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Unformatted text preview: Antibody Structure and the Generation of B-Cell Diversity Lecture 5
Chapter 4 MMI 188 Human Immunology
José V. Torres, Ph.D. Professor Medical Microbiology and Immunology School of Medicine University of California at Davis [email protected] Function of antibodies: To combat infection
• Several different mechanisms • Neutralization • Opsonization • Immunoglobulins = antigen-binding molecules made by B cells • Antibody = the secreted form of an immunoglobulin B cells have membrane-bound Ig on their surface When B cells encounter antigen, they differentiate to plasma cells Plasma cells secrete antibody but do not have Ig on their surface Antibodies produced by plasma cells clear extracellular pathogens and their toxins best source of protective immunity antibody repertoire: 109 specificity of the secreted antibody is the same as that of the B cell receptor The IgG Molecule • two identical heavy chains • two identical light chains • carbohydrates attached to heavy chains • variable region • constant region • flexible hinge Structure • 4 polypeptide chains (2 heavy, 2 light) • Connected by covalent (S-S) and noncovalent bonds • Divalent: 2 antigen-binding sites • Repeating globular domains • C-terminal domains are Constant (C) • N-terminal domains are Variable (V) Edited by Foxit Reader Copyright(C) by Foxit Corporation,2005-2009 For Evaluation Only. Protease cleavage reduction of disulfide bonds
enzyme from papaya Papain digestion:
– 2 Fab fragments (Antigen Binding) – one Fc subunit (Constant region) Fragment Crystallizable
antigen biding site IgG molecule has flexible hinge Heavy chains:
IgM pentamer IgG IgA monomer and dimer IgE IgD determine class Edited by Foxit Reader Copyright(C) by Foxit Corporation,2005-2009 For Evaluation Only. Human Immunoglobulin Classes Differences: • heavy chain C region length • location of disulfide bonds • carbohydrate groups • hinge region in IgG, IgA and IgD • no hinge region in IgM and IgE Light chains:
kappa κ lambda λ
disulfide bond Edited by Foxit Reader Copyright(C) by Foxit Corporation,2005-2009 For Evaluation Only. Major Classes of Antibodies
– Pentamer; First antibody made after infection – Can bind complement; Cannot bind Fc receptors on phagocytes • IgG
– After acute infection, IgG becomes the dominant antibody in circulation – Neutralizes Toxins; Binds complement; Binds Fc receptors on phagocytes (opsonization) • IgA
– Dimer; Protects mucosal surfaces • IgE • IgD evolve to deal with parasite – Mast cell, eosinophil degranulation (allergy, parasites) – Coexpressed with IgM by naïve B cells Edited by Foxit Reader Copyright(C) by Foxit Corporation,2005-2009 For Evaluation Only. Protein domains in immunoglobulins 3 globular portions of similar size each portion has 4 immunoglobulin domains VL and CL domains in light chains VH and CH domains in heavy chain light rectangular-immunoglobulin domain heavy 3D structure of Ig C and V domains β strands of β sheet antiparallel sheets connected by loops Edited by Foxit Reader Copyright(C) by Foxit Corporation,2005-2009 For Evaluation Only. Hypervariable regions of V domains HV loops determine antigenic specificity of antigen binding site Variable domains of both heavy and light chains (VH and VL) each contain three hypervariable loops that determine antigen specificity • Hypervariable loops (HV1, HV2, HV3) are also called complementarity-determining regions (CDR1, CDR2, CDR3) • Regions flanking hypervariable loops are called framework regions (FR1-4) business endmutation happen Epitopes for antibodies are exposed at the surface of pathogens poliovirus Each protein contains several epitopes antigenic determinant or epitope Binding to Multivalent Antigens Two Kinds of Multivalent Antigen
A pathogen can be recognized by multiple epitopes and different antibodies, and/or by repeated epitopes and the same antibody. Antibodies bind epitopes of different shapes Hypervariable loops of antibody V domains vary in shape to fit different epitopes POCKET GROOVE SURFACE Linear and discontinuous (conformational) epitopes Edited by Foxit Reader Copyright(C) by Foxit Corporation,2005-2009 For Evaluation Only. The Production of Monoclonal Antibodies
• Polyclonal antisera = more than 1 specificity or isotype • Monoclonal antibodies are produced from one single clone of an immortalized plasma cell • A hybridoma results from the fusion of a single antibody-producing cell and a myeloma • Hybridoma clones are selected for antigen specificity and can be cultured indefinitely • “Humanized” monoclonal antibodies can be produced by genetic engineering techniques labeling cell Edited by Foxit Reader Copyright(C) by Foxit Corporation,2005-2009 For Evaluation Only. Common Clinical Uses of Monoclonal Antibodies: Fluorescence-Activated Cell Sorting (FACS), Flow Cytometry B cell( 60%) T cell What factors account for antibody diversity?
(How can we recognize so many antigens??) Edited by Foxit Reader Copyright(C) by Foxit Corporation,2005-2009 For Evaluation Only. • During B-cell maturation, before the B-cell encounters its antigen:
– Genetic diversity – Combinatorial diversity – Junctional diversity Genetic Diversity
Germline organization of the immunoglobulin heavy chain and light chain loci • Germline sequence (inherited from egg and sperm) is different from mature B cell (somatic) • One of each segment (V, D, J, C) is selected during recombination Edited by Foxit Reader Copyright(C) by Foxit Corporation,2005-2009 For Evaluation Only. Combinatorial Diversity
Random selection of one gene segment of each type: • Variable (V) • Joining (J) • Diversity (D) only for heavy chain
Constant region determine which Ig you get by splicing Immunoglobulin Diversity Edited by Foxit Reader Copyright(C) by Foxit Corporation,2005-2009 For Evaluation Only. Successful recombination leads to allelic exclusion
Heavy chain loci on both chromosomes begin rearrangement • As soon as one chromosome reaches a productive (in-frame) recombination, the process stops. • From that point on, only the successfully-rearranged locus is used to make immunoglobulins for that particular B cell. The other allele is “excluded”. Light chain loci rearrange after heavy chains • Light chains have four chances to rearrange • κ chain (both copies of chromosome 14), followed by λ chain (both copies of chromosome 22) First twice for K, later twice for lambda, so more kappa • As for heavy chains, once a successful rearrangement is found everything stops. • Immunoglobulins use κ chains more frequently than λ chains (66% κ chain, 33% λ chain) Edited by Foxit Reader Copyright(C) by Foxit Corporation,2005-2009 For Evaluation Only. Mechanism of recombination ensures the correct orientation of gene segments • • • • • RAG (recombination activating genes) complex binds intergenic sequences 12 nucleotide spacer matches 23-nt spacer (12/23 rule) RSS (recombination signal sequences), 7 and 9 before and after spacers DNA is cut; V and J segments are ligated Circular loop containing unused segments excised Junctional Diversity:
Random addition of nucleotides during joining
• P-nucleotides (palindrome) come from imprecise cleavage; • N-nucleotides (non-germline) are randomly added by terminal deoxynucleotidyl transferase (TdT) 7 Edited by Foxit Reader Copyright(C) by Foxit Corporation,2005-2009 For Evaluation Only. What does the final gene looks like? specificity • The VDJ region (left) provides the specificity for antigen recognition • All of the different C regions are still present • This means the B cell can continue to differentiate and switch to different antibody isotypes during its lifetime: from IgM to IgG, for example. • However, different antibody isotypes made by the same B cell will have the same antigen specificity since they have the same VDJ region. B cells are monospecific. Genetics of Antibody Diversity
• After B cell encounters antigen Edited by Foxit Reader Copyright(C) by Foxit Corporation,2005-2009 For Evaluation Only. – The B cell switches from making membrane-bound to secreted immunoglobulin (reversible changes in RNA splicing, processing) – Somatic hypermutation – Isotype switching Switch from Membrane-Bound to Secreted Immunoglobulin
After a naïve (virgin) B cell encounters antigen, alternate RNA processing results in a switch from membrane-bound to secreted immunoglobulin present in surface Alternative RNA processing Edited by Foxit Reader Copyright(C) by Foxit Corporation,2005-2009 For Evaluation Only. Antigen-binding sequences are further diversified after antigen exposure: Somatic Hypermutation
• Random point mutations in V region genes • > 106 times more frequent than normal mutation rate • Further selection of best fit results in affinity maturation of Ig Heavy chain isotype switching produces Abs with different C regions but identical antigen specificities • Recombination between the rearranged VDJ segment and different CH gene segments; • DNA loops out and circular DNA containing the intervening sequences is deleted; • Sequential switching to different isotypes and subclasses can occur during an immune response, but only in one direction. Higher Order Structures: IgM (Pentamer) and IgA (Dimer) Isotype switching at switch regions AID enzyme activation-induced cytidine deaminase Properties of human Ig isotypes Antibodies with different C regions have different functions Function and Properties of Ig Subclasses Gene rearrangement for synthesis of cell surface IgM in B cells Summary: Genetics of Antibody Diversity ...
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This note was uploaded on 02/16/2011 for the course MIC 105 taught by Professor Wheelis during the Spring '08 term at UC Davis.
- Spring '08