Antigen recognition molecules

Transcription

1 Viraphong Lulitanond, Ph.D. Antigen recognition molecules Contents: (4 hours) Immunoglobulin - Structure and function - Immunoglubulin genes T cell receptor (TCR) - Structure and function - TCR genes Generation of antibody and TCR diversity The major histocompatibility complex (MHC) & HLA - Structuret and function - MHC genes Other accessary molecules involved in activation and cellcell interaction Immunoglobulin superfamily Viraphong Lulitanond, Ph.D. 1

2 References Cellular and Molecular Immunology, second ed., 1994, edited by Abul K. Abbas, Andrew H. Lichtman, and Jordan S. Pober Chapter 3,4,5,7,8 Viraphong Lulitanond, Ph.D. 2

3 Immunoglobulins or Antibodies Groups of glycoproteins found in serum, body fluids and surface of B cells Produced from mature B cells or plasma cells So-called gammaglobulins Five major classes of Igs: IgG, IgM, IgA, IgD and IgE. They differ from each other in size, charge, amino acid sequence and carbohydrate content. Viraphong Lulitanond, Ph.D. 3

4 Electrophoresis of serum proteins Immunoglobulin structure Two identical heavy chains and 2 identical light chains linked by disulfide bonds and non-covalent bonds. Light chains :κ (kappa) and λ (lambda) Heavy chains:μ(mu), α (alpha), γ (gamma), δ (delta), ε (epsilon) Viraphong Lulitanond, Ph.D. 4

5 Immunoglobulin structure The heavy and light chains of Ig can be divided into variable and constant regions (domains) based on sequence similarity. Light chain: VL and CL Heavy chain: VH, CH1, CH2, CH3 (CH4 in IgM and IgE). Hinge region located between CH1 and CH2 in certain classes. It contains aa ranging from residues which confers flexible motion to the antigen binding sites. Immunoglobulin structure Certain proteolytic enzymes were used to determined the structure of Ig molecule. Bivalent hapten was used to demonstrate the flexibility of the hinge region of Ig Viraphong Lulitanond, Ph.D. 5

6 Immunoglobulin structure Each domains of the Ig has a common globular structure called immunoglobulin domains or fold VL and VH form the antigen-binding sites Immunoglobulin structure Characteristics of Ig domains - ~110 aa residues in length - Two layers of beta-pleated sheet with 3 or 4 strands of antiparallel polypeptide chains and linked by intrachain disulfide bonds. Viraphong Lulitanond, Ph.D. 6

7 Immunoglobulin structure Variable region consists of three hypervariable regions or complementarity-determining regions (CDR) and framework regions Structure variation in Ig constant region made Ig divide into 5 classes or Isotypes and this differences confer different functional properties on each class. Differences between constant-region genes Isotype; those between two alleles of the same constant genes Allotype; differences in Ag-combining site Idiotype. Viraphong Lulitanond, Ph.D. 7

8 Antibody Functions Antigen receptor Neutralization Activation of complement Opsonization Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) Anaphylactic type hypersensitivity Mucosal immunity Neonatal immunity Antibody feedback regulation Viraphong Lulitanond, Ph.D. 8

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11 IgG It comprises 70-75% of the serum Ig. Major Ig in the 2nd Ab. response. Four subclasses: IgG1, IgG2, IgG3, IgG4. It can activate complement (IgG1 and IgG3). It can transfer across the placenta. IgM It comprises 10% of the serum Ig. Major form: pentamer (jointed by J-chain). Major Ig in primary Ab. response. Most effective class of Ig for complement activation. Viraphong Lulitanond, Ph.D. 11

12 IgA It comprises 15-20% of the serum Ig. Major Ig in the secretion and existed as dimeric IgA attached with secretory piece. Dimeric IgA can be found in serum Two subclasses: IgA1 and IgA2. IgD Less than 1% of serum Ig. Surface IgD is the differentiation marker of B cells. Its function is largely unknown. A very fragile Ig. IgE Trace amounts in the serum. Most are bound to the surface of mast cells and basophils. It plays role in parasitic infection and anaphylactic hypersensitivity. Viraphong Lulitanond, Ph.D. 12

13 Hybridoma Technique for Monoclonal Ab Production Viraphong Lulitanond, Ph.D. 13

14 Polyclonal Ab Monoclonal Ab T cell receptor (TCR) Viraphong Lulitanond, Ph.D. 14

15 TCR:CD3: ζ complex - CD3 and ζ proteins are stably associated with TCR - CD3 consists of γ, δ, ε chains - Extracellular region of γ, δ, ε chains each contains a single Ig-like domains. - The cytoplasmic domains of each CD3 chain and homodimer of ζ chain contain immunoreceptor tyrosine-based activation motifs (ITAMs) - CD3 and ζ proteins are required for cell-surface expression of TCR. Igα and Igβ are associated with membrane Ig and are required for cell-surface expression of Ig. Cytoplasmic tails of Igα and Igβ contains ITAMS Viraphong Lulitanond, Ph.D. 15

16 TCR recognize Ag in the peptide form derived from foreign protein antigens that are physically associated with self MHC molecules on the surface of APC or target cells. The major histocompatibility complex (MHC) is a region of highly polymorphic genes whose products are expressed on the surfaces of a variety of cells. Ag-specific T cells do not recognize antigens in free or soluble form but recognize peptide form of protein antigens that are non-covalently bound to MHC gene products There are two major types of MHC gene products: MHC class I and class II Viraphong Lulitanond, Ph.D. 16

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18 MHC genes are by far the most polymorphic genes present in the genome of every species analyzed. MHC Class I molecule Viraphong Lulitanond, Ph.D. 18

19 MHC Class II molecule Viraphong Lulitanond, Ph.D. 19

20 Pathway of MHC I - restricted presentation of an endogenous antigen TAP - Transporter in Antigen processing Pathway of MHC II- restricted presentation of an exogenous antigen Ii = invariant chain Viraphong Lulitanond, Ph.D. 20

21 CD4 and CD8 : Accessory molecules involved in MHC-restricted T cell Activation D1,D3 = V D2,D4 = H CD4:MHC II (β2 domain) CD8:MHC I (α3 domain) Viraphong Lulitanond, Ph.D. 21

22 Genomic Organization of Human Ig Genes Viraphong Lulitanond, Ph.D. 22

23 Sequence of gene rearrangement, transcription and synthesis of the mouse Ig μ heavy chain Sequence of gene rearrangement, transcription and synthesis of the mouse Ig κ light chain Viraphong Lulitanond, Ph.D. 23

24 Generation of Ab diversity Multiple germline genes Combinatorial diversity Junctional diversity Combinations of H and L chains Somatic Mutations Mechanisms contributing to the generation of primary Ab diversity in the mouse H κ λ Germline genes H gene segments J segments D segments Combinatorial joining V x J x (D) 10,000-40, H-L chain associations H x κ 1-4 x 10 7 H x λ 5-10 x 10 4 Total potential repertoire with junctional diversity Note: Somatic mutations further increase diversity in 2 nd Ab response Viraphong Lulitanond, Ph.D. 24

25 Somatic mutation - primarily involved V gene segments and primarily responsible for the affinity maturation of Abs. - somatic mutation rate is x higher than spontaneous mutations Organization of human TCR genes in the germline Viraphong Lulitanond, Ph.D. 25

26 The Ig Superfamily Viraphong Lulitanond, Ph.D. 26