Imunoglobulins-Basics
- 2. Glycoprotein molecules that are produced by plasma cells in response to an immunogen and which function as antibodies. Antibodies belong to a class of proteins called immunoglobulins Immunoglobulins are Y shaped proteins. Thearms of the Y bind antigens. The tail of the Y is responsible for biological activity Immune serum Ag adsorbed serum 1 2 + - albumin globulins Mobility Amountofprotein
- 3. General Functions of Immunoglobulins Ag binding Can result in protection Valency Effector functions Fixation of complement Binding to various cells Usually requires Ag binding
- 4. Basic Immunoglobulin Structure Immunoglobulins - heterogeneous Myeloma proteins - homogeneous immunoglobulins
- 5. Antibody Structure Antibodies Are Made Up Of: 2 Light Chains (identical) ~25 KDa 2 Heavy Chains (identical) ~50 KDa Each Light Chain Bound To Heavy Chain By Disulfide (H-L) Heavy Chain Bound to Heavy Chain (H-H) First 100 a/a Of Amino Terminal Vary of Both H and L Chain Are Variable Referred To As VL , VH, CH And CL CDR (Complementary Determining Regions) Are What Bind Ag Remaining Regions Are Very Similar Within Same Class
- 7. Repeating Domains of ~110 a/a Intrachain disulfide bonds within each domain Heavy chains 1 VH and either 3 or 4 CH (CH1, CH2, CH3, CH4) Light chains 1 VL and 1 CL Hinge Region Rich in proline residues (flexible) Hinge found in IgG, IgA and IgD Proline residues are target for proteolytic digestion (papain and pepsin) Rich in cysteine residues (disulfide bonds) IgM and IgE lack hinge region They instead have extra CH4 Domain Antibody Structure
- 9. Digestion With Papain Yields 3 Fragments 2 identical Fab and 1 Fc Fab Because Fragment That is Antigen Binding Fc Because Found To Crystallize In Cold Storage Pepsin Digestion F(ab`)2 No Fc Recovery, Digested Entirely Mercaptoethanol Reduction (Eliminates Disulfide Bonds) And Alkylation Showed Enzymatic Digestion Of Antibodies
- 11. Sequencing Of Several Immunoglobulins Revealed 100-110 Amino Terminus, Highly Variable (V) Five Basic Sequence Patterns , , , , IgA, IgD, IgG, IgE and IgM The Above Classes Are Called Isotype Each class can have either or light chains Minor differences Led To Sub-classes For IgA and IgG IgA1, IgGA2 and IgG1, IgG2, IgG3, IgG4 Sequencing Of Heavy Chains
- 12. Characteristics of hinge region Immunoglobulins (with possible exception of IgM & IgE) contain hinge between CH1 & CH2 No homology between AA sequence of hinge & heavy chains AA sequence differs with different classes Comprised of many cysteine and proline residues Cysteine involved in formation of interchain disulfide bonds Proline prevents folding in a globular structure, allowing flexibility between two Fab arms of the Y-shaped antibody; allows open & close to accommodate binding to two epitopes; because it is open, it can be cleaved by proteases (e.g. papain) to generate the Fab & Fc fragments
- 13. Immunoglobulin Fragments: Structure/Function Relationships Fab Ag binding Fc Effector functions F(ab)2 Pepsin Fc Peptides F(ab)2
- 14. Immunoglobulin Fragments: Structure/Function Relationships Fab Ag binding Valence = 1 Specificty determined by VH and VL Papain Fc Fab Fc Effector functions
- 15. Immunoglobulin Fragments: Structure/Function Relationships Ag Binding Complement Binding Site Placental Transfer Binding to Fc Receptors
- 16. IgG IgG1 IgG2 IgG3 IgG4 Subclasses show close overall relation Heavy chains = 1,2,3,4 Highest concentration in serum Plays major role in immune dfns. Small size ppt in surfaces (e.g. cross placental barrier) All normal indiv. have all IgG1>IgG2>IgG3>IgG4 IgG3 has shortest 1/2 life, highest catabolic rate, highest # S-S IgG4 = monoval. no aggl ppt rx., autoab to clot fac Autoab to DNA = IgG1,IgG3 Two H chains, Two L chains (either or but not both) Each H chain = 50 kD Each L chain = 25 kD MW approx. 150 kD, 7S, globulin
- 17. IgG Structure Monomer (7S) IgG1, IgG2 and IgG4 IgG3
- 18. IgG Structure Properties Major serum Ig Major Ig in extravascular spaces The only antibody to cross the placental Fixes complement Binds to Fc receptors Phagocytes - opsonization NK cells ADCC Binds to SPA
- 19. IgM Structure Properties 3rd highest serum Ig First Ig made by fetus and B cells Produced early in the primary response The most efficient Ig Fixes complement Tail Piece Agglutinating Ig Binds to Fc receptors B cell surface Ig
- 20. IgM Structure Pentamer (19S) composed 5 H2L2 units plus one molecule of J chain Extra domain (CH4) J chain CH4 J Chain
- 21. Structural features of IgM Pentamer (5) First Ig produced following immun. Macroglobulin (M) 900 kD, 19S SSS S SS J chain Doesnt have hinge region has additional H domain Has a J chain (one of 2 Ig isotypes) 15 kD
- 22. IgD Structure Monomer Tail piece Tail Piece
- 23. IgD Structure Properties 4th highest serum Ig B cell surface Ig Does not bind complement
- 24. Structural features of IgD Primarily a B cell antigen receptor Long exposed hinge region 170kD, 7S, migrates as fast No interchain S-S bridges in H chains Readily denatured
- 26. IgA Properties 2nd highest serum Ig Major secretory Ig ( saliva, tears, respiratory, intestinal, and genital tract secretions.) Does not fix complement unless aggregated Binds to Fc receptors on some cells
- 27. IgA Structure Serum - monomer Secretions (sIgA) Dimer (11S), sIgA molecule consists of two H2L2 units plus one molecule each of J chain and secretory component(SC or SP) J ChainSecretory Piece
- 28. IgE Structure Monomer Extra domain (CH4) CH4
- 29. Structural features of IgE Sometimes called reaginic ag Mediates allergies (Type I hypersensitivies) 190 kDa, 8S, migrates as fast Contains an extra domain (CH4) which binds to mast cells & basophils May remain attached for long time when ag reappears, cross links IgE on mast cell surface, release mast-cell granules & signs of anaphylaxis
- 30. IgE Structure Properties Least common serum Ig Allergic reactions Parasitic infections Does not fix complement
- 31. Fixation of C1 by IgG and IgM Abs No activation Activation
- 32. B Cell Antigen Receptor (BcR) Ig-Ig- Ig-Ig-
- 33. L & H chain folding to yield 3 CDR in each chain to form walls of ag binding groove