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Immunology Ch4 notes week of 9/12

by: Mariana Vasquez

Immunology Ch4 notes week of 9/12 MICR 4353

Marketplace > University of Texas at El Paso > Biological Sciences > MICR 4353 > Immunology Ch4 notes week of 9 12
Mariana Vasquez
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Ch.4 notes
Dr. Almeida
Class Notes




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This 6 page Class Notes was uploaded by Mariana Vasquez on Thursday February 25, 2016. The Class Notes belongs to MICR 4353 at University of Texas at El Paso taught by Dr. Almeida in Fall 2016. Since its upload, it has received 21 views. For similar materials see Immunology in Biological Sciences at University of Texas at El Paso.

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Date Created: 02/25/16
9/13 Ch.4 Antibody Structure & the Generation of B-Cell Diversity  Plasma cells secrete antibody of the same antigen specificity as the membrane-bound immunoglobulin(Ig) expressed by their B-cell precursor o B-cells are made and mature in the bone marrow  Mature B cells express membrane-bound Ig of single antigen  Resting B-cell has membrane-bound Ig  B-cell meets antigen, binds to surface molecule giving rise to antibody(Ab)-secreting plasma cells o Antibodies(Abs) circulate as major component of plasma in blood and lymph.  Always present in mucosal surfaces  In theory, the body could make up to 10^16 Abs, BUT this is limited due to the number of B-cells present (which are the makers of Abs)  Inability to make Igs(Igs=Abs) makes people very susceptible to certain kinds of infection  Abs molecules have 2 short “light” chains and 2 long “heavy” chains linked by disulfide bonds  Carbohydrate is attached to heavy chain  Hinge region is very flexible; allows the Ab’s “arms” to stretch as needed to properly attach to surface antigens of pathogens. NOTE: NOT ALL Abs have a hinge region  Constant region and variable region(binds to antigen)  There are diff. classes(heavy chain isotypes) of Abs/Igs: o α, β, γ, δ, ε, and μ  All these Igs occur as monomers in their membrane-bound form  In soluble form:  IgD,IgE, and IgG are monomers  IgA may be a monomer or dimer  IgM only forms pentamers o Can be distinguished by diff. length of heavy chains and distribution of N-linked carbohydrate groups o All these Igs occur as monomers in their membrane-bound form  The light chain has only 2 isotypes or classes called λ and κ o No func. Differences have been found btwn Abs w/ κ light chains and those w/ λ light chains o Each Ab only contains 1 of the light-chain isotypes κ and λ, NEVER BOTH o K and λ may be be assoc. w/ any of the heavy chain isotypes (α, β, γ, δ, ε, and μ) o In humans, two-thirds of the antibody molecules contain kappa chains and one-third have lambda chains.  Variability is the ratio of the # of amino acids found at a given position to the frequency of most common amino acid at the common position  Max variability = 400, which is 20^2, where 20 is the number of amino acids usu. Found in proteins such as Igs/Abs  There are 3 hypervariable regions, HV1,HV2, andHV3 flanked by four framework regions (FR1, FR2, FR3, and FR4) o Hypervariable regions are also known as complementaritydetermining regions: CDR1, CDR2, and CDR3. Treating Ab w/papain enzyme will reduce the disulfide bonds, caused by presence of free cysteine not papain activity. This leads to:  Fc-fragment crystallizable(2)  Fab-fragment antigen binding (1) WHAT KIND OF INTERACTIONS OCCUR WHEN ANTIGENS BIND TO ANTIBODIES? Noncovalent interactions always!! (remember Hydrogen bonds-hydrophobic interactions-Ionic interactions-Van der Waals (H-HIV)) Epitopes B and T cells recognize different epitopes on the same antigenic molecule. • They have =/= requirements for antigen recognition Characteristics of B-cell epitopes: • Generally composed of hydrophilic amino acids on the protein that are accessible to membrane-bound or free antibody. • Tend to be located in flexible regions of an immunogen and often display site mobility. • Complex proteins contain multiple overlapping B-cell epitopes, some of which are immunodominant (induces a more pronounced immune response). 2 kinds of multivalent antigen:  Diff. epitopes  Repeated epitopes A linear epitope of a protein antigen is formed from contiguous amino acids. A discontinuous (conformational) epitope is formed from amino acids from different parts of the polypeptide that are brought together when the chain folds.  Must be in native form to bind  Reason why making vaccines is hard; conformations must be known Antigen-binding sites of Abs are rich in aromatic amino acids which can participate in many Van der Waals (temporary, weak electrostatic interactions of opp. Charges due to current position of electrons around the nucleus) and hydrophobic interactions Different Abs may recognize same epitope but the affinity(strength) of the recognition may be different  During infection, the first Abs made have less affinity to the pathogen than those made later on. o IgM has less affinity than later IgG Abs o CD4 T-cells o Recombination process (hyper-mutation of CDRs)- amino acids change to improve Abs  Van der Waals-When you have hydrocarbon pockets, electrons are closest to the protons from the nucleus; attraction occurs, else repulsion occurs o Transient interaction  Production of mouse monoclonal Abs o Monoclonal Ab (mAb) comes from a single isolated B-cell, mAbs recognize same epitope w/same affinity since they are the same o Polyclonal Ab occurs when a variety of B-cells are selected, they polyclonal Abs consequently recognize same epitope w/diff. affinities  Cells don’t last long in culture, so they are “immortalized” by fusing them w/cancer cells o Grown in drug-containing medium so only the hybridized cells survive o Select for antigen-specific hybridoma o Clone selected hybridoma cells 9/15 The flow cytometer allows cells to be distinguished by their cell surface molecules; can be coupled with cell sorting. Tag cell with monoclonal antibody labeled with a fluorophore, that emits light in a sp. wavelength Cells go through micro-fluidity device; micro-tubing only passes one cell at a time, the cell gets a laser shone through it, that a detector notices due to the change in transmittance and cell fluorescence Monoclonal antibodies  4 types of therapeutic monoclonal antibodies: o Mouse, issue is that constant chain in mouse, this could lead to a response if antibody developed in mice injected into human o Chimeric-constant domains where replaced with human but the variable region is murine o Humanized- human constant chain and partially human variable chain o Human monoclonal antibody Rituximab-monoclonal chimeric antibody against CD20, primarily found on surface of B cells. Rituximab destroys B cells, and thus used to treat disease w/overactive/excessive/nonfunc. B cells, such as lymphomas, leukemias, transplant rejection, and autoimmune disorders. Number of diff. Abs that can be made by body is virtually limitless  Ig genes organized diff. from other genes  In germline DNA, mult. Gene segments encode portions of a single Ig heavy or light chain  Germline-line of germ cells that have genetic material that can be inherited o Gene segments in germ cells BUT can’t be transcribed and translated into complete chains until they’re rearranged into func. Genes  Ig genes found in 3 chr, locations in humans o Heavy-chain-chr 14 o K light chain - “” 2 o Lambda light -“”22 Leader peptide-signal peptide that directs mRNA to be translated; every protein made in ER needs this peptide, cytoplasm proteins don’t. During B-cell maturation :genes randomly shuffled by dynamic genetic sys. Capable of making 10 ^6 combos Subsequent process incr. diversity of Ab repertoire of binding sites to >10 ^8 Mature cells have chr. DNA no longer identical to germ-line DNA During B-cell development, arrays of V, D, and J segments are cut and spliced by DNA recombination; process called “Somatic recombination” VJ-joined rearranged DNA in light chain w/1 constant domain VDJ-joined rearranged DNA in heavy chain w/4 constant domains MC is membrane-coding exon, transmembrane motif Somatic recomb. Carried out by enzymes that “cut and paste” DNA  Recombination of V,D, and J is directed by sequences called recomb. Signal sequences(RSS) o 2 types:  Nonamer(9bp)  Heptamer(7bp)  They are separated by spacer (12bp long)  Other is hepatamer and nonamer sep. by 23 bp spacer Requirements of RSS recomb. 12/23 rule Enzymes for V,D, and J recomb called V(D)J recombinase  2 components(only made by lymphocytes, coded in RAG genes): o RAG-1 o RAG-2 Other comp. present in all nucleated cells and have activities to repair dsDNA, bend DNA, or modify ends of broken DNA strands Fig.4.29 Recomb. Process ini. By RAG complex(cuts DNA), when RAG cuts, need to introduce diversity “Changes” at coding joints, making variable regions more diverse RAG complex cleaves heptamer RSS from D and J gene segments to yield DNA hairpins RAG complex opens hairpins by nicking one strand of the DNA , making palindromic P-nucleotides N-nucleotide additions by TdT, strands are paired, unpaired nucleotides are removed by exonuclease and gaps are filled by DNA syn. And ligation to form the coding joint Lots of var. due to the dependence on what is bound; codons are modified, lots of mutation due to recomb. Besides the IgM in every B-cell surface, we have IgD as well (func. Not understood well yet) Developing and naïve B cells use alt. mRNA splicing to make both IgM and IgD “naïve” means not yet exposed to antigen Isotype and class of an Ab determined by its heavy chain Each B-cell makes IG of a single antigen specificity. B-CELLS ARE MONOSPECIFIC! Focuses the response on the encountered antigen.  Transmembrane IgM vs. Secreted IgM o Only diff is that secreted IgM does NOT have a transmembrane motif Somatic hyper-mutation depends on enzyme AID, made only by proliferating B cells Somatic hyper-mutation gives rise to B cells bearing mutant Ig molecules on their surface B cells bearing these mutant high-affinity Ig receptors compete most effectively for binding with…….  Not all vaccines activate CD4 T-cells o Problem, polysaccharides need to be coupled w/peptide to activate CD4 T-cell and isotype switch to have long-term protection, instead of just activating B cells What signal does B-cell receive to know when to do isotype switching? CD4 T-cells provide signal in the form of cytokines


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