An antibody, Immunoglobulin, and its types (Ig G, Ig M, Ig A, Ig D, and Ig E)

 An antibody, Immunoglobulin,                 and its types:

Immunoglobulin	Antibody
Immunoglobulin refers to any types of structurally related proteins in the serum and the cells of the immune system which functions as antibodies.	Antibody refers to a globin protein, which is produced by B cells in response to a particular antigen.
Occurs on the surface of B cells	Freely occurs in the circulation
Has a transmembrane domain in order to be attached to the plasma membrane of B cells	Doesn’t have transmembrane domains
Five classes are Ig G, Ig M, Ig A, Ig D, and Ig E	A particular antibody is specific to a particular antigen
Functions depends on the type of heavy chain	Non-self antigens are recognized by specific antigens and are neutralized by antibodies

 

Nature:

·       Antibody molecules are glycoproteins.

·       Tiselius and Kabat (1938) subjected immune serum to electrophoresis and separated its protein into four major fractions - serum albumin, alpha (α), beta (β), and gamma (γ) globulins.

·       Most immunoglobulins are found in the gamma fraction and some are in beta fraction.

·       Immunoglobulins are a heterogeneous group of proteins and constitute about 20% of plasma proteins.

·       Based on sedimentation studies using ultracentrifuge, it was found that most antibody molecules sediment at 7S (Mol. Weight 150000Da) and some at 19S (Mol. Weight 900 000Da). ''S‟ stands for Svedberg unit = a sedimentation constant of 1 x 10 –13 seconds.

·       Protein molecules that bind specifically with antigen are called antibodies.

·       Proteins with antibody activity are called immunoglobulins.

·       The term immunoglobulin is a structural and functional concept; antibody is a biological and functional concept.

·       Immunoglobulins have been classified into five groups based on physicochemical and antigenic differences – IgG, IgM, IgA, IgD, and IgE.

Structure of Immunoglobulins: 

·       The immunoglobulins are a large group of closely related heterogeneous glycoproteins.

·       An immunoglobulin molecule is about 160 KDa and composed of 2 parts of peptide chains of different sizes.

·       The larger chains are called heavy (H) chains (50-60 KDa) and smaller chains are called light (L) chains (25 KDa).

·       Both the light chains and both the heavy chains are identical.

·       The carboxy (C) terminus domains of the heavy chains are inserted into the lipid bilayer of the B cells surface membrane.

·       The light chains are only half the length of heavy chains and they are linked to heavy chains at its amino (N) terminus by disulfide (-S-S-) bonds, giving a „Y‟ shaped appearance.

·       Two heavy chains are also joined together by disulfide bonds.

·       The tail of „Y‟ is FC (crystallizable fragment can be crystallized in the cold) region is attached to the B cell and the arms called Fab region which binds antigen.

·       The antigen-binding sites are formed by the groove created between light and heavy chains at their N-terminus.

Heavy chains:

·       In general, there are five types of Ig heavy chain denoted by the Greek letters: α, δ, ε, γ, and μ, which determines the class of antibodies such as IgA, IgD, IgE, IgG, and IgM antibodies respectively.

·       Distinct heavy chains differ in size and composition, α and γ contain approximately 450 amino acids, while μ and ε have approximately 550 amino acids.

·       Each heavy chain has two regions, the constant region, and the variable region.

·       The constant region is identical in all antibodies of the same isotype but differs in antibodies of different isotypes.

·       Heavy chains γ, α, and δ have a constant region composed of three domains and a hinge region. Heavy chains μ and ε have a constant region composed of four domains.

·       The variable region of the heavy chain differs in antibodies produced by different B cells, but they are the same for all antibodies produced by a single B cell or B cell clone.

·       The variable region of each heavy chain is approximately 110 amino acids long and is composed of a single Ig domain.

Light chain:

·       Two light chains are either κ (Kappa) or λ (lambda) but never both in one immunoglobulin.

·       The names were derived from Koragold (κ) and Lapori (λ) who were originally described. Each light chain consists of two domains of 110 amino acids.

·       The amino acid sequence of the C-terminus domain is constant (CL) whereas the sequence of the N-terminus is variable (VL) and some regions show great variability, known as hypervariable regions or complementarity determining regions (CDRs) or hot spots, each containing 6-10 amino acids. Between the CDRs there are intervening peptide sequences called framework regions.

·       Three regions show maximum variability i.e. at positions 24-34, 50 to 56, and 89-97.

·       The amino acid sequence in the VL domain of each light chain is different. The molecular weight of each light chain is 25KDa.

·       The presence of the κ or λ light chain varies with species. For example, cattle and horses have 95 % λ, rats and mice have over 95 % κ chain, monkeys have 50 % of each, and humans have 60 % κ chains.

Hinge region:

·       Fab regions of immunoglobulin which bind antigen can swing around the central molecule and it is possible as they are hinged.

·       The hinge region is about 12 amino acids long and located between CH1 and CH2 and the sequence is unique for each immunoglobulin class and subclass. But the μ and ε heavy chains do not have hinge regions.

·       The hinge region of Ig D lacks cysteine residue thus there are no interchain links.

·       This region is hydrophilic and rich in proline residues.

·       When digested with the proteolytic enzyme papain, it cleaves at hinge region by hydrolysis of peptide bonds and produce 3 fragments, 2 ‘Fab’ fragments (sedimentation coefficient is 3.5S) and one FC fragment.

·       When digested with pepsin, the site of cleavage is towards the carboxy-terminal side of the disulfide bond and produces two Fab fragments held together [referred as F(ab) 2] with a sedimentation coefficient of 5S and FC fragment is digested into multiple fragments.

 

IMMUNOGLOBULIN CLASSES

·       The five different forms of immunoglobulin molecules are common to all members of a particular species and referred to as immunoglobulin isotypes or classes.

·       Different forms of the immunoglobulin isotypes are class sub isotype or subclass. They are,

1.      IgG

2.      IgM

3.      IgA

4.      IgE

5.      IgD

 

IMMUNOGLOBULIN G(IgG)

·       This is the major source of immunoglobulin secreted by plasma cells (plasma cells develop from B cells on antigenic stimulation and secretes immunoglobulins) found in the spleen, lymph nodes, and bone marrow.

·       IgG is 7S immunoglobulin with a molecular weight of 160 KDa found the highest concentration in blood.

·       It constitutes about 80 % of all immunoglobulin and about 20 % of plasma proteins.

·       They have the lowest electrophoretic mobility and are found in the γ fraction.

Structure:

·       It has two heavy γ chains and two light chains of either κ or λ types but not both. Fc region has CH1, CH2, and CH3 domains.

·       It is the smallest of all immunoglobulins and can easily escape the blood vessels into the area of inflammation and distribution in extravascular and intravascular compartments.

·       It is found in almost all tissue fluids and secretion except CSF.

·       It is the only immunoglobulin that can pass placental barriers and is found in newborns because of passive immunization.

·       Based on antigenic and structural differences in heavy chains the subclasses of IgG in humans are IgG1 (65-70%), IgG2 (23-28%), IgG3 (4-7 %), and IgG4 (3-4%) and numbers were given in accordance with their decreasing average serum concentration.

·       Interchain disulfide bindings vary between two heavy chains at their hinge region. It is less in IgG1 and more in IgG2, IgG4, and IgG3 in order. In dogs and cats also IgG has been classified into IgG1, IgG2, IgG3, and IgG4. In cattle, IgG is classified into IgG1, IgG2, and IgG3.

·       In horse it is IgG1, IgG2, IgG3, IgG4, IgG5 and IgG6. In sheep, they are classified as IgG1, IgG2, and IgG3.

·       · IgG contains about 3 % carbohydrate. Half-life varies with species.

·       IgG has a high affinity.

Functions

·       Act as antitoxins

·       Form antiviral antibodies

·       Act as precipitins

·       Act as complement-fixing antibodies.

·       Provide passive immunity in newborn animals or birds

·       It is produced later to IgM but provides long-lasting immunity.

·       CH3 domains of the FC region bind to macrophages for biological activity.

 

IgM (MACROGLOBULIN)

·       It is also secreted by plasma cells in the spleen, bone marrow, and lymph nodes.

·       It has a sedimentation coefficient of 19S (900 KDa) and constitutes about 5-8% of all immunoglobulins and giving rise to the second-highest concentration in mammalian serum.

Structure

·       On B cell surface, IgM is a single monomer (180KDa) but the secreted IgM is a pentamer (occasionally hexamer).

·       Five monomers of IgM are linked in a circular fashion by disulfide bonds between heavy chins and one-addition chain rich in cysteine and asparagine called J Chain (15 KDa) joining two monomers to complete the circle.

·       J chain is about 118-125 amino acids long.

·       Each IgM monomer has two heavy μ chains and 2 light chains of either κ or λ.

·       Each heavy chain has an additional domain (CH4) at the C terminus of the FC region.

·       IgM does not have a hinge region and contains about 12 % Carbohydrate.

·       IgM is predominantly present intravascularly (80%) and theoretically the pentamer.

·       IgM has a valence of 10 (each subunit possesses two antigens binding sites) but practically found to be 5 probably due to steric hindrance.

·       IgM found in serum (pentamer) can be disrupted by treating with 2-mercaptoethanol (0.12M) into monomers, as disulfide bonds joining the monomers are broken.

·       This treatment differentiates IgM antibodies from IgG antibodies.

·       IgM is the first antibody produced in the primary response to antigen and half-life is relatively shorter than IgG. Based on peptide mapping and complement-fixing activity IgM is classified into IgM1 and IgM2 in humans but not in other animal species. IgM has high avidity.

Functions:

·       Biologically more active than IgG. A single molecule of IgM can cause immune hemolysis whereas 1,000 IgG are required for the same effect.

·       IgM is 500-1000 times more effective than IgG in opsonization, 100 times more effective in bactericidal action, and 20 times more effective in agglutination.

·       The complement-binding site is present in CH4 and CH3 domains. It acts as complement fixing antibody. It is more active than IgG.

·       It is the first antibody produced in primary immune response.

 

 

IMMUNOGLOBULIN A (Ig A)

·       It is produced by plasma cells present mainly in the intestinal tract, respiratory tract, urinary tract, mammary gland, and skin.

·       Serum concentration is usually lower than IgM (except in humans).

·       It is the major Immunoglobulin found in colostrums, saliva, and tears.

·       IgA has a sedimentation coefficient of 7S with a molecular weight of 160 KDa to 360 KDa.

Structure

·       IgA molecule has two α heavy chains containing 3 constant domains (CH1, CH2, CH3) and either two κ or two λ light chains.

·       IgA occurs in two forms.

1.      Serum IgA

2.      Secretary IgA

·       Serum IgA is a monomer (7S with molecular weight 160 KDa).

·       But secretory IgA (SIgA) usually found in mucosal surfaces and in secretions is a dimer.

·       It is formed by two 7S IgA monomers joined at their carboxy terminus of the Fc region by the “J” chain and also with a secretory component (Sc).

·       “J” chain is synthesized by the same plasma cells.

·       Secretary component is a glycoprotein (71 KDa), synthesized in the epithelial cells of the mucous membrane (not by plasma cells) and present as a coil about the double FC cylinders.

·       Synthesis of Sc is independent of the production of IgA.

·       The dimer IgA (SIgA) is much longer (11S with a molecular weight of 360KDa).

·       The Sc protects the SIgA from proteolysis by the gastrointestinal enzymes.

·       Two IgA subclasses have been identified IgA1 and IgA2.

·       The subclass IgA2 lacks disulfide bonds between the heavy and light chains. It is a minor component in serum but more present in secretions.

·       The subclasses IgA1 and IgA2 were recorded in humans, cats, mouse, sheep, and pigs. IgA possesses about 7% carbohydrate.

Functions:

·       Provide local immunity to the mucosal surface of the respiratory and intestinal tract.

·       It does not fix complement but can activate alternative complement pathways.

·       It helps in phagocytosis and intracellular killing of microorganisms

·       It is a minor component in systemic humoral immunity but plays a major role in mucosal immunity. IgA antibodies found in gut contents or feces are known as copro-antibodies.

 

IMMUNOGLOBULIN E (Ig E)

·       It is synthesized by plasma cells located beneath body surfaces. It has two ε heavy chains and two light chains (either κ or λ).

·       Constant region of heavy chain composed of four domains (CH 1 to CH4).

·       It has a sedimentation coefficient of 8S (molecule weight 190 KDa). IgE has about 12 % carbohydrate.

·       The serum concentration is very low with the shortest half-life.

Functions

·       Reaginic antibody: Provide protection against microbes by degranulation of mast cells. IgE adheres to the cell surface through CH3 and CH4 domains and releases inflammatory mediators.

·       It mediates immediate hypersensitivity reaction (Type-1) e.g. anaphylactic shock, hay fever, asthma, etc.

·       Provide immunity against parasitic worms.

·       It does not fix complement components.

IMMUNOGLOBULIN D (Ig D)

·       It is secreted by plasma cells in the spleen and lymph nodes. It has two δ heavy chains and two light chains (either κ or λ) with a molecular weight of 180 KDa (7S).

·       Mouse IgD lacks CH2 domain and hinge region separates these two domains. But cattle, sheep, pigs, and humans have 3 constant domains CH1 to CH3.

·       It has no interchain disulphide bonds. It is susceptible to proteolytic enzymes and thus could not be detected in serum but found in low concentrations in plasma.

Functions

·       It is not found in all animal species

·       Function is not well established

 

IMMUNOGLOBULINS IN BIRDS

·       Predominant serum immunoglobulin in birds is IgY similar to mammalian IgG,

·       Heavy chain is Epsilon.

·       Chicken possesses the full-sized 180 KDa isoform of age. Ducks and geese possess full-sized and truncated (120KDa) isoforms.

·       A monomeric IgM can be defected in the amniotic fluid of eggs and in one day old chicks.

·       IgA is present in chicken secretions and polymers are common.

·       An avian homolog of IgD has been identified.