Biological Chemistry
L-6: Immunoglobulin Structure August 7, 1997
OBJECTIVES: 1. To understand the major structural features of immunoglobulins 2. To understand the interaction of immunoglobulins with antigens 3. To learn the major classes of immunoglobulins 4. To understand the analytical uses of immunoglobulins READING: Devlin, Chapter 3, pp. 88-97. Marks, Chapter 8, pp. 92-94. CLINICAL CORRELATIONS: Amy Lloyd - Multiple myeloma pp. 80, 94, 96-97 LECTURE: I. Humoral immunity A. B lymphocytes produce immunoglobulins (Igs) 1. One clone of B cells produces Igs with specificity for one antigen a. antigen is the target macromolecule that elicits the immune response 2. Produce Igs on surface and secretory Igs B. Humoral response 1. Surface Igs bind antigen 2. Antigen engulfs the antigen and partially digests it 3. Major Histocompatibility Complex II (MHC II) displays the fragment on the surface 4. Helper T cells cause clonal expansion of that B cell a. Helper T cell specific for that specific MHC II b. Helper T cell secretes interleukins (ILs) which stimulate B cells 5. B cell clonally expands to produce plasma cells a. plasma cells secrete large amounts of Igs 6. Igs bind antigen a. Igs coat antigen and elicit phagocytosis by macrophages or b. Igs bound to antigen initiate the complement cascade 7. Memory B cells remain to allow more rapid secondary response II. Immunoglobulin structure A. Features of an immunoglobulin 1. y-shaped (Devlin Fig. 3.2 pg 90) a. immunoglobulins are bivalent (can bind two antigens) b. multivalency is important to immune reactions 2. 2 light chains (L), 2 heavy chains (H) (Fig. 34-18) a. chains are disulfide linked i. intramolecular disulfide bridges within polypeptide ii. intermolecular disulfide bridges between heavy and light chains and heavy chains b. Heavy chain (53-75 kDa) i. 5 types (IgA alpha, IgD delta, IgE epsilon, IgG gamma, IgM (mu) ii. heavy chain is glycosylated iii. 4 domains, 1 variable, 3 constant c. Light chain ( 23 kDa) i. two types ( or ) ii. 2 domains, 1 variable, 1 constant 3. variable regions make up the antigen binding sites (Devlin Fig. 3.7 pg 95) a. antigen binding site is shared between the H and L chain b. hypervariable regions exist in the variable regions i. antigen binding occurs in the hypervariable region ii. hypervariable regions are at the end of variable domain composed of -pleated sheet B. 3-D structure of immunoglobulins 1. Immunoglobulin chains have domains consisting of 110 amino acid repeating sequences from to separate chains a. domain consists of two supersecondary structures known as immunoglobulin fold i. one fold is from the light and the other from the heavy chain b. Immunoglobulin folds (Devlin Fig. 3.4 pg 93) i. fold is a 2 antiparallel -sheets face to face ii. face to face -sheets are refered to as a -barrel iii. each fold has an intrachain disulfide bridge 2. Antigen binding site (Devlin Fig. 3.7 pg 95) a. formed by six loops i. three from L-chain, three from H-chain b. has a small pocket lined by hyper variable region c. has a large surface area which forms antigen-Ig contact d. Antigen-Ig binding is very tight i. binding site and the antigen are complementary ii. by excluding water, electrostatic interactions are made stronger 3. Hinge region a. flexible b. susceptible to proteolysis C. Immunoglobulins can be divided into functional fragments 1. Proteolysis with papain cleaves IgG into three fragments (Devlin Fig. 3.8 pg 95 is incorrect) a. cleavage occurs at hinge regions (exposed)(Fig. 34-18) b. pepsin cleave Ab into 1 F(ab)2 - F(ab)2 is bivalent c. papain cleave Ab into 2 Fab and 1 Fc region - Fab is monovalent 2. Ig fragments are useful for research D. Bence Jones proteins 1. Immunoglobulin fragments found in the urine 2. -sheet fragments of immunoglobulin folds 3. Symptom of multiple myeloma III. Immunoglobulin classes (Devlin Table 3.1 pg 90) A. Humans have 5 classes of secreted immunoglobulins 1. Differ in heavy chain types a. IgA alpha, IgD delta, IgE epsilon, IgG gamma, IgM mu 2. Differ in size a. IgD, IgE, and IgG are LH2 monomers b. IgM is LH2 pentamer c. IgA can be an LH2 monomer, dimer or trimer d. J-chain (joining) links dimeric subunits 3. Differ in origin and timing of expression a. In immune response, IgM secreted first, IgG follows b. IgA is in intestinal tract, milk, colostrum, sweat, tears c. IgG, IgM, IgD, IgE mostly in blood IV. Antibody-Antigen interaction A. Antigen binding sites are complementary to antigens 1. Hapten occupies only apart of the binding site a. hapten is the smallest recognizable target for the antibody b. epitope is the site on the target protein recognized by the antibody c. small haptens may bind in the pocket d. the antibody-protein antigens binding interface is a flat surface with a small pocket V. Monoclonal Antibodies A. Produced by fusing mouse lymphocytes with mouse myeloma cells 1. mouse immunized with antigen 2. mouse lymphocytes fused with immortal myeloma line a. cells generated by fusion of lymphocytes and myeloma are hybridomas b. selected on HAT media - immortal myeloma cells lack a purine salvage enzyme - cannot grow in HAT media - fusion of myeloma cells with lymphoctes allows gain of purine salvage enzyme 3. Hybridoma is immortal B. Monoclonals are immunoglobulins to a single epitope of the antigen C. Used in many biomedical tests VI. Analytical Uses of Immunoglobulins A. Immunoblots (Westerns) 1. Used to quantitate amount of a protein in crude cellular extracts 2. Immunoblot procedure a. Proteins from SDS-PAGE transferred to a membrane (nitrocellulose or PVDF) b. Membrane incubated with immunoglobulins to protein of interest c.. Membrane incubated with 2 immunoglobulins to the primary antisera i. 2 immunoglobulin has a functional enzyme linked to it d. Detect protein by assaying location and level of activity of linked enzyme B. Enzyme-linked immunosorbant assay (ELISA) (Fig. 5-1) 1. Used to detect small amounts of protein in cell extracts or body fluids 2. ELISA procedure a. Igs to a specific antigen are coated onto a plastic dish b. sample is incubated on Ig-coated plate c. dish incubated with antibodies with enzyme linked d. assay for enzyme activity
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