Structure of Antibodies and BCR PDF

Structure of the Antibodies and BCR BCR is antibody in the B cell work as a receptor if in T cell called TCR can not secret  Serum Antibodies made by activated B-cells Called plasma cells. They are glycoproteins with immune function. (protein attached t...

Structure of the Antibodies and BCR BCR is antibody in the B cell work as a receptor if in T cell called TCR can not secret  Serum Antibodies made by activated B-cells Called plasma cells. They are glycoproteins with immune function. (protein attached to carbohydrate) Antibodies also called immunoglobulins-Ig. Structure is globulin and have immune function – 5 classes: (issotype) IgG Gamma (5) IgM mu (5) IgA alpha (4) IgE epsilon (4) IgD delta (4) name of subissotype (Ig1.Ig2…)  Antibody Structure Functional structure Hinge – biological activity - activate effectors' region mechanisms – Antigen binding site Bind to one specific epitope Hinge Region: – flexibility to binding/angle of antibody kappa or between 2 bound epitopes lambda – allow movement of the arm CH only IgG, IgA and IgD have hinge region (3 constant domains) Gamma, IgM and IgE have no hinge (5 domain) mu, alpha, – (does not mean no flexibility) have 4 domains epsilon, some flexibility between V and C domain delta biological activity Structure of antibody is Y shape has two arm antibody have 4 shape 2 light 2 heavy Bound together by disulfide bound Antibody like any polysaccharide have N terminal in arm have bind site on antibody have 2 binding site and C terminal in the body We can identify the antibody by the heavy chain if I have IgG according to the heavy chain Each antibody specific bind to each epitope Light L heavy H Have domain some antibody have 4 domain in the heavy chain and some have 5 but in light have only 2 domain the domian which bind to antigen called variable domain If I produce 1000 antibody its have the same C but different V We can recognize the antibody by 1. the carbohydrate and 2. the chain The heavy chain is important  Functional structure Antigen binding site – Each antibody (monomer) has only two antigen binding site – Composed from the tip of the variable domain of heavy and light chains region – This region also called Fab region (fragment ability binding) biological activity – activate complement – bind to phagocytes – this is constant region of two identical heavy chains – Bind to Fc receptors on effector cells – Each antibody (monomer) has only one biological active region – This region also called Fc region fragment that can crystallization ) bioligy function complement activation. Phagocytosis  Proteases used to study antibodies Purposes Studying structures In immune diagnosis ‫مادة تضوي بصبغها بال‬antibody In immune therapies – papain (to cut) 2 Fab and 1 Fc fragments – pepsin (to cut bellow the hinge region ) F(ab)2 can bind antigen but no biological activity ?? I use this enzyme to allow the Antibody to inter the cell Genetic engineering (cancer) – single chain has only Variable region Fv (fragment variable) – 25 m  Domain Structure Similar overall polypeptide contain amino acid sequence (~110 aa) & structure – homology (~60%) anti-parallel beta sheets/strands or the structure of aa (110) When you fold it we called beta barrel or beta sandwich structure both of them we called ig domain – disulfide bond beta strands create loops The different in loop 3 loop responsabile for binding Each domain have 3 loop HV  Ig supergene family Conserved structure found frequently in proteins involved in cell-cell interactions Immunoglobulin domain- found in many molecules – Ig, TCR, MHC – ICAM (intracellular adhesion molecule) At least one domain similar to Ig domain (V, C1 or C2)  Hypervariable regions HV The most of the variability resides in the variable regions of different Ig's present in: – three regions called the hypervariable regions variability plot-HV1, HV2, HV3 (determine the binding site of the ag) Antibodies with different specificities have different HV regions – In the same Ig molecule: the light chains HV regions are different from the Heavy chains HV Complementarity determining regions (CDR) HV1 from light and HV1 from heavy The of the HV region of the heavy and light chain combination in one arm of the Ig molecule create: – complementarity determining regions Therefore each arm has 3 CDR CDR present in the two arms of the Ig molecule are identical – CDR1 in the arm 1 of single Ig is similar to CDR1 present on the second arm (bind the same epitope ) framework regions – Refer to the semiconservative a.a. sequence in the variable domain » The regions between the HV regions in the variable region Pocket, groove or extended , surface  Nature of Ag/Ab Reactions Lock and Key Concept (Induce fittnese) Non-covalent Bonds (weak) – Hydrogen bonds – Electrostatic bonds – Van der Waal forces – Hydrophobic bonds Multiple Bonds Weak non-covalent bonds over very short ranges Disrupt: high salt concentrations, extremes of pH, detergents, and high concentrations of pure epitope (if increase antigen or antibody lead to no reaction Reversible , strong  Binding of Ag and Ab 2 type of antigen 1.Conformational or discontinuous epitope ‫برتبط بشكل‬ ‫معين‬ – segments of the protein that are discontinuous in the a.a. sequence but brought together by the 3 dimensional structure 2.Continuous or linear epitope ‫قلب‬ ‫الحب‬ Ab normally recognize Ag in its native state The power bind between Ab and Ag ‫برتبط كيف ما‬ ‫كان‬ – Affinity: Strength of the reaction between a single antigenic determinant and a single Ab combining site (determine the avidity) – Avidity: The overall strength of binding between an Ag with many determinants and multivalent Abs (IgG monomer IgM pentumum  Nature of Ag/Ab Reactions Specificity – The ability of an individual antibody combining site to react with only one antigenic determinant. – The ability of a population of antibody molecules to react with only one antigen. – Levels of specificities Fab on the same Ab (bind the same epitope) Antibody from the same plasma cells (BCR or secreted is the same) Antibodies from same progeny Antibodies secreted vs membrane bound (BCR)b cell receptor Cross Reactivity – The ability of an individual Ab combining site to react with more than one antigenic determinant.(weak binding) – The ability of a population of Ab molecules to react with more than one Ag High Affinity Low Affinity Ab Ab Structure of the T-cell Receptor  TCR on the T cell can not secretes(formate just a receptor) Similar to Ig Fab (Belong Ig super family) Compared to Ig (BCR), TCR – have only one Ag binding site – never secreted (always membrane bond) – extrenal structure fold the same way as BCR folds – have short hinge region with disulfide bond (move is – Very limited) (N terminus and C terminus bind with membrane ) one binding site – have one V domain and one constant domain in each chain V, C. C, C – Less C region possiblities – no real biologic activity – hydrophobic transmembrane – short cytoplasmic tail (can not send signals) – CHO group from C making H. bond with group from C – (Antibody as a receptor) – Consist of alpha beta or gamma dalta – (antibody can not bind with antigen in native format )  TCR Type of TCR found (determine by the chain forming the receptors) – majority: alpha & beta chain (linked by disulfide bond) – minority: gamma & delta chain(linked by disulfide bond)2year (bind with proteins only) – few in circulation (1-5%) – mainly in epithelial tissue: epidermis and small intestine – produced very early in development – structure not crystalized and studied – not MHC restricted in the classical sense – (T cell have molecule to help it without this molecule the T cell became inhibited ) CO receptor CD4 MHC2 helper or CD8 MHC1 cytotoxic – Without this I need a lot of T cell we need at least 2 co receptor without no f Each T cell bear up to 30,000 receptor Antigen recognition: – TCR: short contiguous (continous) epitopes of peptide – often from internal site of Ag – Processed antigen – held by MHC molecules in the groove – Compound ligand for TCR peptide+MHC – Therefore, TCR binds only processed antigen presented by MHC molecule – Hypervariable regions in V contribute to diversity of TCR  TCR Co-receptors: CD4 & CD8 Function – Strengthen binding of TCR: MHC – Increases T-cell sensitivity (long cytoplasmic tail involved in signaling) need less Ag for activation (100 fold less requirement for activation) – Cytoplasmic domain interacts with internal cytoplasmic tyrosine kinase Nature of binding – Bind to invariant part of MHC – Co-receptors binding to MHC alone is weak binding CD4 binds MHC II (helper T cell ) CD8 binds MHC I cytotoxic T cell (highly glycolation rich with Suger Structures – CD4 monomeric hinge D1 and D2 packed tightly - same for D3 and D4 Ig like structure – CD8 is heterodimer two chains  and   CD8  could form homodimer each chain has single Ig-like domain structure linked to membrane by long polypeptide chain – chain is highly glycosylated (keep the structure) » protect from cleavage and keep chain extended Ig Gene Rearrangement always generate B cell against foreign substance when the B cell development became rearrangement in the variable region  Immunoglobulin diversity Diversity: is the total of all the antibodies specificities that an individual is capable of expressing Complete V-domain are generated by somatic recombination – light chain V & J gene segments – heavy chain V, D & J gene segments – exons (coding regions) vs introns (non-coding regions) – Like a.a when form protein – V variable D diversity J joining  TCR has similar gene structure to BCR Two chains (alpha and beta) with 2 V-regions  variable domain made from – V and J segments  variable domain made from – V, D, and J segments Simpler constant domains – why – (One  &  and shorter structural domains)  Diversity 1. Multiple copies of genetically different V (D) J segments Multiple copies of V (D) J different possibilities 2. Combinatorial diversity – Light chain and heavy chain joining independently Rearranged heavy chain combine with rearranged light chain widen the possibilities of combination  Diversity (make the diversity of T cell more than B cell) 3. Flexibility of joining (Junctional diversity) – Recombination steps P-nucleotide formation (palindromic sequences) – Rag proteins catalyzes a single strand cleavage at the point of hairpin loop creating a single stranded tail N-nucleotide addition (highly random ) – can randomly add up to 15 bp) /needs Tdt add nucleotide (RAG enzyme) – Continued addition until pairing occurs with the other gene segment  Diversity 4. Somatic hypermutation (just in B cell ) – occurs in secondary immune organs after cells are activated (exposed to Ag) – occurs in all rearranged Ig V region DNA random mutations – Most productive changes occur in CDR1, CDR2, (bind with MHC CDR3 bind with peptide (hotspots) if occur in FR could cause disruption of the Ab structure – It enhance tight binding Result in affinity maturation  TCR gene rearrangement mechanisms TCR has similar mechanisms of gene rearrangement to BCR – Takes place in Thymus – Recombination controlled by same recombinase enzyme systems defect in recombinase enzyme system? -SCID – More P and N nucleotide addition occur at all recombination junctions – More J segments in alpha chain – CDR1 or CDR2 diversity only comes from choosing different V segment The periphery CDR1 & 2 less diverse to recognize MHC – Most of the diversity occurs in CDR3 (Why?) » CDR3 more diverse in TCR Locate in the center of the binding and have direct contact with Ag more J and more P & N nucleotide  TCR gene rearrangement mechanisms No somatic mutation - why? T-cells can’t risk becoming self reactive because it is a center cell in the immune system While Somatic mutation occurs at all CDR’s in BCR – Creation of self reactive B cell is possible » Fortunately without T cell help- no activation of B cell occurs and self reactive B cell die by apoptosis) – T-cells can’t risk losing MHC restriction Specially at CDR1 and CDR2 (less diverse CDR1 and CDR2) – Over all more diversity in TCR than BCR B-cell development  Lymphocytes are important! We each have 1,000,000,000,000 of them Approximately half are “B cells” and half are “T cells” B lymphocytes & Disease – Leukaemia; Lymphoma; Myeloma – Immunodeficiencies Children congenitally deficient in lymphocytes (“SCIDS”) die very quickly due to overwhelming infections – Autoimmunities – Allergies Stages of B-cell development Antigen independent- – bone marrow Antigen dependent- – bone marrow + periphery Antigen dependent – Terminal development- Peripheral immune organs Bone marrow Stromal cells Provide growth factors that stimulate differentiation and proliferation – IL-7 Stages determined by Ig rearrangement – pro-B-cell Early pro-B cell Late pro-B cell – Pre-B-cell LARGE-pre-B-cell receptor with surrogate light chain small-rearrangement of light chain  Gene Rearrangement and Lymphocytes development General notes – A productive rearrangement of cell R leads to the synthesis of protein products which signal the cell to progress into the next stage if not productive – Non-productive rearrangement lead to either to further rearrangement or cell death - apoptosis if productive rearrangement – need signal to stop rearrangement and proceed with the next chain – Rearrangement always occurs in the locus that has the D gene segment B cell ---- Heavy chain T cell ---- beta chain locus  Early pro-B cell Late pro-B cell – limited self renewal – Rearrange V-DJ – D-J rearrangement - both heavy – one allele at time chain genes – Check for success & Signal – Usually successful – This mean a B-cell receptor – No Ig on surface expression B Cell Development: Part 1 “Stem cell” > “Large pre-B”  Problem? How the receptor expressed with only one chain rearranged – Need complete BCR and invariant chains for surface expression! Solution: “pre-B cell receptor complex” – “surrogate” light chain – rearranged heavy chain  Expression of pre-B-cell receptor Hallmark of Large pre-B cell – Pre BCR (instead of light chain --- a surrogate light chain) But mostly intracellular accumulation of rearranged heavy chain Stops – gene rearrangement making RAG-1 and RAG-2 presence decrease Starts- proliferating – expands population 30-60X – Same specificity  Immature B cell Receptor editing Immature B cell – If productive… check it out on surface Therefore functional B-cell receptor complex appear This now is called immature B cell An immature has a complete BCR complex on the surface  Mature B cell Immature B cell express IgM leaves the bone marrow (Why?) Very close to the rearranged genes The final maturation step is expression of IgD along with IgM on the surface Related to mRNA processing  Small pre-B cell and receptor Immature B celland receptor editing editing No surface -heavy chain If productive… – Start rearrangement of light chain – check it out on surface isotypic exclusion - has preference Therefore functional B-cell – -usually first; -second receptor complex appear » ratio: humans 65:35 – Now called immature B cell – Usually successful! – An immature has a complete – More chances BCR complex on the surface (2 types of light chain  &  and two alleles for each one from each parents) – If not productive…..? B Cell Development: Part 2 “Small pre-B” > “Mature B”  B cells - the early days Pro-B cells: HC gene rearrangements Pre-B cells: LC gene rearrangements Immature B cells: express surface IgM; exits the BM; if “sees” an antigen, then eliminated (negative selection) Mature B cells: express sIgM & IgD; circulates and now ready to “do their thing” The Thymus & T Lymphocyte Development  The Thymus as The Primary Site of T-cell Production Bone marrow/ Fetal liver  T-cells precursor Dendritic cells  T-cells NK cells Commitment to the T-cell Lineage Number of New T-cells produced per day In health, thymus atrophy is okay because T-cells are long lived  The Thymus Consists Of Two Main Cellular Elements: Haemopoietic bone marrow/fetal liver derived precursors, which are developing into T-cells Non-haemopoietic cells called STROMAL CELLS, which drive the development of T-cell precursors Both elements are required for normal T-cell production – Cellular Expansion of Early T-cell Precursors in Thymus Likely to be mediated by soluble growth factors CD4-8- Thymocyte Thymic Epithelial Cell Express receptors for: Producers of: Interleukin-2 (IL-2) Interleukin-7 Interleukin-7 (IL-7) Interleukin-15 Interleukin-15 (IL-15) Stem Cell Stem Cell Factor (SCF) Factor  Cellular Expansion of Early T-cell Precursors in Thymus ▪ A role for Stem Cell Factor and IL-7: studies on mice lacking IL-7Receptor and c-Kit Gene Knockout Mouse Thymus: Wildtype Mouse Thymus: Genes encoding IL-7receptor and c-Kit deleted Less than 120 million 1 million thymocytes thymocytes T-cell Development Can Be Separated Into Distinct Stages Lack Expression Increasing Maturity Dual Expression Of CD4 and CD8 CD8 expression CD4 expression only only ‘CD8 single ‘CD4 single positive’ positive’ Two Main Checkpoints in T-cell Development Maturation from the CD4-8- to CD4+8+ Stage – Events include: Commitment to the T-cell lineage Rearrangement of genes encoding the T-cell receptor Negative selection Cellular expansion Maturation from the CD4+8+ to the CD4+ or CD8+ Stage Negative selection Positive selection  Rearrangement of T-cell Receptor Genes T-cell Receptor  chain gene Variable (V) Diversity (D) Joining (J) Constant (C) regions regions regions regions Fully rearranged gene TCR  protein  Selecting Cells Bearing Fully Rearranged TCR Chain Genes: A function of the pre-T cell receptor Pre-T cell receptor Complex Expressed only on CD4-8- thymocytes Consists of TCR protein, CD3, and pre-T Signalling through pre-TCR is essential for the generation of CD4+8+ thymocytes Precursor undergoing TCR Unsuccessful rearrangement rearrangement Cell Death Successful TCR rearrangement Pre-T 1. Stops TCR Rearrangement CD3 (Allelic exclusion) 2. CD4, CD8 expression Signalling 3. Cellular expansion 4. TCR rearrangement Second stage: The Need For Positive and Negative Selection in Thymus Unlike B-cells, T-cells can only recognise processed antigen presented by self Major Histocompatibility Complex (MHC) molecules Millions of CD4+8+ thymocytes, each expressing an Antigen TCR complex of differing T-cell Presenting specificity Cell Because rearrangement of TCR genes is random, these specificities could be: USELESS: Don’t recognise peptide/MHC at all USEFUL: Recognise peptide/MHC of low affinity or avidity HARMFUL: Recognise peptide/MHC at high affinity or avidity Need to: Retain USEFUL specificities (POSITIVE SELECTION) Remove HARMFUL specificities (NEGATIVE SELECTION CD4- CD4+ Positive Selection of CD4+ and CD8- CD8+ CD8+ Cells TCR binds to PRE-TCR MHC Class II on thymic epithelium TCR binds to MHC Class I on thymic epithelium Loss of CD4 Expression Loss of CD8 Expression CD4+ CD4- CD8- CD8+ Conclusions The thymus is essential for T-cell development The most immature thymocytes are CD4-8-, they develop to the CD4+8+ stage by signalling through the pre-T-cell receptor CD4+8+ thymocytes express the T-cell receptor. These cells must be sorted, or SELECTED to allow only cells which can bind MHC in a non-autoreactive way to survive.  Tumor cells found in same spot as normal counterparts leukemia lymphoma Myeloma Allow study of – stages of B cell development – Receptors for homing – Lead to better diagnostics and therapy!