Antibody Structure


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Talk on my lab rotation with Steve Kleinstein. I looked at the framework regions of antibodies in an attempt to characterize conserved positions.

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  • Lethal FR mutations hinder detecting selection
  • Likely to re-encounted the same antigens.These mutated sequences help provide an efficient secondary response.
  • Variable regions areN-terminus of each chainCDR mutations may improve Ab affinity,FW mutations may destabilizeNon-tolerated fraction important b/c reduced presence of R mutations might look like selection (in terms of Ab binding ability)
  • The CDRs are predisposed to mutation while the FWs are not.Codon bias.R:S sequences numbered on the order of dozensWhy don’t we just take the 50%? X number of sequences (small number), etc. Now we have Y sequences…etc
  • Talk a little about where the data came from.Next move onto numbering
  • Log2(20) ~ 4.32Lead in to dividing into two groups: conserved and not…
  • Log2(20) ~ 4.32Lead into non-functional (expect conservation to not matter, good control)
  • variable (V), diversity (D) and joining (J) segmentsCould analyze which of the 9 reachable-in-one-mutation amino acids are found
  • Antibody Structure

    1. 1. CBB Spring Rotationwith Steven Kleinstein<br />Conserved Residues ofan Antibody’s V Region<br />Daniel Gadala-Maria<br />7 June 2010<br />
    2. 2. 1° immune response:mostly germline antibodies<br /> Somatic Hypermutation& Affinity Maturation<br />2 ° immune response:<br /> sequences with point mutations<br />Ag<br />Ag<br />Mutations improve antibody affinity<br />D F Gadala-Maria 7 June 2010<br />
    3. 3. Antibodies have several domains<br />4 protein chains<br />2 Light chains<br />1 Variable domain (VL) each<br />1 Constant domain (CL) each<br />2 Heavy chains<br />1 Variable domain (VH) each<br />3 Constant domains (CH) each<br />VH<br />VH<br />VL<br />VL<br />CH<br />CH<br />CL<br />CL<br />CH<br />CH<br />CH<br />CH<br />D F Gadala-Maria 7 June 2010<br />
    4. 4. VH & VL are divided into 2 parts<br />ComplementarityDetermining Regions (CDRs)<br />Mutated to improvebinding to antigen (Ag)<br />Positive selection of certain mutants<br />Framework Regions (FWs)<br />Provide structure and support<br />Fraction of mutations are not tolerable<br />Ag<br />VH<br />VH<br />VL<br />VL<br />D F Gadala-Maria 7 June 2010<br />
    5. 5. Estimate that ½ of replacement mutations are lethal in FW<br />Shlomchik et al (1989)1, using two methods:<br />Use replacement:silent mutation ratio<br />Expect R:S of ≈2.9 if no selection<br />Observe R:S of 155/107 ≈ 1.45 in FW<br />Estimate ≈ ½ R Mutations lethal in FW<br />Categorize and count replacement mutations<br />79 positions categorized as into:21 invariant, 27 conservative, 31 non-conservative<br />Estimate of 0.48 R Mutations lethal in FW<br />1Shlomchik M,Litwin S, and Weigert M. (1989) The influence of somatic mutation on clonal expansion. Prog. Immunol. 7:415-423.<br />D F Gadala-Maria 7 June 2010<br />
    6. 6. Could just line them up and count, if we had a good alignment…<br />Many more sequences available!<br />IMGT (the international ImMunoGeneTics information system)<br />Lefrancet al. (2003) IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Igsuperfamily V-like domains. Dev Comp Immunol. 27:55-77.<br />D F Gadala-Maria 7 June 2010<br />
    7. 7. FWs 2 & 3 show much conservation<br />D F Gadala-Maria 7 June 2010<br />
    8. 8. Can we categorize positions?<br />20/ 56 = 0.3571<br />36/ 56<br />D F Gadala-Maria 7 June 2010<br />
    9. 9. Loose ends & future directions<br />Could use more sequences (readily available),especiallynon-functional (not so common)<br />Correct bad alignments for FW1<br />Looked at just V, but could do V(D)J <br />Other methods for assessing % lethal<br />Plot conserved positions onto crystal structure<br />D F Gadala-Maria 7 June 2010<br />
    10. 10. Thank you for listening!<br />Special thanks to:<br />Steve Kleinstein<br />Mohamed Uduman<br />Chris Bolen<br />Uri Hershberg<br />D F Gadala-Maria 7 June 2010<br />