免疫球蛋白的结构与功能的关系.ppt

Immunoglobulin Structure- Function Relationship 免疫球蛋白的结结构与功能的关系 Signalling antigen receptors on B cells - bifunctional antigen-binding secreted molecules（B 细胞表面受体和分泌的抗体） Structural conservation and infinite variability - domain structure（结构 上不仅保守而且无限可变的）. The Immunoglobulin Gene Superfamily （免疫球蛋白的超家族） The immunoglobulin fold （免疫球蛋白的折叠） Framework and complementarity determining regions - hypervariable loops （框架结构和可变区） Modes of interactions with antigens （与抗原相互作用的模型） Effector mechanisms and isotype role of the Fc. （Fc 区的作用） Multimeric antibodies and multimerisation Characteristics and properties of each Ig isotype Ig receptors and their functions Immunoglobulin Structure-Function Relationship Cell surface antigen receptor on B cells B 细胞表面受体和分泌的抗体 Allows B cells to sense their antigenic environment Connects extracellular space with intracellular signalling machinery Secreted antibody （抗体） Neutralisation （中和作用） Arming/recruiting effector cells （激活或者诱导功能细胞） Complement fixation （帮助机体对抗原的清除） Immunoglobulin Structure-Function Relationship Immunoglobulins are Bifunctional Proteins Immunoglobulins must interact with a small number of specialised molecules - （免疫球蛋白必须与特殊分子相互作用） Fc receptors on cells （细胞表面的Fc受体） Complement proteins （辅助蛋白） Intracellular cell signalling molecules （细胞内信号转导分子） whilst simultaneously recognising an infinite array of antigenic determinants. （同时能够识别无限抗原族） Structural conservation and a capacity for infinite variability in a single molecule is provided by a DOMAIN structure. （结构上不仅保守而且无限可变的- 抗体结构域） 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（Ig 结构域源于一个原始基因，复制，多元化，修饰等） Ig domains are not restricted to immunoglobulins （Ig 结构域不仅 仅局限于免疫球蛋白）. The most striking characteristic of the Ig domain is a disulphide bond - linked structure of 110 amino acids long（Ig结构域最明显 的特点是其双硫键，连接了110个氨基酸）. Immunoglobulin domains 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 Antibodies are Proteins that Recognize Specific Antigens 抗体能够特异性的识别抗原 Epitopes（抗原决定簇 ）: Antigen Regions that Interact with Antibodies Consequences of Antibody Binding 抗体结合效应 CL VL S S S S S S S S CH3 CH2 CH1 VH FcFab F(ab)2 Domains are folded, compact, protease resistant structures Domain Structure of Immunoglobulins 免疫球蛋白的结构域 Pepsin cleavage sites - 1 x (Fab)2 & 1 x Fc Papain cleavage sites - 2 x Fab 1 x Fc Light chain C domains k or l Heavy chain C domains a, d, e, g, or m 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 CH3 CH2 Fb Fv Fv Fv Fb Fv Hinge Elbow CH3 CH2 Fb Fv Flexibility and motion of immunoglobulins Hinge Fv Fb Fab CH3 CH2 CH1 VH1 VL CL Fc Elbow Carbohydrate 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 b barrel of 7 (CL) or 8 (VL) polypeptide strands connected by loops and arranged to enclose a hydrophobic interior Single VL domain Unfolded VL region showing 8 antiparallel b-pleated sheets connected by loops. NH2 COOH S S The Immunoglobulin Fold Immunoglobulins must interact with a finite number of specialised molecules - Easily explained by a common Fc region irrespective of specificity - whilst simultaneously recognising an infinite array of antigenic determinants. In immunoglobulins, what is the structural basis for the infinite diversity needed to match the antigenic universe? Immunoglobulins are Bifunctional Proteins Amino acid No. Variability 80 100 60 40 20 20406080100120 Cytochromes C Variability of amino acids in related proteins Wu & Kabat 1970 Amino acid No. Variability 80 100 60 40 20 20406080100120 Human Ig heavy chains FR1FR2FR3FR4CDR2CDR3CDR1 Distinct regions of high variability and conservation led to the concept of a FRAMEWORK (FR), on which hypervariable regions were suspended. Framework and Hypervariable regions Amino acid No. Variability 80 100 60 40 20 20406080100120 Most hypervariable regions coincided with antigen contact points - the COMPLEMENTARITY DETERMINING REGIONS (CDRs) Hypervariable regions Hypervariable CDRs are located on loops at the end of the Fv regions Space-filling model of (Fab)2, viewed from above, illustrating the surface location of CDR loops Light chainsGreen and brown Heavy chainsCyan and blue CDRsYellow The framework supports the hypervariable loops The framework forms a compact b barrel/sandwich with a hydrophobic core The hypervariable loops join, and are more flexible than, the b strands The sequences of the hypervariable loops are highly variable amongst antibodies of different specificities The variable sequences of the hypervariable loops influences the shape, hydrophobicity and charge at the tip of the antibody Variable amino acid sequence in the hypervariable loops accounts for the diversity of antigens that can be recognised by a repertoire of antibodies Hypervariable loops and framework: Summary Antigens vary in size and complexity Protein: Influenza haemagglutinin Hapten: 5-(para-nitrophenyl phosphonate)-pentanoic acid. 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 CH3 CH2 Fb Fv Fv Fv Fb Fv Hinge Elbow CH3 CH2 Fb Fv Flexibility and motion of immunoglobulins 30 strongly neutralising McAb 60 strongly neutralising McAb Fab regions60 weakly neutralising McAb Fab regions Human Rhinovirus 14 - a common cold virus 30nm Models of Human Rhinovirus 14 neutralised by monoclonal antibodies 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 Antibody + complement- mediated damage to E. coli Healthy E. coli Electron micrographs of the effect of antibodies and complement upon bacteria Non-covalent forces in antibody - antigen interactions Electrostatic forcesAttraction between opposite charges Hydrogen bondsHydrogens shared between electronegative atoms Van der Waals forces Fluctuations in electron clouds around molecules oppositely polarise neighbouring atoms Hydrophobic forcesHydrophobic groups pack together to exclude water (involves Van der Waals forces) Why do antibodies need an Fc region? Detect antigen Precipitate antigen Block the active sites of toxins or pathogen-associated molecules Block interactions between host and pathogen-associated molecules The (Fab)2 fragment can - Inflammatory and effector functions associated with cells Inflammatory and effector functions of complement The trafficking of antigens into the antigen processing pathways but can not activate Structure and function of the Fc region CH3 CH2 IgA IgD IgG CH4 CH3 CH2 IgE IgM The hinge region is replaced by an additional Ig domain 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 Monomeric IgM IgM only exists as a monomer on the surface of B cells Cm4 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 Cm4 Cm3 Cm2 Cm1 N.B. Only constant heavy chain domains are shown Cm3 binds C1q to initiate activation of the classical complement pathway Cm1 binds C3b to facilitate uptake of opsonised antigens by macrophages Cm4 mediates multimerisation (Cm3 may also be involved) Cm4 Cm3 Cm2 Cm1 N.B. Only constant heavy chain domains are shown Polymeric IgM IgM forms pentamers and hexamers C C C C C C Multimerisation of IgM Cm4 Cm3 Cm2 C C Cm4 Cm3 Cm2 C C Cm4 Cm3 Cm2 C C Cm4 Cm3 Cm2 C C Cm4 Cm3 Cm2 C C s s s s s s C C s s 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. 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 IgM facts and figures Heavy chain:m - 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 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:d - 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 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. J C C S S S S C C S S S S C C s s 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 Epithelial cell JCC S S S S C C S S S S C C ss Secretory IgA and transcytosis B J C C S S S S C C S S S S C C s s JCC S S S S C C S S S S C C ss JCC S S S S C C S S S S C C ss pIgR & IgA are internalised Stalk of the pIgR is degraded to release IgA containing part of the pIgR - the secretory component JCC S S S S C C S S S S C C ss 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 IgA facts and figures Heavy chains:a1 or a2 - 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 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:e - 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 The high affinity IgE receptor (FceRI) a chain b chain g2 SS SS SS Ce1Ce1 Ce2 Ce2 Ce3 Ce3 Ce4 Ce4 Ce1 Ce1 Ce2 Ce2 Ce3 Ce3 Ce4 Ce4 The IgE - FceRI interaction is the highest affinity of any Fc receptor with an extremely low dissociation rate. Binding of IgE to FceRI increases the half life of IgE Ce3 of IgE interacts with the a chain of FceRI causing a conformational change. IgG facts and figures Heavy chains:g 1 g 2 g3 g4 - 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): IgG15 - 12IgG2 2 - 6 IgG3 0.5 - 1IgG4 0.2 - 1 % of Ig in serum:IgG145 - 53IgG2 11 - 15 IgG3 3 - 6IgG4 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 + 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 Cg2 domain Fcg receptors ReceptorCell typeEffect of ligation FcgRIMacrophages Neutrophils, Eosinophils, Dendritic cells Uptake, Respiratory burst FcgRIIAMacrophages Neutrophils, Eosinophils, Platelets Langerhans cells Uptake, Granule release FcgRIIB1 B cells, Mast CellsNo Uptake, Inhibition of stimulation FcgRIIB2 Macrophages Neutrophils, Eosinophils Uptake, Inhibition of stimulation FcgRIIINK cells, Eosinophils, Macrophages, Neutrophils Mast cellsInduction of killing (NK cells) High affinity Fcg receptors from the Ig superfamily: The neonatal Fcg receptor The FcgRn is structurally related to MHC class I In cows FcgRn 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 FcgRnHuman MHC Class I