Lecture 6: Antibody Structure and Function PDF

MICI 3115 Lecture 6: Antibody Structure and Function (textbook Parham 5E – chapter 4; Kuby 8E - chapter 3)  Antibodies (Immunoglobulins) Glycoproteins on the surface of B cells and secreted by plasma cells. Present in blood serum and secreted fluids such as saliva and...

MICI 3115 Lecture 6: Antibody Structure and Function (textbook Parham 5E – chapter 4; Kuby 8E - chapter 3)  Antibodies (Immunoglobulins) Glycoproteins on the surface of B cells and secreted by plasma cells. Present in blood serum and secreted fluids such as saliva and milk. Activate the classical complement pathway. Act as opsonins to enhance phagocytosis. Neutralize toxins and viruses. Function as antigen receptors for B cells.  The B Cell Receptor Membrane-bound immunoglobulin (mIg) molecules have very short cytoplasmic tails and so are not able to interact with intracellular signaling molecules. The antigen-binding receptor on B cells consists of mIg and the disulfide- linked Ig-α/Ig-β heterodimer. The cytoplasmic tails of both Ig-α and Ig-β interact with intracellular signaling molecules such as tyrosine kinases. Plasma cells secrete antibody of the same antigen specificity as that of the antigen receptor of the B-cell precursor  Immunoglobulin Structure Monomers (IgA, IgD, IgE, IgG, and IgM) are composed of 2 identical heavy chains (5 classes designated α, δ, ε, γ, µ) and 2 identical light chains (2 classes designated κ and λ), held together by disulfide bonds and noncovalent interactions. Globular domains of ~110 amino acids are formed by intrachain disulfide bonds. V region of heavy and light chains is characterized by a variable amino acid sequence. C regions of heavy and light chains have a relatively constant amino acid sequence. Glycosylation affects antibody stability and its interactions with other proteins.  Immunoglobulin Structure Light chains contain 1 VL and 1 CL domain. Heavy chains contain 1 VH domain and 3 or 4 constant domains, designated CH1-4. V region domains at the amino-terminal portion of heavy and light chains form the antigen-binding site. Three hypervariable regions within the V region show even greater amino acid sequence variability and form the antigen-binding site while framework regions outside of the hypervariable regions exhibit much less amino acid variability. The hypervariable regions of antibody V domains lie in discrete loops at one of the of the domain structure  Proline-rich amino acid sequences between the CH1 and CH2 domains form a flexible hinge region in IgA, IgD, and IgG molecules.  The CH2 domain of IgM and IgE has hinge region-like properties.  Immunoglobulin Structure-Function C region domains at the carboxyl-terminal portion of the heavy chain mediate biological effector function. Differences in the various heavy chain constant region domains determine antibody half-life, distribution, complement-fixing ability, and Fc receptor binding. The carboxyl terminal domain of membrane-bound and secreted antibody is different in both structure and function. Different classes of membrane-bound immunoglobulin molecules are expressed by B cells at different stages of their development.  Proteolysis of IgG Enzymes (papain, pepsin) can cleave IgG molecules into specific fragments. Papain-mediated digestion of an IgG molecule yields 2 identical Fab fragment (fragment antigen- binding) and 1 Fc fragment (fragment crystalizable). Pepsin-mediated digestion of an IgG molecule yields a divalent F(ab’)2 fragment and a pFc’ fragment.  Immunoglobulin Epitopes Antibodies are immunogenic and produce an antibody response when injected into other species. Isotypic epitopes are located in constant regions and define heavy chain classes/subclasses and light chain types/subtypes within a species. Allotypic epitopes are located in constant regions and may vary from individual to individual. Idiotypic epitopes are located within heavy and light chain variable regions (both hypervariable and framework regions) and are defined by the unique amino acid sequences that determine specificity. Note that determinant is a synonym for epitope, i.e. means the same thing.  /epitopes /epitopes /epitopes  Immunoglobulin Effector Functions Opsonization – interactions of antibodies with Fc receptors on phagocytes promotes phagocytosis. Complement activation (via the classical pathway) – antibody-mediated activation of complement is responsible for the inactivation/removal/killing of pathogens. Antibody-dependent cell-mediated cytotoxicity (ADCC) – antibody (IgG) acts as a receptor to enable recognition and killing of target cells by natural killer (NK) cells with Fc receptors (CD16). Transcytosis – passage of antibodies across epithelial layers delivers certain classes of antibody (primarily IgA) to mucosal surfaces. Induction of mast cell degranulation – this function is initiated by Fc receptors for IgE. The five different human Ig classes ε2λ2  IgG Consists of 2 identical 50 kDa γ and 2 identical 25 kDa κ or λ chains. There are 4 IgG subclasses in humans, each with unique biological properties. Most abundant immunoglobulin in serum and extravascular spaces. Neutralizes viruses/toxins, opsonizes microbes, activates classical complement pathway, mediates ADCC reactions, and crosses the placenta during pregnancy.  IgM Expressed as a monomer (2 µ chains, 2 κ or λ chains) on the surface of B cells and secreted by plasma cells as a pentamer containing a J chain. Only 5 of 10 antigen-binding sites on pentameric IgM are able to physically interact with large antigens. First immunoglobulin class made by newborns and in a primary immune response. Efficient agglutinator of particulate antigens and activator of the classical complement pathway. Concentration is low in fluid between cells due to its large size, which prevents easy movement across blood vessel walls. J chain in the pentamer allows transport of IgM across epithelial mucosa. Electron micrograph of pentameric IgM molecules  IgA Present as a monomer (2 α chains, 2 κ or λ chains) in serum and as a dimer (contains a J chain and secretory component) in mucosal secretions. Dimeric IgA is formed in plasma cells by the addition of a J chain. Secretory component is added to the IgA dimer during its passage through glandular epithelial cells, to protect the secreted IgA molecule from proteolysis. Dimeric IgA defends mucosal surfaces from microbial attack by inhibiting pathogen adherence. IgA-antigen complexes are trapped in mucus and eliminated by mechanical action. IgA in breast milk provides newborns with mucosal immunity to environmental pathogens.  IgE Exists as a monomer consisting of 2 ε chains and 2 κ or λ chains. Only small amounts of IgE are present in serum, except in allergy or parasitic infections. Binds high affinity Fcε receptors on mast cells and basophils. Crosslinking membrane-bound IgE causes mast cell and basophil degranulation, leading to acute inflammation, as well as atopic (Type I) allergic responses. Important role in host defence against certain species of parasitic worms.  IgD Exists as a monomer, consisting of 2 δ chains and 2 κ or λ chains. Amount of IgD is very low in serum due to its susceptibility to proteolysis; most IgD is on the surface of B cells. IgD is also a mucosal Ig, mainly present in the airways, saliva, and tear fluid. IgD activates basophils (and mast cells). Physical properties of human Ig isotypes Specialized functions of human Ig isotypes +  Immunoglobulin Superfamily Many molecules associated with immune function exhibit the immunoglobulin-fold domain structure, i.e., 110 amino acid residues arranged in antiparallel sheets of β- pleated strands and are therefore members of the immunoglobulin superfamily. Immunoglobulin superfamily members include antibody, Ig-α/Ig-β, the T cell receptor, Fc receptors, CD4, major histocompatibility complex molecules, and various cell adhesion molecules. This family of genes is thought to have originated through mutation and duplication from a single primordial gene encoding a polypeptide of 110 amino acids.  Monoclonal Antibodies Derived from a single B cell clone and are therefore specific for a single epitope, as opposed to polyclonal antibodies that are produced by many different B cell clones and so bind many different epitopes. Produced by immortal hybridoma cells created by fusing an antigen-specific B cell with a transformed (cancerous) myeloma cell. The hybrid cells are selected by growth in special tissue culture medium that allows only the hybridoma cells to grow. Hybridoma clones that produce a monoclonal antibody with the desired antigenic specificity are propagated in tissue culture using bioreactors for use as diagnostic, imaging and therapeutic agents. Production of a mouse monoclonal antibody Monoclonal antibodies as treatments for disease Learning Objectives: 1) Describe and draw the structure of all the antibody classes including heavy and light chains. 2) Explain which regions of antibodies are responsible for antigen specificity and effector functions. 3) Describe the complementarity determining regions, Fc, Fab, and hinge regions of antibodies. 4) Differentiate isotypic, allotypic and idiotypic determinants/epitopes of antibodies. 5) Paraphrase the effector functions of antibodies (opsonization, ADCC, complement activation, transcytosis, and mast cell degranulation), and which antibody class contributes to each of them. 6) Recall that antibodies are part of a greater protein family encompassing many molecules regulating the immune system (Immunoglobulin family). 7) Describe how monoclonal antibodies are produced in the laboratory.