Immunoglobulin Structures & Antibodies Functions PDF

Immunoglobulin structures & antibodies functions Learning Objectives On completion of this session you should be able to 1) Describe the basic structure of immunoglobulins. 2) Explain the diverse functions of antibodies in immune defence. 3) Define antibody response upon encountering antigen...

Immunoglobulin structures & antibodies functions Learning Objectives On completion of this session you should be able to 1) Describe the basic structure of immunoglobulins. 2) Explain the diverse functions of antibodies in immune defence. 3) Define antibody response upon encountering antigens. Ig Structure 1) Structure and function are interlinked. 2) Immunoglobulins or antibodies have 2 functions: a) To recognise antigen b) To engage effector mechanisms to dispose of antigen 3) Recognition sites vary between antibodies and constitute the variable region or V region 4) Antibodies of the same class elicit the same effector mechanisms through the constant region or C region Ig Structure 1) Antibody molecules are Y-shaped and consist of 2 heavy (H) and 2 light (L) chain polypeptides held together by disulphide bonds 2) M.W. is approximately 150 kDa 3) The 2 heavy chains (M.W. 50 kDa) and 2 light chains (M.W. 25 kDa) are identical 4) Light chains can be either kappa (k) or lambda (l) 5) No functional difference between k and l chains and ratio in humans is 2: 1 and 20:1 in mice Ig Structure Ig Structure 1) Constant region, heavy chains (m, d, g, a, e) determine the class and function of antibody either IgM, IgD, IgG, IgA or IgE 2) H and L chains comprise both variable and constant domains 3) Each chains consists of 110 amino acid sequence repeats 4) L chains have 2 sequences and H chains 4 sequences and these correspond to structural domains 5) N-terminal domains vary considerable in sequence and are designated the VH and VL domains Ig Structure 1) VL and VH domains form the V region of the antibody 2) C-terminal domains (CL, CH1, CH2, CH3) are constant for each isotype and form the C region of the molecule 3) H and L chains associate such that VH and VL domains are paired, as are the CL and CH1 domains and CH3 domains 4) CH2 domains do not interact as carbohydrate is present between them 5) Antibodies are glycosylated (glycoproteins) Ig Structure 1) Antibody molecules can be cleaved into three portions with proteases 2) Papain cleaves the H chains on the N-terminal side of the S=S bonds in the hinge region 3) This releases 2 x Fab fragments (fragment antigen binding) and 1 x Fc fragment (fragment crystallisable) 4) Pepsin cleaves and antibody on the C-terminal side of the S=S bond releasing 1 x F(ab’)2 fragment 5) Pepsin cleaves the Fc fragment into several peptides Ig Structure – Hinge Region The polypeptide chain linking CH1 and CH2 domains forms a flexible hinge 1) This allows the angle between the two Fab regions to vary 2) Some flexibility is also found at the junctions between the V and C domains 3) This enables both Fab arms to bind sites different distances apart and for Fc portions to engage effector mechanisms Ig Structure - Domain Structure 1) V and C domains form similar structures consisting of b-pleated sheets arranged as a b-sandwich 2) A b-sandwich is 2 b-sheets lying face-to-face 3) b-sandwiches are stabilised by S=S bonds 4) The V domain has a 4-strand + 5-strand structure and the C- domain a 4 strand + 3-strand structure 5) These structures constitute the immunoglobulin fold which is found in many proteins including the T cell receptor & MHC proteins (immunoglobulin superfamily)  Antigen Binding Sites 1) Made up from the VH and VL domains 2) V region has b-pleated sheet structure 3) How is variability produced? 4) Amino acids comprising V-domains show 3 areas of variability designated the hypervariable regions, HV1, HV2 and HV3. 5) For both chains these regions approximately comprise residues: HV1 28 - 35 HV2 49 - 59 HV3 92 - 103 Antigen Binding Sites 1) Remaining residues in V region are less variable and termed the framework regions (FR1, FR2, FR3, FR4) 2) FR regions form b-strands and the characteristic structure 3) Residues in HV regions form loops BC, C’C” and FG that are adjacent in folded protein 4) When VH and VL domains associate, HV loops create the antigen binding site Antigen Binding Sites 1) As they form a surface complementary to the antigen, they are also referred to as the complementarity-determining regions or CDRs (CDR1, CDR2, CDR3) 2) The combination of different H and L chains increases the diversity of antigen binding sites, a concept called combinatorial diversity Antigen:Antibody Interactions 1) The part of an antigen to which an antibody binds is called the epitope 2) Epitopes can be continuous or discontinuous 3) Antigen:antibody interactions are mediated by topological and chemical factors 4) Interactions are stabilised by non-covalent bonds (electrostatic, hydrogen bonds, Van der Waals and hydrophobic interactions) 5) B cells can recognise lipid, polysaccharide, proteins, small molecules Antibody Function 1) Five classes (isotypes) of antibody IgM, IgD, IgG, IgA, IgE 2) Differ in: no & location of S=S bonds no of glycans no of C domains length of hinge region 3) Two subclasses (allotypes) of IgA - IgA1 and IgA2 4) Four subclasses (allotypes) of IgG - IgG1, IgG2, IgG3, IgG4 IgG 1) Most abundant isotype in serum and extracellular fluid (80% immunoglobulin) 2) Monomer of MW 150 kDa 3) Four subclasses numbered according to serum concentration: IgG1 9 mg/mL IgG2 3 mg/mL IgG3 1 mg/mL IgG4 0.5 mg/mL 4) Subclasses vary in hinge region and number and position of disulphide bonds between heavy chains IgG IgG 1) IgG neutralises or opsonises pathogens and activates complement 2) Subclasses have differing biological activities 3) IgG2 and IgG4 are poor activators of complement and poor opsonins – function as neutralising antibodies 4) IgG1 and IgG3 activate complement and phagocytosis – promote inflammatory responses 5) All IgG subclasses cross the placenta via binding to FcRn IgM 1) Accounts for 5 - 10% serum immunoglobulin 2) Monomeric form on B cell surface 3) Pentamer in serum (MW 900 kDa) linked by S=S bonds between CH4 and CH3 domains 4) Each pentamer has one J chain required for polymerisation 5) IgM is first antibody class to be released during an immune response IgM 1) Antibodies are low affinity but pentameric structure allows strong binding 2) Bind to viruses and cellular targets and cause neutralisation by agglutination 3) Very efficient activators of complement 4) Low concentrations in extracellular fluid but IgM is found in external secretions  IgA 1) 10 - 15% total serum immunoglobulin 2) Predominant immunoglobulin in external secretions (breast milk, saliva, tears, mucous of respiratory, GU and GI tracts) 3) In external secretions IgA is a dimer or tetramer comprising a J-chain and secretory component (MW 150 - 600 kDa) 4) 5 - 15 g IgA is secreted daily 5) IgA functions primarily as a neutralising antibody 6) Weak opsonin and activator of complement 7) Two subclasses have similar functions but IgA1 is most predominant  IgE 1) Low serum concentration (0.3 µg/mL) 2) Monomeric immunoglobulin (MW 190 kDa) 3) Mediate hypersensitivity reactions via binding to Fce receptors on mast cells and basophils 4) IgE triggers release of chemicals that induce reflex reactions (coughing, sneezing, vomiting) IgD 1) B cell surface associated IgD mediates B cell activation 2) Only functions as BCR Antibody Responses Three kinds of antibody response occur 1. Initial IgM production from B1 cells and marginal zone B cells T cell- independent (TI) response Occurs within 48 hours Shared with innate response Predominantly low affinity IgM antibodies Antigens stimulating TI responses comprise repetitive protein/carbohydrate sequences Antibody Responses 2. Early T cell dependent (TD) response From marginal zone and follicular B cells Clonal expansion of B cells High levels of antibody produced Mainly IgM, some IgG Produced 3 – 4 days after infection Antibody Response 3. Germinal Centre Reaction TD response Forms germinal centre in B cells follicles 1-3 weeks to develop fully IgG, IgA and/or IgE produced Secreted by long-lived plasma cells in bone marrow or other site Viable for many years Humoral Response – Primary vs Secondary Summary 1. Antibodies have a Y-shaped structure with variable regions that bind to antigens. 2. Different antibody classes have unique features and functions. 3. Three kinds of antibody response occur.