Antigens and Epitopes (MDSC 321-14) PDF

ANTIGENS So…. What can the immune system “see” Infection (bad “foreign”) Gut Flora (good “foreign”) Cancer...

ANTIGENS So…. What can the immune system “see” Infection (bad “foreign”) Gut Flora (good “foreign”) Cancer (bad “self”) Autoimmunity (good “self”) can see infection, can also see normal gut ora, this doesnt cause disease, its a part of our barrier, but its still not us can be foreign and good can also have cancer, not foreign but bad autoimmunity, IS sees our tissue, its good tissue iS can see anything and its largely based on shape, if it has shape, can recognize it, whether its good or bad is based on context ANTIGENS Whereas the innate immune system recognizes conserved pathogen associated molecular patterns (PAMPS), the adaptive immune system recognizes antigens. innate saw broad general patterns, adaptive sees smething precise, these precise things are antigens antigen = piece of something that is recognized by adaptive IS ANTIGENS Antigens, what are they? some of these terms were invented in immunology before we knew big picture ANTIGENS Antigen (Ag): Any molecule that can interact (specifically) with the immunoglobulin (Ig) receptor of B-cells (or the T-cell receptor complexed with MHC). any molecule that speci cally recognized by an antibody or immunoglobulin (larger collective term of anjtibody, binds at the end of antibody arm, what ab sticks to) tiny piece of target that is recognized either by immunogloublin for b cells or by the t cell receptor by t cells ANTIGENS IS can recognize any shape, when we think of immunoglob, can recognize metal ions, as long as it has 3d structure, antibody can stick to it enzymes --> lock and key idea, same with antibody antibody arm has 3d binding pocket, if target can t in it, it will stick dont have immune responses against food we eat, our poretins, not bc we cant see thwm if u have peanut allergy, antibodies see peanuts, antibody can do it, in most of us it doesnt just bc u have shape doesnt mean ur good at building immune response have subgroup of antigens knowns as immunogens, these can elecit immune response Antigen (Ag): Any molecule that can interact (specifically) with the immunoglobulin (Ig) receptor of B-cells (or the T-cell receptor complexed with MHC). But not all molecules induce immunity – what’s up with that? Immunogens……. ANTIGENS What is an immunogen? ANTIGENS Immunogen: A molecule that induce a specific immune response. adaptive immune response adaptive = speci c, di erentiate s b/w closely related molecules instead of pamp, have molecule that elcits speci c immune response, this is immunogen Antigen: Any molecule that can interact (specifically) with the immunoglobulin (Ig) receptor of B-cells (T-cell receptor complexed with MHC). ANTIGENS All immunogens are antigens. Not all antigens are immunogens. group of antigens that generate immune response = immuogen this is what matters, cant focus on how IS sees eveyrthing, focus onw hat it actually reacts to these are immunogens  allows us to understand mols in a context ex. proteins tructure such as agella, is a ligand for TLR, this is a PAMP ANTIGENS ◦ at the same time, if u grow bacteria in dish and use lab techniques, and use antibodies to stain bands on gel, now have antibodies binding same protein that was PAMP ◦ if its recognized by antibody, now its antigen ◦ put agella into vaccine and injected it and give it to b cells in vivo, will generate immune response ‣ now its not just antigen, its immunogen ‣ exact same protein can be neither an antigen or immunogen if it binds to TLR, if we use it on bench when we're not generating immune resposne, now its antigen but not immunogen bc doesnt drive response ‣ put it in vaccine and give it to somebody and they make Flagellin antibodies, now this mol is immunogen AND antigen ◦ terms are context related, have antibodies that see dna, ex. of lupus ‣ dna recognized by TLR as well ‣ same mol can be PAMP and antigen and immunogen ‣ some antigens are good at being immunogen, something about them that makes IS angry ‣ all of these can be seen by IS but some make it angry Ligand for macrophages in culture - TLR5 (PAMP) Immunogen – No Antigen - No Western blot - detected by antibodies Immunogen – No Antigen - Yes Ligand for B-cells in vivo Immunogen – Yes Antigen - Yes ANTIGENS What makes a good immunogen? ANTIGENS - immunogenicity Humoral Immunogens (B-cells) – Proteins >> Polysaccharides >> Lipids or Nucleic Acids Cell Mediated Immunogens (T-cells) – Proteins, some lipids, some glycolipids. – Proteins are not recognized directly, peptides processed from the protein are seen in association with MHC molecules, lipids with an MHC-like molecule CD1 ‣ b cell making antibodies, can see anthing ‣ have preference tho, like proteins most ‣ makes sense, if we're seeing 3d shape, proteins are complex, then sugars wqhich are repeating steuctural units, and the.n lipids or NA which are repeating ‣ lots of diversity nad structures in protein, as u go down this preference, mol get more simpke ‣ more complicated = more likely by b cells ‣ t cells only see proteins with some exceptions, more speicifcally see only peptides (piece of proteins), 8-15 a.a, t cell does not ever see target directly, has to be shown with MHC ‣ b cells see anytghin with 3d shape, t cells only see petide and only when its presented by MHC  ANTIGENS – 4 properties of an immunogen 1. Foreignness – To serve as an immunogen, a molecule must be seen as “non- self”. The degree of immunogenicity is dependent upon the degree of foreignness. The greater the phylogenetic distance between species typically the greater the chance of immunogenicity. – E.g. Bovine Serum Albumin injected into chickens or goats. ‣ foreigness = if its not me, wont like it probably, not alway ex. foods, but in general, if its di from me, im gonna see that as a problem, and build IR against it ‣ if its a self protein, hopei ly we've taught IS about it, reason why we dont have autoimmuntiy is bc we're toleraized to our cells ‣ more di = more likely to get response Exceptions ‣ exception = across species, theres mol that are well conserved ex, chicken dna looks like human dna, molecular strucutre is same just di pattern, phopshate backbone, NA ‣ have strucutal proteins ex. collagen, our collagen is not di than chicken, cow , goat, sheep ‣ inject cow collagen into mouse, look s like its mouse collagen, mouse wont see it as foreign ‣ hard to make IR agaisnt it then – Highly conserved molecules like collagen or cytochrome c may not be immunogenic even in distant species. – Some self molecules, normally sequestered from the immune system, will raise an immune response (E.g. sperm or lens tissue) ‣ also have immune privleged body sites, antigen or immunogen is in the animal that they came from. physically separated from immune system ‣ ex. interior comparmtnet of eye, can inject things into space immediately in front of lens and wont reject it ‣ IS cant get in there, doesnt matter how foreign it is, it will live there ‣ cna be in theory recognized but if it cant contact IS, wont matter  ANTIGENS – 4 properties of an immunogen 2. Molecular Size – There is a correlation between size and immunogenicity. – The best immunogens are in the range of 100,000 Da. – Small molecules 5-10,000 Da are generally poor immunogens. – Minimally they must be large enough to be processed. ‣ size = antigens are sometimes too small, antigens have to be big enough to have structure and shaoe but t cells have to se something on mhc, target has to be big enough for dendritic cell to eat it digest it rpocess it and put it back on MHC ‣ if its too small, cell cant phagocyorse that ‣ goldlocks zone, too big, cant eat it, too small, cant show it ‣ protwein thats big enough to chew up into big peptides and put onto mhc is good  ANTIGENS – 4 properties of an immunogen 3. Chemical Heterogeneity – Size alone will not make a good immunogen. – Synthetic homopolymers are not immunogenic regardless of size. – Large co-polymers can be immunogenic, adding aromatic amino acids increases the chance. – Proteins with more complexity in primary structure and those showing secondary, tertiary, and quaternary structure increase immunogenicity. ‣ if it has unique surfaces and di erences in chem residues on surface, IS will like it ‣ can be like foreign mol and its right size and entirely hydrophobic, wont see it, theres no contrast or di erences, no heterngoetity on surface ‣ wont nd unique shape to grab onto ‣ needs to have some structure ‣ proteins have primary aa sequence, fold into helixes and hseets, make seprate peptides, etc. ‣ lots of 3d structures, lots of di erences, lots of heterogenity = good immunogens  ANTIGENS – 4 properties of an immunogen 4. Degradability – Proteins must be degraded to be presented by MHC molecules to activate T-cells. Factors that influence this process affect immunogenicity. insoluble > soluble (more likely to be phagocytosed & processed) large > small (more processing, more epitopes) L-amino acids > D-amino acids (works with processing enzymes) ‣ target has to be able to be degraded ‣ if we make target with wrong a.a = d-a.a, enzymes cant take amide bonds apart, can onternalize it but cant shop it i up, cant put it on mhc, not on mhc, t cells wont see it ‣ need to be able to process it ‣ insoluble better thaj spoluble bc can phagocyrose it, chunks cell can eat and digest ANTIGENS if we can apply theese pricniples to antigen, can turna ntigen into immunogen do this with vaccines, picka. target, might not be one that IS wants to respond to, might be not insoluble, not right size, but vaccines still work this is bc we take target/antigen we want and make it more immunogenic How can we make an antigen more immunogenic? ANTIGENS - adjuvants From the Latin - adiuvare (to help) Substances that when injected with Ag serve to enhance the immunogenicity of the Ag. This leads to a higher antibody titer and longer lasting immune response. Are not specific to an antigen but can be used with many different antigens add something to it, in vaccines, add an adjuvant this is a chemical or compound or mix we add to antigen to make it more immunogenic and more appealing to IS doesnt change antigen, antigen is still antigen, immune system will still see antigen doesnt in uence IR, makes target more appealing to IS not speci c to target, if u get u shot or tetanus, have same antigen, IS will make antibodies to u or tetanus, still see right target but these reprogram ther way body sees antigen ANTIGENS – adjuvant mechanism A. Stimulate Immune Response Freund's complete adjuvant, containing muramyl dipeptides from the cell walls of heat killed Mycobacteria , stimulate macrophage activity. The increase in IL-1 helps activate Th (helper) cells Synthetic polyribonucleotides and bacterial LPS stimulates nonspecific lymphocyte proliferation. stimulate innate IR innate helps adaptive --> need to be able display antigen, phagocytose and put it on mHC, need to provide help to immune system, costimulatory signals exist (ex. cytokines), in ammation = enhance how we can get antigens into lymph node so IS can see it things we can do to stimualte IR do this by including things in adjuvant such as Tol like receptor ligands, not speci c, wont get IS to recognzie LPS, but activate innate cells, make macs phagoytsoe better, present antigen better, provide costimulatory help Some stimulate local chronic inflammation and granuloma formation (Freund's complete).  ANTIGENS – adjuvant mechanism B. Prolong Exposure to Ag Alum and Freund's adjuvant bind and precipitate the Ag to keep it in the system longer and allow for slow released of Ag. Can increase the time of exposure from a few days to a few weeks. Precipitation also increases the size of the Ag to facilitate ex. u shot, are made up 2 proteins HA and NA, no u ivrus or dead virus, no viral rna, just 2 proteins and adjuvant if u inject just 2 proteins, will get lost in uid between ur cells, be cleared from body phagocytosis. invented adjuvants that keep proteins avaialble for few days, do this by causing them to form aggregates, to rpecipiate, to form gelatinous mass, this ball stays under skin for a few days in doing this, create depo e ect, all cells in area can nibble awayand clear the debris, and doing so, are able to pick up antigen better if u leave it in jelly ball, macs need ot get rid of it, clear material from tissue, so whill pagocytose it preipatitin gets it to be bigge r= optimal size rpolonges exposure, keeps it for few days to couple weeks, allwos IS to get time and nd it geenrates help signal, by activating IS, gett beteter phag, better cell presentation, also get help, get signal that hey this thing i ate is dangeorus, need ot respond to it instead of ignore it adjuvant changes thhis context, if u put HA under the skin without adjuvant, body goes so what, theres no infection or virus, why do i care? now put adjuvant as well as HA, adjuvant has TLR ligands, body gets angry, gets in ammation, tells IS that this important, need to respond to it, IS is based on no go or go signals, in absence of help, even if T cell sees it, immune system wont respond. we have to get help signals  ANTIGENS – adjuvant mechanism C. Co-stimulatory Signal Th cells when stimulated by Ag require a second co-stimulatory signal. Freund's adjuvant, LPS, and other factors up regulate co-stimulatory signal systems. ANTIGENS – adjuvant mechanism havent changed antigen or target,but by putting adjuvant there, body is more prone to nd it and respond to it signalling through TLR ex. adjuvant a, activates IS, better phagocytosis and better antigen presentation depo e ect will enhance phagocytosis, will precipitate it and keep it in tissue so get better presentation bc its uptaking (adjuvant b) adjuvant c, activated cells will express mol the t cells are looking for for help, the context that says this a problem, need to respond Nature Reviews Microbiology 5, 505-517 (July 2007) ANTIGENS – epitopes dont see entire antigen, sees a piece of antigen ANTIGENS – epitopes Lymphocytes do not recognize an entire antigen Lymphocytes recognize small, discrete sites on macromolecules called antigenic determinants or epitopes. Epitopes seen by B-cells and T-cells differ in several fundamental ways. antigen = forest, oen spot on entire protein that adaptive IS is seeing is one large yellow tree dont see entire forest, see one tree = epitope these are small structural piece in larger protien or sugar that binds to t cell recpeotr or binds to immunoglob binding sites (end of arms) the epitopes on a given protein that t cell and b cell sees are pundamentally di , see them in di ways  ANTIGENS – epitopes not created equal, IS will nd one or tow favs, can make antibodies for these surfaces, but will only ahve antibodie that will see 2-3 of these epitopes, these are optimal, on surface, exibel, right size, etc., these are called immunodominant, will dominate immune response ◦ optimizing for the thing that is bound and recognized best, if ur a pathogen, will do best to make sure immunodiominant epitopes are not important to me, might even put out on bacterial surface a very good epitope, bc uf they stick to that, wont impede function, set up decoys, set up things IS wants to se but dont bllock my function ◦ pathogens have spots that are intentionally immunodominant so IS fails to recognize it ◦ one of the classic examples = HIV, has GP 120, large glycoprotein, b cells love it, its sugars, protein, 3d shape, outside of virus, ts every criteria but most of that protein doesnt do shit for HIV, doesnt stick to target, doesnt allow infection, the stem does that holds up big protein (thats how it gets into cells), but by having juicy globular protein on top, IS ignores stem, we never block it, as a result, HIV can keep evading immunity ◦ when we're making vaccines, wanna avoid what paothgen wants us to do,instead getting a better and more intelligent vaccine design, wanna ure out what we need to target Predicted HPV L1 Protein B-cell Epitopes ProImmune.com each antigen can have multiple epitopes this is HPV viral protein, predicted 3d model of the surface, protein itself could be target of vaccines, IS sees discrete bindign sites on protein each shaded area = di spot antobdy is know to bind, each is di antibody one will bind dark blue and wont stick to anything else, doenst know anything else about protein even within own immune response, can make 12 di antibodies that see 12 di parts of same protein, parts cna also be overlapping, only one antibodiy at a time will stick but these are just surfaces, if surface has unqie shape antibodiy can dock to, can bind to it multiple eptioes on given antigen ANTIGENS – epitopes B-cell epitopes see proteins, sugar, nuecleuci acid, see 3d shapes, see anything, this is truly the lego bloc, it needs to t together otherwise it doesnt stick  ANTIGENS – epitopes (B-cells) B-cells bind Ag directly via cell surface immunoglobulin (Ig) Ag can be almost anything – sugars, lipids, proteins, nucleic acids, heavy metals … For Ag in solution – Epitopes must be topographically accessible on the native molecular surface (hydrophilic) (exceptions) – Epitopes must be flexible and mobile for agglutination (often located on bends and loop structure of protein) – Epitopes can be sequential or nonsequential (conformational) rules b cell have to follow: typically epitopes have to be outside of protein, b cell cant get to middle of protein, b cell sees outside surface, needs to be accessible to outside world --> large globular protein, lots of a.a buried in middle, b cell cant get there, sees full protein, sees outside surface of that, needs to be accessible to outside world epitope has to be exible, need to stick in bidning pocket, b cells love side chaisn and loops, sugar side chains, a.a that have side chain, they like extra shapes that they can bend and t into binding pockets can be iether sequential or nonsequential, b cell can recognize 3d shape of soemthing or if u stretched protein out, can see a.a sequence, doesnt speci cally matter most b cells see conformational shape, but if u streteched protein out, could generate antibodies b cell only cares about seeing shape or 3d structure  ANTIGENS – epitopes (B-cells) Epitope size defined by the binding site of the Ab. Complementary binding between Ag-Ab limits epitope size. Typically 6-7 amino acids (aa) or sugars can fit into the deep pocket structures of linear epitope binding sites. Conformational epitopes of globular proteins cover much greater space on flatter surface binding sites of Ab. Conformational epitopes may consist of 15-22 aa. Complex proteins may contain multiple overlapping B-cell epitopes. Not all epitopes induce a response IMMUNODOMINANCE size of bindign pocket determiens how big epitope can be 6-7 a.a why can a virus excape imune response? just has to change one of those a.a, only 6 a.a and one changes, 20% of surface has changed, these are speci c binding sites, theyre not tolerable to change, very speci c binding sites true 3d shape that has contributions from 2 di loops on protein, maybe it gets bigger, 15-20 aa. proteins are in hundreds of aa, this is still tiny IS will nd one or two favorite binding sites, could make ab against all surfaces, if u watch IS, by the end of it, will have ab that only see 2-3 epitopes, theres a preference, these are optimal, exible, right size, on surface, these are IMMUNODOMINANT, these dominate IS, optimizing for the thing that is bound and recognized best ANTIGENS – epitopes (B-cells) ◦ orangw and blue make up arms of antibodiy ◦ purple petide is sititng ina ntigen bindign pocke,t this is epitope, this is piece of larger protein b cell sees ◦ this is entirely determiend by how it ts http://proteopedia.org/wiki/images/6/64/Rituxan2.jpg ANTIGENS – epitopes (B-cells) ◦ look at protein structure, can have round socket at top of protein structure, target is gonna t into pocket, entire determiend by how well side chaisn t into space ◦ cylinder, lies through groove ◦ get entirely at surfaces that bind basically at end of ab arm ◦ targets with internal surface ex. enzymes that have catalytic site inside and domains on ab arm, if they t into that space, cant allow it to stick ◦ better the physical t, the better these things hang on  ANTIGENS – epitopes (B-cells) Linear (sequential) vs. Conformational (non-sequential) ◦ can see linear or conformational, this explaiosn how it sees protien or sugar as structure rather than sequence ◦ sees shape, shape can be represented by multiple loops from dif fparts of protien that end up in same 3d shape, antibody may be binding to all of these loops, loops together maing a surface, ab might be binding to little bit of loop 1, 2 and 3 ◦ if u took protein apart, those antiboides wont stick anymore bc surface is gone ◦ at the same time, pieces of proteins can be continuous a.a sequences = linear epitope ◦ if ab sticks to linear epitope, even if u unfold sequence, can still see same ab sequence ◦ there are both the linear epitopes, sequential, all on one piece of molecule or conformational that have di pieces of mol that end up in 3d space Arch Dis Child 2004;89:238-243 doi:10.1136/adc.2002.013250  ANTIGENS – epitopes (B-cells) Linear (sequential) vs. Conformational (non-sequential) ◦ type of epitope determines how it will work in lab ◦ linear epitopes,when we unfold it or chop it up, ab still sees red domain bc in tact ◦ if ab sees red domain based on 3d folded shape, then when we unfold or cut it into small pieces, red domain is gone, piece is missing, ab is not gonna work anymore Epitome - Database of Structurally inferred Antigenic Epitopes in Proteins ANTIGENS – epitopes (B-cells) http://xray.bmc.uu.se/kurs/BSBX2/practicals/practical_2/practical_2.html ◦ can be primary a.a, a helix, peptide, or multi domain proteins ◦ if it has a shape, itll bind ANTIGENS – epitopes T-cell epitopes  ANTIGENS – epitopes (T-cell) Recognize only protein (and some glycolipid) epitopes T-cells do not recognize soluble native Ag Recognize only Ag that has been processed and whose peptide fragments are presented in association with Major Histocompatibility Complex (MHC) molecules. T-cell epitopes are generally oligomeric peptides of 7-20 amino acids in size ─ Ag binding cleft of MHC defines Ag expression ─ MHC Class I typically 9-11 aa ◦ require antigen presenting cells ◦ only see peptides, some glycoproteins, piece sof a protein ◦ have to be displayed on MHC, t cell is seeing peptide but also MHC, mHC = pedestal that has protein sititng on it ◦ will se eepdestal and prtotien ─ MHC Class II typically 11-17 aa ◦ will see both arget and sself protien holding it ◦ 2 avors of t cells, killer and helper ◦ see 2 di types of MHC ◦ have slighlty di shpapes, bind d peptides, one is clsoed pocket, 9-11 a.a, other one is a hotdog in a bun, can ahng out ends a biit and is lot more oeixlbe in length  ANTIGENS – epitopes (T-cell) Ag processing required to generate peptides Ag is seen as part of a trimolecular complex TCR-Ag-MHC Peptides may be internal and must be amphipathic ─ must have hydrophobic regions to bind MHC ─ must have hydrophilic regions to bind TCR ◦ 3 moelcule complex that comes together, TCR, MHC, and peptide ◦ all have to itneract ◦ peptides must have both hyodrtphobic and philic domains, if enitrely philic, and put that on mhc, will wash o with any uid in cell ◦ part that sitkcs ot mhc will sitkc to hydropbihc parts of mhc, wont let go, wont expose phobic parts to extracell env, stuck there ◦ rest of the peptide has to be good with aq env, has to be philic ANTIGENS – epitopes (T-cell) ◦ x has to stya stuck to mhc, t cell will see mhc and peptide ANTIGENS – epitopes (T-cell) MHC I MHC II ◦ 1 = shows stu to killer cells, cancer and viruses ◦ mhc 2 = what phags have, shows t cells outside world, eat, process,a ndput it on mhc ◦ have di shapes ◦ mhc has closed nest and ends ◦ mhc 2 = peptides can hang out ◦ if peptide is doing 2 jobs, sticking to mhc and presenting to TCR, there must be 2 surfaces to that epitope ◦ t cell epitopes have functional domains  ANTIGENS – epitopes (T-cell) MHC binding site of the Ag is called the AGRETOPE, binds via hydrophobic amino acids TCR binding site called the EPITOPE, binds via hydrophilic amino acids Immunodominant T cell epitopes are determined in part by what set of MHC molecules are expressed and what TCR are expressed by an individual. ◦ agretope = sitcks to mhc, hydrophibc, will t into the next/hot dog pocket of mhc, shape selection that binds to mhc ◦ other is the epitope, piece t cells see ◦ t cell has agretope and eptipoe ◦ b has only eptiope ANTIGENS – epitopes (B & T-cell) B-cells T-cells Membrane Ig and Membrane TCR, Antigen interaction antigen antigen, MHC Soluble Antigen? Yes No Additional Molecules No MHC, CD4/CD8 Required Chemical nature of Protein, lipid, Protein antigen polysaccharide, Accessible, Accessible or hydrophilic, mobile, Epitopes internal, linear, sequential or amphipathic conformational ◦ b cells = membrane immunoglob and antigen come together for b cell to reocgnize, for t, its peptide, mhc, and tcr, and target ◦ b cell cant ssee thinhfs insie of protien but t cell does bc mac eats it and processes it  ANTIGENS – epitopes (B & T-cell) B-cells T-cells Membrane Ig and Membrane TCR, Antigen interaction antigen antigen, MHC Soluble Antigen? Yes No Additional Molecules No MHC, CD4/CD8 Required Chemical nature of Protein, lipid, Protein antigen polysaccharide, Accessible, Accessible or hydrophilic, mobile, Epitopes internal, linear, sequential or amphipathic need chemicals to be compatible with each other to form a tight association, not creating covalent linkages, bc of this, if something can bind tighter, can displace rst thing, these are dynamic interactions, when antibody binding conformational bacteria or virus to neutralize it, it can fall o if speci city not good enough ANTIGENS Antigen (Ag) – Antibody (Ab) Interactions the association is maintained by number of noncovalent forces, acting at same time in theory, di antigens will rely on di forces to greater exent, some will be more polar, non-polar, etc., this determines what antibody will stick to it need perfect match (nearly) betweens urface of antibody and surface of epitope how do we measure strength/how tightly things stick together? do this empirically, measure how quickkly something falls o antiboidy tough to measure this, set up chemical reactions where we determine how many mol within mix are associated with each other, how many are free and separate, can calculate how likely something is to stay stuck to antibody set up simple reactions that allow us to measure bidning of one binding site (one arm of antibody) with single epitope (single binding target)  ANTIGENS – Ag-Ab interactions Ag-Ab interactions, like enzyme-substrate interactions, involve highly specific, reversible binding between molecules Specificity is determined by multiple low affinity non- covalent bonds requiring specific fit between Ag & Ab. These bonds include; – ionic bonds (electrostatic) – hydrogen bonds – van der Waals interactions – hydrophobic bonds ANTIGENS – Ag-Ab interactions  ANTIGENS – affinity Affinity is the strength of the sum total of non covalent interactions between a single Ag binding site on an Ab and a single epitope. Dictated by the Ag/Ab fit create chemical rxn/set up where we measure amount of antibody-antigen Association Constant Ka= [Ab-Ag] aggregates, can measure this by size know how big target is, know how big antibody is, if theyre stuck together, its the sum of those sizes divide that by concentration of free antibodiy and free antigen if things stick together well and come apart infrequently, numerator will be big, amount of free mol will be small [Ab][Ag] if on the pther hand, things dont sitkc together well, fall apart frequently, numerator will be small, product on bottom will be large get a number, this is arbitrary units, gives us a measure of how well this sticks typically, the bigger the number, the tigheter things are sticking quite a range, several logs di erence between good and high a nity/tight Where sticking target than one that is more weakly associated massive range in how well antibodies will stick to target how do we measure this? ◦ did this through di usion assays – [Ab-Ag] concentration of Ag-Ab complexes – [Ab] concentration of free Ab – [Ag] concentration of free Ag low affinity Ab-Ag binding → Ka = 104-105 M-1 high affinity Ab-Ag binding → Ka = 1010-1011 M-1  ANTIGENS – affinity Affinity is the strength of the sum total of non covalent interactions between a single Ag binding site on an Ab and a single epitope. Dictated by the Ag/Ab fit Association Constant Ka= [Ab-Ag] [Ab][Ag] Where – [Ab-Ag] concentration of Ag-Ab complexes – [Ab] concentration of free Ab – [Ag] concentration of free Ag low affinity Ab-Ag binding → Ka = 104-105 M-1 high affinity Ab-Ag binding → Ka = 1010-1011 M-1  ANTIGENS – affinity Affinity is the strength of the sum total of non covalent interactions between a single Ag binding site on an Ab and a single epitope. Dictated by the Ag/Ab fit Association Constant Ka= [Ab-Ag] [Ab][Ag] Where – [Ab-Ag] concentration of Ag-Ab complexes – [Ab] concentration of free Ab – [Ag] concentration of free Ag low affinity Ab-Ag binding → Ka = 104-105 M-1 high affinity Ab-Ag binding → Ka = 1010-1011 M-1  this is a nity, how well one epitope sticks to one bidning site, how strong that single interaction is, antibodies arey shaped tho, have 2 arms or even ANTIGENS – affinity more (ex. IgM with 10), can bind lots of epitopes biologically thats more important, doesnt matter how good 1 hand is at binding, more about how well ab sticks to target wanna know how well u hang onto something, how well does 1 arm stick onto something, but in real world, use both hands, this is biological strength, not about how well 1 hand hangs on, biologically interested in how well both arms bind, chemistry we're interested in one theres a di erent term then, biologically more interested in avidity, how well A B A B does ab stick to its target B this is both how well does each arm hang on as well as how many arms Ag Concentration are engaged (holding onto antigen) --> this from a bio point of view is more important, if each arm is weak and doesnt hang on, but have 2 arms grabbing ag, when one lets go other is still hanging on, gives u chance to grab back on before other hand lets go can have low a nity (low strength in each hand) being compensanted by more than 1 arm hanging on A Time di usion membranes, take beaker, put semi permeable membrane in beaker, put compounds in one side vs other, based on size of holes in membrane, one can move, other mol cant, antigen small, antibody big, antibody cant move, antigen can to give us something that allows us to measure how much antigen there is, put radioactive tag on it, if u let it sit on bench, both sides of membrane will have same radioacgivity, law of brownian motion, things will reach eq, for a small anitgen, that barrier isnt there, will move through it, both sides will have same radiation if u measure amount of radiation in side a vs b, get a pattern where a has no radiation and overtime will reach sustained point, b will start higher and go down until it reaches level of a Radiolabeled Ag if we now introduce antibody to mix, and put that on one side, it cant move across, but if a molecule from b moves over and binds to antibody, wont be counted in equation, not free mol of antigen, set up will try to reach eq with free antigen, want same number on both sides, antibody will absorb some antigen out of equation, over time, mol will bind to antibody, these come out of math, remaining dots will reach eq Semi-permeable Membrane in doing this, one side has more radiation than other, if u were to measure this, a has more radiation bc has free moving antigen AND bound antigen di between b and a is amount of antigen thats bound can directly measure the amount of antigen trapped by antibodies, if these antibodies bind tightly, will bind even more b, if its weak interaction and b keeps falling o , theres too much free on one side, will move back across, less bound antibody, can measure dynasmically how much antigen sticks to ab and how tightly it sticks there bigger di = more being bound by antibody A B A B B Ag Concentration Ag bound by the Ab A Time  ANTIGENS – avidity The strength of multiple interactions between multivalent antibody and antigen is called Avidity. High avidity can compensate for low affinity. – E.g. IgM usually has a lower affinity than IgG, but the higher avidity of IgM, resulting from its multivalency, enables it to bind Ag effectively.  ANTIGENS – affinity vs. avidity Affinity → Strength of binding between a single Ag epitope and an Ab. Dictated by the Ag/Ab fit Avidity → The function of the combined strength of binding affinity and the Ab/Ag valency. Dictated by affinity and the number of Ab/Ag bindings a nity = strength of SINGLE BINDING SITE NOT ANTIBODY determined by how well does ag and ab t together avidity = COMBINED STRENGTH of ALL BIDNING SITES on SINGLE ab molecules determined by how well each site hang on as well as number of times ab is sticking to its target ANTIGENS – affinity vs. avidity Low High Affinity Affinity when we're measuring somthing with free epitopes, seeing how well does one epitope bind within the binding pocket of ab, how well does shape t binding pocket, this is a nity, strength of interaction within biology, within ghting infection, below is happening, antibody with 2 arms can hang onto 2 sites at same time, IgM, 10 arms, can hang onto more targets, each one of those binding sites is a little bit weaker than schematic on left, its ok bc has lots of bidning sites, will overcome this, biologically this is what our IS designs Low High if it cant have high a nity, makes high a nity, if it can make high a nity, it makes these little Y shaped antibodies with fewer binding sites ◦ as an IS, willing to trade o a nity for avidity Avidity Avidity ANTIGENS – affinity vs. avidity ◦ whats advantage? ◦ if we're talking about mol with only 1 binding site, better bind tight, if it falls o will lose it,odds of randomly bumping into again are low ◦ if both arms are binding and 1 arm lets go, its ok bc still hanging on with other arm, chance that arm can grab back on before losing molecules ◦ a nity = one single binding pocket, one epitope ◦ avidity = how well do mol stick together with as many bindign sites they have ◦ as we add more binding sites, avidity goes up ◦ arms are identical, antibody has 1 binding site and it puts it on all arms  ANTIGENS – cross-reactivity Different Ags may share common epitopes – Ab specific for the common epitope will bind both Ag concept of cross-reactivity, can have ab that are designed to recognize something speci c ex. this years u strain, the Na on the outside is a typical target, its in this years u shot, will make ab that will recognixze this years Na, when we have immune response to vaccine and take ab and check them, will stick well to this years u strain as well as last years (but not as good) adaptive is very speci c, can tell di between 2 us yet ab will see both, this is bc old u looks similar to this years, ab raised against an epitope could still recognize something else that looks similar, doesnt know what its actually seeing, its based on shape, as long as shape is similar, will stick ex. u, 2 Na are di molecules, di sequences but do same job, cut sialic acid residues for virus to escape, in general will look similar, can understand this there are other scenarios where mol wont look alike but by chance have same chemical properties Epitope may be different but share common chemical properties. – Ab will bind 2 different epitopes. ANTIGENS – cross-reactivity side chain can carry charge, in one ag its represented in an a.a and in another its represented by sugar, if ab does recognition by how well it ts http://sarsv.co.cc/cct-assignments/micro/cross-reactivity.html in biding pocket based on chemical itneraction, similar charges will look same to ab can have di scenarios where ab on left is recognizing its proper epitope, perfect t in bidning pocket if same ab ran into ag-b, this is di molecule, but has side chain thats the same or similar, ab will still recognize in ag-c, di molecule, not perfect t but gets into binding pocket, ab will stick, stick more weakly but will work if ab doesnt know what its bidning, its just looking at chemical signature and its t in binding pocket this is useful bc IS is evolved that we can ght o last years u and this years u also exist, have cross protection, body can respond to similar pathogens creates problems tho, things that we design IS against (pathogens) sometimes look like us, can lead to autoimmunity, can make ab thats good at ghting pathogen and if we can clear it, if theres something in us that looks similar, it will stick,doesnt have to be pathogen, just wants something to t in its binding pocket ANTIGENS – cross-reactivity ABO Blood Groups Molecular Cell Biology. 4th edition abo blood groups in humans, these are sugar groups on rbc, all of us start o with core sugar molecule = o antigen, all start with this based on genetics, can modify sugar, some ppl will have galINAc transferase, put extra group (acetylgalactosamine) on end, not all of us do it, get A antigen, u are blood group A if u express gal transferase, put galactose on end, now ur B some of us have neither, and stay as O, some have both and now ur AB all of rbc have sugars on surface, some will have A, B, O, or both this creates problem for blood transfusions if u are blood group O, already have ab that recognize A and B, if u give blood from someone with blood type A or B, ab will attack it, have autoimmune response ANTIGENS – cross-reactivity ABO Blood Groups if ur blood type A and give u B, u have antibodies against b, will attack it how do u get ab against things u never see? dont need to see blood type b, saw something that looke dlike blood type b, if u have blood type a, have ab against blood type b, if ur blood type b, have ab against a, if ur ab, have antibodies against ntohing bc express both, if ur O, have ab against everythin if ur A, express A antigen, b express B antigen, AB express both, O express none dont reject blood type O bc dont have targets on surface, this is why theyre universal donors if ur O, will attack everybody's blood unless u get O where do these ab come from? ANTIGENS – cross-reactivity ABO Blood Groups Anti-A antibodies Originate from influenza virus, whose epitopes are similar to α-D-N-galactosamine on the A glycoprotein Anti-B antibodies Glycoproteins on Gram-negative bacteria, such as E. coli, that resemble the α-D-galactose on the B glycoprotein. as soon as we're born, will make ab that reject other blood group even if u have never seen them bc they look like pathogen and ab are cross reactive sugars on O antigen resemble pathogens, a AB (EXPRESSED BY B), a sometimes sugar group on rbc looks similar to protein in in uenza, if u have had u, u if ur blood type a and cant make ab against that in uenza sugar, thats 1 fought o u, have ab that recognize residues on u virus, those look like sugar on whole complex of in uenza so will make ab against all other parts blood gorup antigens of virus, any one antigen can have 20 di epitopes, 20 di docking sites, if ur blood type a and got u, dont make that antibody, ignore that and dont just make the one ab that sees that sugar, attack everythhing else, attack everything else, dont make ab that can reject ur blood group miss one ab speci city but make all others if ur a b and dont know what a looks like, this is foreign, will attack this part newborns, can u give newborns with blood type a a mismatched blood of u, now have antibodies against it type, if they never get u at this stage, in theory can receive other blood groups bc havent made ab against u  ANTIGENS – Ag precipitation Precipitation occurs when Ag-Ab interactions result in the formation of a lattice structure. Lattice formation requires at least a bivalent Ab and at least bivalent or polyvalent Ag. Precipitin reactions can be measured by adding increasing amounts of Ag to a constant Ab biologically, want ab to bind at multiple sites, this comes with few advantages, increases the di culty of ripping ab o of target but can do concentration. cool things immunoprecipitation/ ag precipitation, if u have ab that can bind with both arms and have target that can have di ab to stick to it, can cross link these, can make crystal like structure, as long as target has multiple binding sites (2 di binding sites) and ab can each bind 2 targets, can make these complexes good for lab jobs, if u have protein and wanna pull it out of solution, can do this if ur a virus, if we do this to u, ur trapped in crystal allows us to understand how well ab and antigens interact, for precipitation need minimum of bivalent ab (2 arms), cant do this with 1 arm ab, if u have 10 aka igM even better, need a POLYVALENT antigen, antigen with at bottom, have 1 type of ab, not precipitating very well multiple bidning sites on it  ANTIGENS – Ag precipitation Excess in either Ab or Ag inhibits precipitation. Maximal precipitation is achieved in a zone of optimal Ag to Ab concentration called the Zone Of Equivalence. This is not to be confused with the concept of equivalent amounts of Ag and Ab. can determine concentrations as well, this is bc we add ab in solution, as we increase amount of ab, will hit sweet spot where precipitation happens zone of equivalence = very rarely occurs when concentration of ag and ab are the same, this is the point at which we get max precipitation ANTIGENS – Ag precipitation concentration curve, amount of ag added across horizontal, amount of ab on vertical as we add ag, we get to sweet spot where we get max precipitation, if theres too much ag, precipitants fall apart, start ripping things apart, breaks it up too much ab ex. too little ag, precipitants dont form, need just the right mix too little ag = ab have to ght overr it too much ag = ab dont have to share, dont form large complexes, each ab has own ag in middle, each ab is fully occupied with ag and its sharing with other ab, get large lattice crystal structures can do assay in eld, can know who has ab and how many u have against target, we can take ur blood and add increasing concentration of target until precipitant forms, can calculate how many ab u have ANTIGENS – Ag precipitation take serum (liquid part of blood), put in tube, have set concentration that we know precipitate u ag, see tubes 5 and 6 have white crystal at bottom,t his is right amount of ab still use this bc it measures true biological impact, dont care speci cally about what ab sees, does it see overall target, each of us bind slightly di ab but still see target this is also cheap vs. 1.2 million dollar machine that tells u how well 1 ab hangs onto virus, no electricity, no re dgeration, simple, can get lots of info quick http://www.microbeworld.org/component/jlibrary/?view=article&id=1284 IMMUNOGLOBULIN (antibodies) concept of innate immunity has 1 gene for each receptor, have 30 receptors that see PAMPs, whereas adaptive cant have genes for each receptor, needs rearrangement of gene pieces when we look at ab, this is accurate rep of it, the binding sites are all that stick to target, the rest does immune jobs, rest activates complement, binds to macs, this doesnt have to be diverse, if this ab sees u and want it to stick to mac, bottom green bit is same as ab that sees e coli that can also stick to mac business part that does immunology doenst have to change, only has couple jobs to do, diversity has to be at bidning sites from evolutionary point, dont need to change whole mol, just need to make bidning pockets unique this is premise of gene rearrangement IMMUNOGLOBULIN - diversity Antibodies can recognize and bind >108 different Ag in a specific fashion. How is this much diversity and specificity possible? high degree of speci city that this ab wont necessarily recognize next years u, this is lots of diversity, from a biology pov, how is this possible? how can u explain that body can see anything, ehrlich had side chain theory, arguing that theres proteins on side of cell that can bind target, form around shape, remember shape and make more arguing while theres cell that expresses bunch of things, depedning on which one sticks to target, will make more of that IMMUNOGLOBULIN - diversity Theories – Selective (Side Chain) Theory Put forth in 1900 by Paul Ehrlich to explain the specificity of an Ab response. Ab producing cells express multiple “side chains” of various Ag specificities → side chain theory. Engagement of one of these “side chains” with Ag results in the production and secretion of many more side chains with identical Ag specificity. this was revolutionary, this is exact what b cells do, have antibodies, each cell has one receptor, that receptor is speci c, although concept was somewhat right, actual scheme was entirely wrong, reason why it fell apart was bc how do u get billion receptors on one cell? math doesnt add up in his defense, he didnt know about dna typical ab is 660 a.a IMMUNOGLOBULIN - diversity Theories – Selective (Side Chain) Theory Ag A Side Chain Ag B Ab Producing Cell Side Chains Side Chains Against Against Ag A Ag B IMMUNOGLOBULIN - diversity Theories – Selective (Side Chain) Theory One problem... How do you generate enough diversity in the Ig repertoire ? IMMUNOGLOBULIN - diversity Theories – Selective (Side Chain) Theory There are >108 Ig specificities Each Ig is ~ 660 aa → 1980 bp of DNA 1980 bp * 108 = 1.98 x 1011 ~ 2 X 1011 bp to encode the Ig repertoire The human diploid genome = 5.6 x 109 bp The Ig gene locus would be 35 X bigger than the entire human genome. math doesnt work out  IMMUNOGLOBULIN - diversity Theories – Instructional Theory Developed in the 1930’s and 40’s to account for the enormous diversity within the Ig repertoire. Ag itself had a role in generating Ig specificity. The Ag serves as a template around which the Ig molecule was folded and mutated → The Ig took a shape complementary to that of the Ag. Few genes would be needed to account for diversity as a single molecule could assume many specificities. this was meant to get over this problem, as we started to learn about geneitc inheritance, something that codes for things, put together that its impossible to have that much diversity, need other mechanism proposed we have plastic type receptors, they fold around target, have receptor that can see not everything but can see wide variety of things, when they engage, they reprogram and take on new shape, this was advantageous bc could get away with few number of genes to make this starting pool of ameboid like receptors make generic ab type molecule, depending on what it binds, it will fold itself around it to make bidning pocket, can stick to binding site with high a nity if generic ig mol sees pointy binding site, will fold around it, have point binding pocket that has high a nity to it if now it sees rectangular binding site, will fold around that and change to have rectangular binding pocket IMMUNOGLOBULIN - diversity Theories – Instructional Theory Ag A Generic Ig Molecule Ag B Ag A Specific Ig could adapt to what we could see, this is unlimited diversity, if we put it on Ag B Specific Ig cell, if we put it on a cell, it doesnt matter what antigen it sees, cell will do job and reprogram itself and make appropriate ab based on what it engaged explained problem but then ppl found other probnlem, how do we account for speci city that exists BEFORE exposure? take cells out of ur blood and put antigen on them, 1 in 40 000 cells willa ctually respond to antigen, not reforming receptor around target, each already have something they see when theyre born, inherent speci cty already exists need system that explains unlimited diversaity but pre inherited speci city IMMUNOGLOBULIN - diversity Theories – Instructional Theory Generic Ag Receptor Ag A Ag B Ab Producing Cell Ag binding determines specificity acquired by generic Ag receptor Ig Specific For Ag A Ig Specific For Ag B IMMUNOGLOBULIN - diversity Theories – Instructional Theory Another problem... How do you account for specificity prior to Ag exposure ?  IMMUNOGLOBULIN - diversity Theories – Two Gene Theory Proposed by Dryer and Bennett (1965) as a solution to the problems of the selective and instructional models. Two genes encode the Ig molecule → one for the Variable Region one for the Constant Region. Many different copies of the variable gene (accounts for diversity) but only a few constant genes (one for each isotype). The few constant genes could be combined with any of the numerous variable genes to generate an Ig repertoire with huge diversity. suggested that part of ab doesnt see target so why have diversity there, if stem of ab activates complement, should be same for all ab otherwise wont all activate complement one gene codes for structural pieces of ab, di smaller gene codes for bidning pocket have billions of copies of tiny gene and only handful copies of structural gene, body will take one of these random tiny genes and stick on the structual gene of ab, if u take di structural gene and di binding gene, get di ab have one gene that codes for variable gene and one for constant, have lots of di copies for variable, each one di , only couple for constant, one for IgM, 1 for IgA, can take any of these constants, add new variable gene to it, get new ab one gene codes for tip of the arms (variable gene), one gene that codes for rest of mol (constant gene), lots of variable, handful of constant, randomly put together, make ab this concept although couldnt prove it at time was exactly what happens IMMUNOGLOBULIN - diversity Theories – Two Gene Theory Variable Gene Constant Gene 1000’s of copies 1 copy / isotype Rearrangement of genes IMMUNOGLOBULIN - diversity Theories – Two Gene Theory Confirmed by Tonegawa and Hozumi (1976). Analysis of Ig genes from myeloma and embryonic cells demonstrated DNA differences. The Ig DNA of myeloma cells was “rearranged” as compared to the “germline” DNA of the embryonic cells. IMMUNOGLOBULIN - diversity Theories – Two Gene Theory Myeloma Cell Embryonic Cell  Chain Probe Variable Constant Region Region Isolate Isolate DNA Myeloma Embryonic RNA Cell Cell Restriction 32P Digest label  chain mRNA Electrophorese IMMUNOGLOBULIN - diversity Theories – Two Gene Theory Embryonic V1 V2 Vn C DNA Restriction Restriction Restriction Enzyme Enzyme Enzyme Myeloma Embryonic Myeloma V1 V2 C DNA Restriction Restriction Enzyme Enzyme  IMMUNOGLOBULIN - diversity Theories – Two Gene Theory Additional sequencing identified a separate constant gene for each Ig isotype and subclass. The gene coding the variable region was found to be made up of multiple gene segments. Light Chain Variable Region ( or ) » Comprised of a variable (V) and a joining (J) gene segment. Heavy Chain Variable Region » Comprised of variable (V), joining (J), and diversity (D) gene segments.  IMMUNOGLOBULIN - diversity Theories – Two Gene Theory Proposed by Dryer and Bennett (1965) as a solution to the problems of the selective and instructional models. Two genes encode the Ig molecule → one for the Variable Region one for the Constant Region. Many different copies of the variable gene (accounts for diversity) but only a few constant genes (one for each isotype). The few constant genes could be combined with any of the numerous variable genes to generate an Ig repertoire with huge diversity. IMMUNOGLOBULIN - diversity Theories – Two Gene Theory Variable Gene Constant Gene 1000’s of copies 1 copy / isotype Rearrangement of genes IMMUNOGLOBULIN - diversity Theories – Two Gene Theory Confirmed by Tonegawa and Hozumi (1976). Analysis of Ig genes from myeloma and embryonic cells demonstrated DNA differences. The Ig DNA of myeloma cells was “rearranged” as compared to the “germline” DNA of the embryonic cells. IMMUNOGLOBULIN - diversity Theories – Two Gene Theory Myeloma Cell Embryonic Cell  Chain Probe Variable in the 70s, no ability to PCR dna, cant amplify it or sequence it, how cna u understand genetics? gured out that they needed to nd cell that makes lots of antibodies bc if it makes alot of ab must have alot of rna for ab Constant Region could use b cell cancer, myeloma, cancer where patients have vast majority of wbc are clones of each other, all one b cell, have same ab, as a result, all express huge amounts of rna coding for single ab speci city Region removes diversity from equation proposed that this rna must represent the genes that are glued together to make protein rna is complementary to dna, so can use rna to look in the genome for any genes that look like ab gene, if u can nd them, can tell where they are, if theyre close or far set up experiment, have b cell cancer and embryonic cell (not making ab, cant possibly rearrange dna, would give rise to another indiv and that person needs Isolate harvested rna and dna from these cells Isolate DNA to have all di ab choices), whatever chromosome dna should look like would be preserved in this cell get dna from b cell cancer, if this is b cell thats making ab, if theres separate variable and constnat genes, must be fused together in dna Myeloma Embryonic RNA stem cell = embryonic cell should not have them rearranged, would be on separate pieces of dna Cell Cell chopped these up into short pieces of dna, also harvested rna that codes for cell surface receptors, enzymes for cytoplasm, but bc its cancer and producing lots of ab, bulk of rna is for ab, enriched for it without amplifying aything take rna, put radioactive label on it, this will be their probe, this will stick to dna that would code it, take these pieces, run them out in gel in myeloma cell, when we put probe, will nd one piece of dna, everything in that ab is found in one short piece of dna, the ab binding pocket, stem, all of it in one piece in stem cell, this proble labelled 2 pieces of dna, 2 fragments contain parts of antibody, proof that variable gene existed on one dna fragment and this constant on another when u become a b cell, u physically move ur dna and put them on same piece Restriction 32P Digest label  chain mRNA Electrophorese IMMUNOGLOBULIN - diversity Theories – Two Gene Theory Embryonic V1 V2 Vn C DNA Restriction Restriction Restriction Enzyme Enzyme Enzyme enzyme used to cut in a few spots there was a cut site between variable and the constant, enzyme will cut at 3 spots, will end up with 2 pieces of dna in b cell cancer, dna had been rearranged, genes had been attached to constant, everything else had been lost including the cut site, when they put cell on gel, all of this was on one piece of dna, proof that genomic dna had changed its organization and structure to become a b cell entirely di than innate iS, tlr-4 is just its own gene, doesnt need dna to do anything to express gene, for b cells and t cells do same thing, take small gene pieces, shu e aorund, reattach together to make working antibody or t cell receptor fundamental concept of gene rearrangemnet in lymphocyte Myeloma Embryonic Myeloma V1 V2 C DNA Restriction Restriction Enzyme Enzyme  IMMUNOGLOBULIN - diversity Theories – Two Gene Theory Additional sequencing identified a separate constant gene for each Ig isotype and subclass. The gene coding the variable region was found to be made up of multiple gene segments. Light Chain Variable Region ( or ) » Comprised of a variable (V) and a joining (J) gene segment. multiple pieces of ab genes, light chain = shorter piece of arm, have a variable and joining gene and constant gene and we have to shu e all 3 pieces heavy chain = full stem, have variable, joining, and diversity gene along with constant thought that it was variable and constant but variable itself was made from smaller gene Heavy Chain Variable Region peices that had to be rearranged » Comprised of variable (V), joining (J), and diversity (D) gene segments. IMMUNOGLOBULIN (antibodies) Either λ lambda) or κ (kappa) green = heavy yellow = light variable bits = binding pockets, in light chain, this is variable and joining gene that have to make binding pocket of yellow bit followed by constant for heavy = variable, joining, diversity, that make up binding pocket and constant gene have 100s and 1000s have variable, joining, and diversity gene Always starts as for constant, have a handful, have 1 type of ab, IgM, IgE, etc., each one has 1 gene, this changes green bit of ab light chains have 2 avors (kappa or lambda depending on constant) for heavy, depending on species, 7-9, each one codes for di avor of ab IgM → later G,E,A IMMUNOGLOBULIN - rearrangement need to rearrange multiple gene pieces, with all vertebrate, always start with heavy chain, goes rst, when heavy chain works, then we cans tart with light chain need to bring 3 separate gene pieces together to make them work in heacy: variable, diversity, joining rst join diversity and joining together, if thats successful, bring in variable and then transcribe constant light chain: pick one variable and joining, bring them together, can make light chain for ab to work, both of these events have to be successful, need to make 2 rearranged peptides that work together to make y shaped structure IMMUNOGLOBULIN - rearrangement Ig locus organization – κ or λ light chain kappa and lambda are interchangeable for light chain, dont do separate jobs, use both for humans, for sheep its di , for mouse its di dont worry aobut kappa or lambda, b. cell can use either and gives it more diversity this is one of the light chains, have variable genes, each gene has leader sequence, allows u to start transcription, multiple copies of these in genome depending on species, about 40 variable domains then have joining, have 1-5 copies of those, and then single constant gene to make working kappa light chain, pick one variable and one joining and bring them together IMMUNOGLOBULIN - rearrangement Ig locus organization – heavy chain have variable genes, each with leader sequence to start transcription, have 40 then have diversity genes and then joining genes sngle constant gene for each type of ab in this case, mew is for IgM IMMUNOGLOBULIN - rearrangement How does “rearrangement” lead to increased diversity? there has to be a strategy, not just that we have a billio little genes that we put on big constant gene, theres a reason why we rearrange IMMUNOGLOBULIN - rearrangement Multiple V(D)J gene segments depending on species, are in 304-0 range for variable, no diversity on light chain, 5-6 on joining, typically 1 avor of constant for each ab strong indication that something else happens bc this isnt enough IgM IgD IgG1 IgG2 IgG3 IgG4 IgE IgA1 IgA2  IMMUNOGLOBULIN - rearrangement Diversity generation Heavy Chain 46 V Genes X 23 D Genes X 6 J Genes = 6348 Different H Chains Light Chain  Chain 38 V Genes X 5 J Genes = 190 Combinations  Chain 33 V Genes X 5 J Genes = 165 Combinations 355 Different L Chains Total Heavy and Light Chain Combinations 6348 X 355 = 2.25 x 106 Different Immunoglobulin Molecules 100 fold less than what we need, doesnt provide exibility, this is saying that ab can only recognize what dna tells it to recognize, everything here is a gene piece thats already in genome, over evolution, pathogens wouldve gured out how to avoid the variable genes we have in dna, more evidence that there are other processes here  IMMUNOGLOBULIN - rearrangement Junctional Diversity Although combinatorial mechanisms generate the bulk of the Ig repertoire in mouse and man, additional Ig variation is created through junctional diversity. Variation in the V(D)J joint contributes greatly to Ig diversity as this sequence corresponds to the third hypervariable loop of the V domain. Junctional diversity is generated at the time of rearrangement by three principle mechanisms; Junctional Flexibility P-nucleotide Addition real reason we have gene rearrangement is that process of joining gene pieces up gives us chance to put in more diversity, these are not clean, we get edges fuzzy and do it on purpose edges, junctions between genes correspond directly to binding pocket, if we can mess up N-nucleotide Addition joining sequneces a bit, can get more diversity in pocket third hypervariable loop aka CDR3, most important spot in binding pocket, this is where those junctions lie putting diversity here makes hypervariable loop hypervariable, this creates diversity, its so important that we have 3 mechanisms we use to put diversity in and all 3 can act at same time at least in heavy chain dont rely on any one process, this is central to entire structure of our entire adaptive IS collectively this process = junctional diversity, made up of 3 sub-processes, one os junctional exibility, other is p-nucleotide addition, other is n-nucleotide, caveat is that n- nucleotide only works on heavy chains, not light chains, other work on both chains  IMMUNOGLOBULIN - rearrangement Junctional Diversity – junctional flexibility Junctional flexibility refers to the imprecise joining of the V(D)J gene segments. Through endonuclease activity several nucleotides may be deleted from the cut ends of the gene segments being joined. This process may result in shortened V, D or J and often produces “nonproductive” or out of frame coding joints. this is simplest, simply dont cut or dont join genes at the edge of gene segment, have variable gene segment in dna, has an end, dont use proper end of gene when we attach it back together use endonucleases, when we cut dna, they nibble back a bit, lose a bit of our variable gene and joining gene, when we stick two together, have new a.a sequence bc lost some nucleotides, this creates diversity tradeo of this tho, if we remove nucleotides in anything other than multiple of 3, risk changing the reading frame of nucleotide sequence codons work in threes, remove one nucleotide, frame shifted everything, odds are we get stop codon and that ab's not gonna work 1/3 of gene rearrangements actually work, 2/3s are non-functional, cell cant use them, so it is proof that this is truly random IMMUNOGLOBULIN - rearrangement Junctional Diversity – junctional flexibility if we look at gene structure in dna, have joining in blue, variable in red, yellow = rest of dna, this is signal that says genes start and stop here, this is useless dna, wont be part of ab, this is rest of chromosome RSS CACTGTG G TGG ACT AGG J always gonna make cut at right spot, but once we make cut, we can randomly remove a couple nucleotides from the V and J, using the same exact gene segments, can have perfectly symmetrical rearrangement = cut right at end of gene at both, remove nothing, stick them back together to get 1 a.a sequence but also can remove some nucleotides using endonuclease, this creates other scenarios, can remove 3 for example and everythings ok but a.a sequence is di bc missing nucleotide, now this is di than og loop, binds di target and di antigen can remove 6 nucleotides, brand new sequence V GAG GAT GCT CC CACAGTG RSS alot of the time, dont remove multiple of 3, remove something non-divisible by 3, this creates frame shift, put stop codon in, ab wont work in grand scheme of thing, this strat allows us unlimited diversity, risking few not working to ensure we get unlimited diversity for ab GAG GAT GCT CC G TGG ACT AGG GAG GAT GCT CC G TGG ACT AGG Glu Asp Ala Thr Arg Glu Asp Trp Thr Arg GAG GAT GCT CC G TGG ACT AGG GAG GAT GCT CC TGG ACT AGG GAG GAT GCT CC G TGG ACT AGG GAG GAT GCT CC G TGG ACT AGG Glu Asp Gly Thr Arg Glu Val Asp STOP GAG GAT G GG ACT AGG GAG GAT TGGCT GACCC TAG G  IMMUNOGLOBULIN - rearrangement Junctional Diversity – P-nucleotide addition Cutting of the hairpin loop at the end of a gene segment is imprecise. Resolution of the hairpin often results relocation of nucleotides from one strand to the other in a reverse sequence. The nucleotides deficient from the donor strand are filled-in complementary to the extended strand. The resulting palindromic sequence is referred to as P-nucleotides. when making cuts, never leave sticky ends of dna open in cell, dont want them adhering to things, when we cut chromosome, create hairpin, loop sense strand back onto anti- sense strand and join them o dna goes and makes 180 turn and starts back on itself when we open that hairpin, need to break hairpin to stick 2 pieces of dna together and sometimes dont do it in right spot, when we miss doing it symmetrically, unfold dna and create small repeating sequence, this is palindromic, these additional nucleotides that get added are palindromic nucleotides aka p-nucleotides this works on heavy and light chain  IMMUNOGLOBULIN - rearrangement Junctional Diversity – P-nucleotide addition 5’ T-G-C-T-C-C cut from chromosome, close the hairpin,dont want it sticking to other things, untilwe're ready to join it back up, we close the dna strand 3’ A-C-G-A-G-G to join it back up, need to re-open hairpin, sometimes we open in right spot but most of the time we dont, in this case we nice before the g's, the strand then unfolds, these 2 g's Cleavage of are now new in sequnce, not part of og sequence, sequence didnt read ccgg, these are new nucleotides, when we build complementary strand (standard dna repair), we create palindromic sequence ccgg, no matter how long it is, will always be palindrome, read same forward as it does on other strand doing this, introduced 2 new nucleotides that change a.a sequence hairpin by endonuclease once we add them, might nibble some back again with endonucleases, these processes dont happen in isolation, have mech that cause diversity everytime we join genes up 5’ T-G-C-T-C-C G-G 3’ A-C-G-A Complementary nucleotides added by DNA repair enzymes 5’ T-G-C-T-C-C G-G 3’ A-C-G-A G-G C-C P-nucleotides  IMMUNOGLOBULIN - rearrangement Junctional Diversity – N-nucleotide addition only works on heavy chain, nontemplated nucleotide addition express an enzyme, TdT, this enzyme comes in, nds open ends while we're rearranging heavy chain, adds up to 15 random nucletodies, completely new suquence, these are brand new nucleotides, not palindrome, not shortened versions, bc theyre non-templated Up to 15 nucleotides may be added to the end of we call them N cant work with light chain bc heavy chain rearranges rst, when that is successful, turn o TdT, downregulate it, gone yb the time we rearrange light chain this is simplest of the bunch the gene segments through the action of terminal from there we use dna repair, ll in other strand, use it as template and ll missing bits, gives brand new sequences this doesnt happen in. isolation deoxynucleotidyl transferase (TdT). These nucleotides are non-templated and thus are termed N-nucleotides. TdT is expressed during H chain rearrangement and is down-regulated before L chain recombination. N-nucleotides are predominantly a feature of the VDJ joint of H chains (although some are found in L chain VJ joints). IMMUNOGLOBULIN - rearrangement Junctional Diversity – N-nucleotide addition 5’ T-G-C-T-C-C 3’ A-C-G-A-G-G TdT adds N-nucleotides 5’ T-G-C-T-C-C A-T-C 3’ A-C-G-A-G-G DNA repair enzymes add complementary nucleotides 5’ T-G-C-T-C-C A-T-C 3’ A-C-G-A-G-G T-A-G N-nucleotides IMMUNOGLOBULIN - rearrangement Junctional Diversity – P- and N-nucleotides V Gene Segment D Gene Segment 5’ T-G-C-T-C-C G-T-G-G-A-C 3’ every new b cell does this di ernetly, get unlimited diversity then still doesnt give ab tho, this is how we rearrange chromosome, need to take these genetic changes and need functional working moleculle few things have to happen C-A-C-C-T-G 5’ need to get diversity right and in frame, need to do it twice, heavy AND light chain need 3’ A-C-G-A-G-G to work, cell also needs to know if it was in frame or out of frame, need more mechanisms 5’ T-G-C-T-C-C G A-C G-T-G-G-A-C 3’ P- Addition 3’ A-C-G-A-G C-C-T-G 5’ 5’ T-G-C-T-C-C G C-C-A-T A-C G-T-G-G-A-C 3’ N- Addition 3’ A-C-G-A-G C-C-T-G 5’ Repair enzymes add 5’ T-G-C-T-C-C G C-C-A-T A-C G-T-G-G-A-C 3’ complementary sequences and ligate 3’ A-C-G-A-G -G-C-G-G-T-A-T-G-C-A- C-C-T-G 5’ fragments P N P IMMUNOGLOBULIN - rearrangement want to make sure we do this in very careful and tightly regulated way, think about grand scheme, this is dangerous process, we're actively cutting up chromosomal dna, shu ing it and glueing it back together, lots of things can go wrong, we know it does can have b cell cancers, if we're reattaching genes and put enhancer that leads to high levels of transcription associated with ab against pro-tumor gene, all of a sudden that cell becomes cancerous, end up with myelome or lymphoma, happens frequently, this is the risk if we dont get it right to do it right, have step wise check system, each thing that happens needs to be validated before next step, if anything goes wrong, kill cell right away, dont risk it developing problems in doing this, go through several devt stage with b cell, each stage is de ned by how immunoglobulin or ab genes look germline is still a stem cell, when theyre full rearranged theyre a b cell, and theres stages in between for the b cell to survive, it has to successfully make 1 heavy and 1 light chain, if it doesnt, b cell wont develop and will die  IMMUNOGLOBULIN - rearrangement B-cells are produced continually throughout life. Ig rearrangement begins early in B-cell development Ig rearrangement is tightly regulated in a sequential, stepwise fashion. Stages of B cell development defined by the progression of Ig rearrangement events. Failure to successfully rearrange both H and L chain genes results in a blockage of B-cell development. what causes dna breaks and the rearrangement of dna? express 2 speci c enzymes for this, this isnt random break in dna, we have enzymes that know exactly where cut site is, cut dna, they discard the dna thats in between the genes, hold them together, bring them close and force them to stick back together dont just cut dna and let it oat around in our nucleus, these are stabilized structures so that we can put them back together  IMMUNOGLOBULIN - rearrangement Rearrangement is initiated RAG complex. Recombination Activating Gene – 1 (RAG-1) Recombination Activating Gene – 2 (RAG-2) The RAG complex recognizes the boarders of the gene segments and facilitates cleavage of the DNA need both of these genes, these are the ones that see where the edge of the v-gene is and where the intervening dna starts or the D or the J, can nd that pattern, know where cut site is, facilitate in cutting and stabilizing pieces if ur missing either o