig-receptor protects the hinge region of dimeric IgA molecules from early proteolytic cleavage. The polymeric receptors also interact with J chain of IgM molecules and enable IgM transportation into mucus secretion across cell membrane.

Figure 7: Diagrammatic illustration of formation of secretory IgA molecules as it transport through epithelial cells mucosal membrane.

Secretory IgA are effector molecules at the mucosal sites of various organs. Mucosal sites are the major sites to encounter most pathogenic agents. Binding of Secretory IgA to multiple epitopes of large antigens such as viral or bacterial surface antigens can block the attachment and colonization of infections agents on the mucosal sites. Secretory IgM provide line of defence against several virus and bacteria such as influenza, polio, reovirus and Vibrio cholera, Salmonella, Neisseria gonorrhoeae respectively. An infant’s immune system is not fully developed during the first months; therefore, secretory IgA molecules through breast feeding can maintain the health of baby by protecting it against infections.

b. Immunoglobulin D

Immunoglobulin D is expressed on mature B cells surface and like IgM, it’s a major membrane bound antibody molecules. With no biological effector functions, IgD helps in stimulating B cells by antigens. IgD exist in a monomer form with δ as heavy chain component and 150 KDa MW (figure 8). It constitutes 0.2% of total serum immunoglobulin with 0.03mg/ml serum concentration. In humans, there is no sub-class of IgD molecule. IgD, probably function in activating or suppressing

lymphocytes. They cannot cross placenta, activate compliment system or cause mast cell granulation release.

Figure 8: General structures of immunoglobulin molecules, IgD, IgE and IgG in their monomer form while polymeric forms of IgA and IgM.

c. Immunoglobulin E

Pransitz and Kustner, 1921, suggested allergic reactions are a response to serum component presence.

When a non allergic person is injected intradermally with the serum of allergic person and again injected at the same site, develop a flare reaction called P-K reaction due to IgE activity. IgE antibody mediated hypersensitive reactions develop asthma, anaphylactic shock, high fever and hives as symptoms. Although the serum concentration is 0.3 μg/ml for IgE, it is a potential and critical biological effector molecule existing in its monomer form (figure 9). Binding of receptor bond IgE molecule to allergen and with its Fc region to the membrane receptors on mast cells, natural killer cells and basophils leads to antibody dependent cell mediated cytotoxicty (ADCC) by the release of granules from mast cells and basophils (granulocytes) releasing pharmacologically active mediators leading to allergic manifestations. IgE molecules also provide anti-parasitic defence system.

Figure 9: Monomer form of IgE

d. Immunoglobulin G

About 80% of total serum Ig consists of IgG, thus, considered as most abundant class of Ig in serum.

The molecular weight is 150 KDa, and structure comprised of two identical heavy chains of γ type and two light chains (λ and κ). Immunoglobulin class IgG categorized into four sub-classes IgG1, IgG2, IgG3 and IgG4 numbered and differentiated in their serum concentration and γ chain sequences.

The four sub-classes of immunoglobulin G are structurally differentiated on the basis of hinge region size, position and number of disulfide bonds between the heavy chains (figure 10, 11) and thus, effects the biological functions between sub-classes such as:

1. IgG1, IgG2 and IgG4 can cross the placenta and critically important for neonatal immunity development in foetus.

2. Immunoglobulin acts against pathogen and their toxins; besides stimulating complementary system of the body.

3. The IgG forms complex with the microorganism and activate the complement system, IgG3 is the most potent activator followed by IgG1 and IgG2 as complement activator.

4. IgG1 and IgG3 are excellent opsonins which enhances the phagocytosis of other innate immune cells such as Natural Killer (NK) cells, macrophages etc by binding with the Fc receptors on their surface. IgG4 and IgG2 have less affinity towards Fc region receptors, therefore, does not contribute much in opsonisation.

Figure 10: General structure of IgG immunoglobulin molecule sub classes IgG1 (2 bonds) and IgG2 (4 bonds) which differ in number and arrangement of disulfide bonds.

Figure 11: General structure of IgG immunoglobulin molecule sub classes IgG3 (11 bonds) and IgG4 (4 bonds) which differ in number and arrangement of disulfide bonds.

e. Immunoglobulin M

The total serum concentration of IgM is 1.5 mg/ml and constitutes 5-10% of total serum

lymphocytes. On antigen binding B cells differentiate to plasma cells secreting pentameric form of IgM of molecular weight 900 KDa (monomeric MW is 180 KDa). The pentameric form of IgM is held together by their carboxyl terminal Fc region, more specifically, Cμ4/Cμ4 and Cμ3/Cμ3 domains by virtue of disulfide bonds. This combined structure of five monomeric forms offers ten antigen binding sites facing towards periphery of the pentamer molecule while effector Fc region at the centre.

Besides, disulfide bonds between heavy chains of monomers; a polypeptide chain called J (Joining) chain is linked to the two monomer of IgG with the help of disulfide bonds (figure 12).

Figure 12: Serum immunoglobulin IgM always present in pentameric form containing a J chain.

This additional Fc linked J chain binds to the cysteine residues of the carboxyl terminal ends of two of the ten μ chains. Just before pentamer formation of IgM molecules, a J chain polymerizes the monomer forms (figure 12). There is no sub-class of immunoglobulin M.

On antigenic stimulation of B lymphocytes in primary immune response first immunoglobulin class produced is IgM. It is the first immunoglobulin molecule produced by newborn. Serum IgM contribute maximum valency for antigen binding than any other isotypes. Although IgM molecules can bind 10 molecules of hapten but in case of larger antigens and due to steric hindrance, only 5 can bind simultaneously. The pentamer form of IgM is potentially efficient than other isotypes in virtue of its multivalent activity against multidimentional antigens. Briefly, considering RBCs as multidimensional antigen, its incubation with antibodies leads to agglutination. In comparison to IgM any other class/ isotype will require 1000 times more molecules to achieve the same agglutination

status. IgM is most efficient in complement system activation. A Pentameric IgM molecule is non diffusible because of its large size. Like IgA molecules, it also forms complex with secratory components which allow IgM transportation through epithelial lining to mucosal surfaces and thus, act as an important secretory immunoglobulin.

2. Allotypic determinants

Although isotypic determinants inherit in all members of species, some genes exhibit multiple allelic forms encoding amino acids differences in some members of species called allotypic determinants or allotropes (figure 13). Allotopes has been characterized for all four IgG sub class or γ chain, one IgA or α chain and one κ chain. The γ heavy chain markers are referred as Gm markers. Depending on the allelic number, class and sub class types total 25 Gm markers are identified, for instance G4m (4a), G1m (1), G3m (11) etc. Among IgA subclass, only IgA2 has allotropes, for eg. A2m (1) and A2m (2).

These allotropes exist for κ chain (κm(1), κm (2) and κm (3)). Allotropic determinants usually differ in one to four amino acid residues.

Figure 13: Allotypic determinants differentiated by comparing same immunoglobulin class among different inbred strains.

The amino acid sequences at the variable domain of heavy and light chains play dual function one as an antigen binding site and via conformational changes in variable region as antigenic determinants also referred as idiotope or idiotypic determinants (figure 14). In some individuals, the idotypic determinants are the sequence of variable region outside antigen binding site while an idiotype may be the actual antigen binding site also.

Figure 14: Idiotypic determinants differentiated by comparing same immunoglobulin class against different epitopes.

B cells of same clone produces antibodies are characterized by identical variable regions and thus have same idiotype. Multiple idiotopes are present on individual antibody molecules, and the sum total of all idiotopes referred as idiotype of antibody.

7. Summary

Immunoglobulin is glycoprotein molecules with four polypeptide Y-shaped structure. The divalent Y- shaped immunoglobulin structure is related to their structural and functional variations, antigen binding specificity, diversity and genetic control. Any foreign molecule can elicit an immune response in the host body, and the response is specific and diverse. This diversity is attributed to the recognization and binding of diverse antigenic determinants or epitopes to antibody molecules with

different affinity and specificity. Immunoglobulin repertoire is a general term used for the sum of all antibodies with different specificities. The immunoglobulin is a Y-shaped polypeptide structure comprised of two identical heavy chains and two similar light chains. Each heavy chain has one variable domain (VH) and three to four constant domains (CH1, CH2, CH3 or CH4). Teach light chain has one variable (VL) domain and one constant domain (VC). These polypeptides are interconnected and stabilized by the disulfide bonds. Some immunoglobulin have hinge regions connecting variable domain and constant domain n of heavy chains, also contributing to the flexibility of the immunoglobulin which aid in effector functioning. Among different means factors creating broad range of immunoglobulin repertoire, somatic hypermutaion in the genetic segments of the variable domain act as a critical factor in determining the extraordinary diversity of immunoglobulin. Besides, SHM which means point changes in the genetic material of rapidly proliferating activated B cells, different gene rearrangements and combinations of hypervariable regions (HV) or CDRs (complementary determining regions) of heavy chain variable region also called as Class switching, also contribute to diversity of immunoglobulin. Each isotype has separate expression of constant domain genes, hence, immunoglobulin isotype are dependent on the constant region of heavy chain.

Based on the constant region domain differences, immunoglobulin are classified into five isotype- IgA, IgD, IgE, IgG and IgM, all differing in their immunological functions and under different conditions and different timings respond differently to same epitope or antigenic determinants.