Immunoglobulin Structure & Function Assigned Reading Performance Ojectives Key terms Key Concepts Content Outline Short Answer Questions

Angels & Devils

Assigned Reading Chapter: 5 pp107-142 Janis Kuby’s Immunology 3rd Ed

Content Outline Basic Structure B Cell Receptor Antigenic Determinants of Igs Immunoglobulin Superfamily Ig Classes Monoclonal Antibodies (MABS) Immunoglobulin Sequencing Studies Immunoglobulin Fine Structure

Background

Tiselius

Electrophoresis

General Structure of Immunoglobulins Electron microscopy Crystallography Hinge region light & heavy chains binding site domains fragments

IgG Crystal

X - Ray Crystallography Crystallized Antigen or Antigen - Antibody Complex Electron Density Map of the Molecule Three Dimensional Images

Antibodies contain a flexible hinge region EM evidence Location Chemical Structure Advantages

Antibody Flexibility EMs of same conc of antibody mixed with different conc of antigen Ag-Ab complexes form different shapes Ag is inflexible AB MUST BE FLEXIBLE

Ag-Ab Complexes

Immunoglobulin flexible hinge

Hinge Prolines

Blocks or binds to antigens over a wide range of concentrations Advantages of hinge Rotation around central point Bind antigens at various distances Bind various ratios of antigens Blocks or binds to antigens over a wide range of concentrations

Structure of IgG

IgG: Domains

IgG: Enzyme hydrolysis

Ag-Ab complexes after Pepsin

B Cell Receptor BEFORE IMMUNIZATION AFTER IMMUNIZATION Ig genes are organized & Igs assembled Anchored on membrane as specific receptors for immunogen AFTER IMMUNIZATION different expression of Ig genes lose anchor>> secreted different class of Ig secreted

Antigenic Determinants of Igs Immunoglobulins are immunogenic Inject human antibodies into a mouse Mouse develops anti-Human antibodies Use these antibodies to probe structure of immunoglobulins Three classes of anti-antibodies Isotypes Allotypes Idiotypes

Antibody Classes: Outline 5 classes of antibodies Concentration of classes Structure and function of IgG IgM IgA IgD IgE

Immunoglobulin Classes Immunoglobulin A IgA, mA Immunoglobulin D IgD, mD Immunoglobulin E IgE, mE Immunoglobulin G IgG, mG Immunoglobulin M IgM, mM

Immunoglobuin concentrations

Ig Superfamily

IgG: Physicochemical Properties Molecular weight Sedimentation constant 7S Total 146,000 Heavy chain 51,000 Isotypes 4 Heavy chain domains 4 Carbohydrate (%) 2-3 half life (days) 21

Structure of IgG

IgG: Domains

IgG subclasses

IgG: Functions Bind antigen in tissue fluids Promote destruction of antigen activates complement Promote phagocytosis binds to Fc receptors on macrophages Passive protection of fetus binds to receptors on placenta transferred to fetal circulation

Functions of IgG Domains VH + VL Antigen binding CH1 Complement binding c4b CH2 complement binding C1q CH3 Macrophage binding,Fc receptor CH2 +CH3 neutrophil & K cell binding, bind to placental syncytiotrophoblast

Primary vs Secondary Response

IgM:Physicochemical Properties Molecular weight Sedimentation constant 19 S Total 900,000 Heavy chain 65,000 Isotypes 1 Heavy chain domains 5 Carbohydrate (%) 12 half life (days) 5

IgM: Structure

IgM: Heavy chains

IgM: Functions Antigen receptor for B cell Short term protection binds tightly to antigen (high avidity)

IgA:Physicochemical Properties Serum Secretory Molecular weight Sedimentation constant 7S 11S Total 160,000 385,000 Heavy chain 52-56,000 52,56,000 Isotypes 2 2 Heavy chain domains 4 4 Carbohydrate (%) 7-11 7-11 Half life (days) 6 6

Structure of IgA

IgA dimers

EM of IgA

Secretion of IgA

IgA: Functions Protects body surfaces against invading microbes Secretory IgA - transported to epithelium secreted in mucus, milk & tears Blood IgA - transported to liver binds to hepatocytes secreted into bile

J chain Small polypeptide chain found in polymeric classes of antibodies Acts as a clasp to join prototype subunits produced by same cell that makes antibodies

J chain structure

J chain: Beta sheets

J Chain: Barrel

IgE: Physicochemical Properties Molecular weight 188,000 Sedimentation constant 8S Total 188,000 Heavy chain 72,500 Isotypes 1 Heavy chain domains 5 Carbohydrate (%) 12 half life (days) 2

Structure of IgE

Molecular model of IgE

IgE: Functions Mediates inflammatory response Immunity to parasitic worms Mediates many allergies (type I)

IgD Physicochemical Properties Molecular weight Sedimentation constant 7S Total 184,000 Heavy chain 70,000 Isotypes 1 Heavy chain domains 4 Carbohydrate (%) 9-14 half life (days) 3

IgD Structure

IgD Hinge region

Substructure of IgD

Mouse IgD

Mouse IgD

IgD Function Activation of B cells by antigen IgD : membrane antigen receptor Indicator of Mature B cells low levels of IgD & IgM in Bone marrow high levels of IgD & IgM in peripheral lymphoid organs

DONE!!!

Performance Objectives

Key Terms allotypes, allotypic determinants, Am determinants, Gm determinants, Km determinants, Bence Jones proteins, Antibodies, immunoglobulin classes, antibody subclasses, constant region (C), Fab fragment, Fab' fragment, F(ab')2 fragment, Fc fragment, Framework residues (FR), Heavy chain,

hinge regions, chimeric antibodies, hybridoma, hypervariable regions (HVRs), complematrarity determining regions(CDRs) Hypoxantinine-Aminoopterin-Thymidine medium (HAT),idiotypes, idiotypic determinants, immunoglobulin domains immunoglobulins, isotype, isotypic determinants,

joining chain (J), Light chain, monoclonal antibodies, multiple myeloma, Secretory Iga, Segmental flexibility, variable regions (V)

Key Concepts Distinguish between the overall structure and the fine structure of immunoglobulins Describe the variable and constant regions of immunoglobulins light and heavy chains Explain the structural organization of the variable regions of an immunoglobulins light and heavy chains

Differentiate between Hypervariable regions and complementarity determining regions Contrast monoclonal antibody production vs polyclonal antibody production Discuss the differences in the biological effector functions of the different classes of immunoglobulins

Diagram the procedure for monoclonal antibody production Construct a table comparing the biological characteristics of the five classes of immunoglobulins Construct a table comparing the biochemical and biophysical characteristics of the five classes of immunoglobulins

Short Answer Questions

The analysis of IgG molecules after hydrolysis by pepsin and papain led to similar yet different results. Explain. What is the difference between and immunoglobulin and a myeloma protein? Why were myeloma proteins and Bence Jones proteins critical to the early studies on antibody structure? What has largely replaced them?

Differentiate among, complementarity determining regions, hypervariable regions and framework regions. Explain the statements: "Antibodies can be antigens" and "Antibodies can be used to characterize antibodies". Discuss the terms immunoglobulin isotypes, immunoglobulin allotypes and immunoglobulin idiotypes and give examples of each.

Compare conventional antibody production with monocloanl antibody production and recombinant antibody production. What are antibody domains? Why can't light chains be used to classify antibodies? The function of an immunoglobulin molecule is related to its structure. Explain.

Distinguish between idiotypes and anti-idiotypes. Different cellular fusion products can potentially occur in a hybridoma fusion. What are the potential products? Why are they a problem? How can you obtain the appropriate combination? Distinguish between idiotypes and anti-idiotypes.