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Structural Biology and Functions of Immunoglobulins © Dr. Colin R.A. Hewitt 2005-2006
<ul><li>Signalling antigen receptors on B cells - bifunctional antigen-binding  secreted molecules </li></ul><ul><li>Struc...
<ul><li>Cell surface antigen receptor on B cells </li></ul><ul><ul><ul><li>Allows B cells to sense their antigenic environ...
Immunoglobulins are Bifunctional Proteins <ul><li>Immunoglobulins must interact with a small number of  specialised molecu...
<ul><li>Structural conservation  and  a capacity for infinite variability in a  single molecule is provided by a  DOMAIN  ...
The genes encoding Ig domains are not restricted to Ig genes. Although first discovered in immunoglobulins, they are found...
 
Domains are folded, compact, protease resistant structures Domain Structure of Immunoglobulins Light chain C domains    o...
CH3
CH3 CH2
CH3 CH2 CH1
CH3 CH2 CH1 VH1
CH3 CH2 CH1 VH1 VL
CH3 CH2 CH1 VH1 CL VL
CH3 CH2 CH1 VH1 CL VL
Hinge CH3 CH2 CH1 VH1 VL CL Elbow
Fv Flexibility and motion of immunoglobulins C H 3 C H 2 Fb Fv Fv Fb Fv Hinge Elbow C H 3 C H 2 Fb Fv
Hinge Fv Fb Fab CH3 CH2 CH1 VH1 VL CL Fc Elbow Carbohydrate
View structures
The Immunoglobulin Fold The characteristic structural motif of all Ig domains Barrel under construction A barrel made of a...
Unfolded V L  region showing 8 antiparallel  -pleated sheets connected by loops. The Immunoglobulin Fold NH 2 COOH S S
View structures
<ul><li>Immunoglobulins must interact with a finite number of  specialised molecules - </li></ul><ul><li>Easily explained ...
Cytochromes C Variability of amino acids in related proteins Wu & Kabat 1970 Amino acid No. Variability 80 100 60 40 20 20...
<ul><li>Distinct regions of high variability and conservation led to the concept  of a  FRAMEWORK  (FR), on which hypervar...
Hypervariable CDRs are located on loops at the end of the Fv regions Hypervariable regions
Space-filling model of (Fab) 2 , viewed from above, illustrating the surface location of CDR loops  Light chains Green  an...
<ul><ul><li>The framework supports the hypervariable loops </li></ul></ul><ul><ul><li>The framework forms a compact    ba...
Antigens vary in size and complexity Protein: Influenza haemagglutinin Hapten: 5-(para-nitrophenyl phosphonate)-pentanoic ...
Antibodies interact with antigens in a variety of ways Antigen inserts into a pocket in the antibody Antigen interacts wit...
View structures
 
Fv Flexibility and motion of immunoglobulins C H 3 C H 2 Fb Fv Fv Fb Fv Hinge Elbow C H 3 C H 2 Fb Fv
30 strongly neutralising McAb 60 strongly neutralising McAb Fab regions 60 weakly neutralising McAb Fab regions Human Rhin...
Electron micrographs of Antibodies and complement opsonising Epstein Barr Virus (EBV) Negatively stained EBV EBV coated wi...
Antibody + complement- mediated  damage to E. coli Healthy E. coli Electron micrographs of the effect of antibodies and co...
Non-covalent forces in antibody - antigen interactions Electrostatic forces Attraction between opposite charges Hydrogen b...
Why do antibodies need an Fc region? <ul><li>Detect antigen </li></ul><ul><li>Precipitate antigen </li></ul><ul><li>Block ...
Structure and function of the Fc region Fc structure is common to all specificities of antibody within an  ISOTYPE (althou...
Monomeric IgM IgM only exists as a monomer on the surface of B cells C  4 contains the transmembrane and cytoplasmic regi...
C  3 binds C1q to initiate activation of the classical complement pathway C  1 binds C3b  to facilitate uptake of opsoni...
Multimerisation of IgM 1. Two IgM monomers in the ER (Fc regions only shown) 2. Cysteines in the J chain form disulphide b...
Antigen-induced conformational changes in IgM Planar or ‘Starfish’ conformation found in solution. Does not fix complement...
IgM facts and figures Heavy chain:   - Mu Half-life:  5 to 10 days % of Ig in serum: 10 Serum level (mgml -1 ):  0.25 - 3...
 
IgD facts and figures IgD is co-expressed with IgM on B cells due to differential RNA splicing Level of expression exceeds...
IgA dimerisation and secretion  IgA is the major isotype of antibody secreted at mucosal sufaces Exists in serum as a mono...
Secretory IgA and transcytosis ‘ Stalk’ of the pIgR is degraded to release IgA containing part of the pIgR - the secretory...
IgA facts and figures Heavy chains:  1  or  2 - Alpha 1 or 2 Half-life:  IgA1 5 - 7 days IgA2 4 - 6 days Serum levels ...
IgE facts and figures IgE appears late in evolution in accordance with its role in protecting against parasite infections ...
The high affinity IgE receptor (Fc  RI) The IgE - Fc  RI interaction is the highest affinity of any Fc receptor with an ...
IgG facts and figures Heavy chains:  1  2  3   4 - Gamma 1 - 4 Half-life:  IgG1  21 - 24 days  IgG2   21 - 24 days...
Carbohydrate is essential for complement activation Subtly different hinge regions between subclasses accounts for differi...
Fc   receptors Receptor Cell type Effect of ligation Fc  RI Macrophages Neutrophils,  Eosinophils, Dendritic cells  Upta...
The neonatal Fc   receptor The Fc  Rn is structurally related to MHC class I In cows Fc  Rn binds maternal IgG in the c...
Molecular Genetics of Immunoglobulins
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Antibody Structure &amp; Function

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Antibody Structure &amp; Function

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  1. 1. Structural Biology and Functions of Immunoglobulins © Dr. Colin R.A. Hewitt 2005-2006
  2. 2. <ul><li>Signalling antigen receptors on B cells - bifunctional antigen-binding secreted molecules </li></ul><ul><li>Structural conservation and infinite variability - domain structure. </li></ul><ul><li>The Immunoglobulin Gene Superfamily </li></ul><ul><li>The immunoglobulin fold </li></ul><ul><li>Framework and complementarity determining regions - hypervariable loops </li></ul><ul><li>Modes of interactions with antigens </li></ul><ul><li>Effector mechanisms and isotype – role of the Fc. </li></ul><ul><li>Multimeric antibodies and multimerisation </li></ul><ul><li>Characteristics and properties of each Ig isotype </li></ul><ul><li>Ig receptors and their functions </li></ul>Topic 1 Immunoglobulin Structure-Function Relationship Outline of Lectures
  3. 3. <ul><li>Cell surface antigen receptor on B cells </li></ul><ul><ul><ul><li>Allows B cells to sense their antigenic environment </li></ul></ul></ul><ul><ul><ul><li>Connects extracellular space with intracellular signalling machinery </li></ul></ul></ul><ul><li>Secreted antibody </li></ul><ul><li>Neutralisation </li></ul><ul><li>Arming/recruiting effector cells </li></ul><ul><li>Complement fixation </li></ul>Immunoglobulin Structure-Function Relationship
  4. 4. Immunoglobulins are Bifunctional Proteins <ul><li>Immunoglobulins must interact with a small number of specialised molecules - </li></ul><ul><li>Fc receptors on cells </li></ul><ul><li>Complement proteins </li></ul><ul><li>Intracellular cell signalling molecules </li></ul><ul><li>- whilst simultaneously recognising an infinite array of antigenic determinants. </li></ul>
  5. 5. <ul><li>Structural conservation and a capacity for infinite variability in a single molecule is provided by a DOMAIN structure. </li></ul><ul><li>Ig domains are derived from a single ancestral gene that has duplicated, diversified and been modified to endow an assortment of functional qualities on a common basic structure. </li></ul><ul><li>Ig domains are not restricted to immunoglobulins. </li></ul><ul><li>The most striking characteristic of the Ig domain is a disulphide bond - linked structure of 110 amino acids long. </li></ul>Immunoglobulin domains
  6. 6. The genes encoding Ig domains are not restricted to Ig genes. Although first discovered in immunoglobulins, they are found in a superfamily of related genes, particularly those encoding proteins crucial to cell-cell interactions and molecular recognition systems. IgSF molecules are found in most cell types and are present across taxonomic boundaries Ig gene superfamily - IgSF
  7. 8. Domains are folded, compact, protease resistant structures Domain Structure of Immunoglobulins Light chain C domains  or  Heavy chain C domains  or  C L V L S S S S S S S S C H 3 C H 2 C H 1 V H Fc Fab F(ab) 2 Pepsin cleavage sites - 1 x (Fab) 2 & 1 x Fc Papain cleavage sites - 2 x Fab 1 x Fc
  8. 9. CH3
  9. 10. CH3 CH2
  10. 11. CH3 CH2 CH1
  11. 12. CH3 CH2 CH1 VH1
  12. 13. CH3 CH2 CH1 VH1 VL
  13. 14. CH3 CH2 CH1 VH1 CL VL
  14. 15. CH3 CH2 CH1 VH1 CL VL
  15. 16. Hinge CH3 CH2 CH1 VH1 VL CL Elbow
  16. 17. Fv Flexibility and motion of immunoglobulins C H 3 C H 2 Fb Fv Fv Fb Fv Hinge Elbow C H 3 C H 2 Fb Fv
  17. 18. Hinge Fv Fb Fab CH3 CH2 CH1 VH1 VL CL Fc Elbow Carbohydrate
  18. 19. View structures
  19. 20. The Immunoglobulin Fold The characteristic structural motif of all Ig domains Barrel under construction A barrel made of a sheet of staves arranged in a folded over sheet A  barrel of 7 (C L ) or 8 (V L ) polypeptide strands connected by loops and arranged to enclose a hydrophobic interior Single V L domain
  20. 21. Unfolded V L region showing 8 antiparallel  -pleated sheets connected by loops. The Immunoglobulin Fold NH 2 COOH S S
  21. 22. View structures
  22. 23. <ul><li>Immunoglobulins must interact with a finite number of specialised molecules - </li></ul><ul><li>Easily explained by a common Fc region irrespective of specificity </li></ul><ul><li>- whilst simultaneously recognising an infinite array of antigenic determinants. </li></ul>In immunoglobulins, what is the structural basis for the infinite diversity needed to match the antigenic universe? Immunoglobulins are Bifunctional Proteins
  23. 24. Cytochromes C Variability of amino acids in related proteins Wu & Kabat 1970 Amino acid No. Variability 80 100 60 40 20 20 40 60 80 100 120 Amino acid No. Variability 80 100 60 40 20 20 40 60 80 100 120 Human Ig heavy chains
  24. 25. <ul><li>Distinct regions of high variability and conservation led to the concept of a FRAMEWORK (FR), on which hypervariable regions were suspended. </li></ul>Framework and Hypervariable regions <ul><li>Most hypervariable regions coincided with antigen contact points - the COMPLEMENTARITY DETERMINING REGIONS (CDRs) </li></ul>FR1 FR2 FR3 FR4 CDR2 CDR3 CDR1 Amino acid No. Variability 80 100 60 40 20 20 40 60 80 100 120
  25. 26. Hypervariable CDRs are located on loops at the end of the Fv regions Hypervariable regions
  26. 27. Space-filling model of (Fab) 2 , viewed from above, illustrating the surface location of CDR loops Light chains Green and brown Heavy chains Cyan and blue CDRs Yellow
  27. 28. <ul><ul><li>The framework supports the hypervariable loops </li></ul></ul><ul><ul><li>The framework forms a compact  barrel/sandwich with a hydrophobic core </li></ul></ul><ul><ul><li>The hypervariable loops join, and are more flexible than, the  strands </li></ul></ul><ul><ul><li>The sequences of the hypervariable loops are highly variable amongst antibodies of different specificities </li></ul></ul><ul><ul><li>The variable sequences of the hypervariable loops influences the shape, hydrophobicity and charge at the tip of the antibody </li></ul></ul><ul><ul><li>Variable amino acid sequence in the hypervariable loops accounts for the diversity of antigens that can be recognised by a repertoire of antibodies </li></ul></ul>Hypervariable loops and framework: Summary
  28. 29. Antigens vary in size and complexity Protein: Influenza haemagglutinin Hapten: 5-(para-nitrophenyl phosphonate)-pentanoic acid.
  29. 30. Antibodies interact with antigens in a variety of ways Antigen inserts into a pocket in the antibody Antigen interacts with an extended antibody surface or a groove in the antibody surface
  30. 31. View structures
  31. 33. Fv Flexibility and motion of immunoglobulins C H 3 C H 2 Fb Fv Fv Fb Fv Hinge Elbow C H 3 C H 2 Fb Fv
  32. 34. 30 strongly neutralising McAb 60 strongly neutralising McAb Fab regions 60 weakly neutralising McAb Fab regions Human Rhinovirus 14 - a common cold virus 30nm Models of Human Rhinovirus 14 neutralised by monoclonal antibodies
  33. 35. Electron micrographs of Antibodies and complement opsonising Epstein Barr Virus (EBV) Negatively stained EBV EBV coated with a corona of anti-EBV antibodies EBV coated with antibodies and activated complement components
  34. 36. Antibody + complement- mediated damage to E. coli Healthy E. coli Electron micrographs of the effect of antibodies and complement upon bacteria
  35. 37. Non-covalent forces in antibody - antigen interactions Electrostatic forces Attraction between opposite charges Hydrogen bonds Hydrogens shared between electronegative atoms Van der Waal’s forces Fluctuations in electron clouds around molecules oppositely polarise neighbouring atoms Hydrophobic forces Hydrophobic groups pack together to exclude water (involves Van der Waal’s forces)
  36. 38. Why do antibodies need an Fc region? <ul><li>Detect antigen </li></ul><ul><li>Precipitate antigen </li></ul><ul><li>Block the active sites of toxins or pathogen-associated molecules </li></ul><ul><li>Block interactions between host and pathogen-associated molecules </li></ul>The (Fab) 2 fragment can - <ul><li>Inflammatory and effector functions associated with cells </li></ul><ul><li>Inflammatory and effector functions of complement </li></ul><ul><li>The trafficking of antigens into the antigen processing pathways </li></ul>but can not activate
  37. 39. Structure and function of the Fc region Fc structure is common to all specificities of antibody within an ISOTYPE (although there are allotypes) The structure acts as a receptor for complement proteins and a ligand for cellular binding sites C H 3 C H 2 IgA IgD IgG C H 4 C H 3 C H 2 IgE IgM The hinge region is replaced by an additional Ig domain
  38. 40. Monomeric IgM IgM only exists as a monomer on the surface of B cells C  4 contains the transmembrane and cytoplasmic regions. These are removed by RNA splicing to produce secreted IgM Monomeric IgM has a very low affinity for antigen C  4 C  3 C  2 C  1 N.B. Only constant heavy chain domains are shown
  39. 41. C  3 binds C1q to initiate activation of the classical complement pathway C  1 binds C3b to facilitate uptake of opsonised antigens by macrophages C  4 mediates multimerisation (C  3 may also be involved) Polymeric IgM IgM forms pentamers and hexamers C  4 C  3 C  2 C  1 N.B. Only constant heavy chain domains are shown
  40. 42. Multimerisation of IgM 1. Two IgM monomers in the ER (Fc regions only shown) 2. Cysteines in the J chain form disulphide bonds with cysteines from each monomer to form a dimer 3. A J chain detaches leaving the dimer disulphide bonded. 4. A J chain captures another IgM monomer and joins it to the dimer. 5. The cycle is repeated twice more 6. The J chain remains attached to the IgM pentamer. C C C C C C C  4 C  3 C  2 C C C  4 C  3 C  2 C C C  4 C  3 C  2 C C C  4 C  3 C  2 C C C  4 C  3 C  2 C C s s s s s s C C s s
  41. 43. Antigen-induced conformational changes in IgM Planar or ‘Starfish’ conformation found in solution. Does not fix complement Staple or ‘crab’ conformation of IgM Conformation change induced by binding to antigen. Efficient at fixing complement
  42. 44. IgM facts and figures Heavy chain:  - Mu Half-life: 5 to 10 days % of Ig in serum: 10 Serum level (mgml -1 ): 0.25 - 3.1 Complement activation: ++++ by classical pathway Interactions with cells: Phagocytes via C3b receptors Epithelial cells via polymeric Ig receptor Transplacental transfer: No Affinity for antigen: Monomeric IgM - low affinity - valency of 2 Pentameric IgM - high avidity - valency of 10
  43. 46. IgD facts and figures IgD is co-expressed with IgM on B cells due to differential RNA splicing Level of expression exceeds IgM on naïve B cells IgD plasma cells are found in the nasal mucosa - however the function of IgD in host defence is unknown - knockout mice inconclusive Ligation of IgD with antigen can activate, delete or anergise B cells Extended hinge region confers susceptibility to proteolytic degradation Heavy chain:  - Delta Half-life: 2 to 8 days % of Ig in serum: 0.2 Serum level (mgml -1 ): 0.03 - 0.4 Complement activation: No Interactions with cells: T cells via lectin like IgD receptor Transplacental transfer: No
  44. 47. IgA dimerisation and secretion IgA is the major isotype of antibody secreted at mucosal sufaces Exists in serum as a monomer, but more usually as a J chain-linked dimer, that is formed in a similar manner to IgM pentamers. IgA exists in two subclasses IgA1 is mostly found in serum and made by bone marrow B cells IgA2 is mostly found in mucosal secretions, colostrum and milk and is made by B cells located in the mucosae J C C S S S S C C S S S S C C s s
  45. 48. Secretory IgA and transcytosis ‘ Stalk’ of the pIgR is degraded to release IgA containing part of the pIgR - the secretory component Epithelial cell J C C S S S S C C S S S S C C s s B J C C S S S S C C S S S S C C s s J C C S S S S C C S S S S C C s s J C C S S S S C C S S S S C C s s pIgR & IgA are internalised J C C S S S S C C S S S S C C s s IgA and pIgR are transported to the apical surface in vesicles B cells located in the submucosa produce dimeric IgA Polymeric Ig receptors are expressed on the basolateral surface of epithelial cells to capture IgA produced in the mucosa
  46. 49. IgA facts and figures Heavy chains:  1  or  2 - Alpha 1 or 2 Half-life: IgA1 5 - 7 days IgA2 4 - 6 days Serum levels (mgml -1 ): IgA1 1.4 - 4.2 IgA2 0.2 - 0.5 % of Ig in serum: IgA1 11 - 14 IgA2 1 - 4 Complement activation: IgA1 - by alternative and lectin pathway IgA2 - No Interactions with cells: Epithelial cells by pIgR Phagocytes by IgA receptor Transplacental transfer: No To reduce vulnerability to microbial proteases the hinge region of IgA2 is truncated, and in IgA1 the hinge is heavily glycosylated. IgA is inefficient at causing inflammation and elicits protection by excluding, binding, cross-linking microorganisms and facilitating phagocytosis
  47. 50. IgE facts and figures IgE appears late in evolution in accordance with its role in protecting against parasite infections Most IgE is absorbed onto the high affinity IgE receptors of effector cells IgE is also closely linked with allergic diseases Heavy chain:  - Epsilon Half-life: 1 - 5 days Serum level (mgml -1 ): 0.0001 - 0.0002 % of Ig in serum: 0.004 Complement activation: No Interactions with cells: Via high affinity IgE receptors expressed by mast cells, eosinophils, basophils and Langerhans cells Via low affinity IgE receptor on B cells and monocytes Transplacental transfer: No
  48. 51. The high affinity IgE receptor (Fc  RI) The IgE - Fc  RI interaction is the highest affinity of any Fc receptor with an extremely low dissociation rate. Binding of IgE to Fc  RI increases the half life of IgE C  3 of IgE interacts with the  chain of Fc  RI causing a conformational change.  chain  chain  2 S S S S S S C  1 C  1 C  2 C  2 C  3 C  3 C  4 C  4 C  1 C  1 C  2 C  2 C  3 C  3 C  4 C  4
  49. 52. IgG facts and figures Heavy chains:  1  2  3  4 - Gamma 1 - 4 Half-life: IgG1 21 - 24 days IgG2 21 - 24 days IgG3 7 - 8 days IgG4 21 - 24 days Serum level (mgml -1 ): IgG1 5 - 12 IgG2 2 - 6 IgG3 0.5 - 1 IgG4 0.2 - 1 % of Ig in serum: IgG1 45 - 53 IgG2 11 - 15 IgG3 3 - 6 IgG4 1 - 4 Complement activation: IgG1 +++ IgG2 + IgG3 ++++ IgG4 No Interactions with cells: All subclasses via IgG receptors on macrophages and phagocytes Transplacental transfer: IgG1 ++ IgG2 + IgG3 ++ IgG4 ++
  50. 53. Carbohydrate is essential for complement activation Subtly different hinge regions between subclasses accounts for differing abilities to activate complement C1q binding motif is located on the C  2 domain
  51. 54. Fc  receptors Receptor Cell type Effect of ligation Fc  RI Macrophages Neutrophils, Eosinophils, Dendritic cells Uptake, Respiratory burst Fc  RIIA Macrophages Neutrophils, Eosinophils, Platelets Langerhans cells Uptake, Granule release Fc  RIIB1 B cells, Mast Cells No Uptake, Inhibition of stimulation Fc  RIIB2 Macrophages Neutrophils, Eosinophils Uptake, Inhibition of stimulation Fc  RIII NK cells, Eosinophils, Macrophages, Neutrophils Mast cells Induction of killing (NK cells) High affinity Fc  receptors from the Ig superfamily:
  52. 55. The neonatal Fc  receptor The Fc  Rn is structurally related to MHC class I In cows Fc  Rn binds maternal IgG in the colostrum at pH 6.5 in the gut. The IgG receptor complex is trancytosed across the gut epithelium and the IgG is released into the foetal blood by the sharp change in pH to 7.4 Some evidence that this may also happen in the human placenta, however the mechanism is not straightforward. Human Fc  Rn Human MHC Class I
  53. 56. Molecular Genetics of Immunoglobulins
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