Antibody Structure
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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.

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

Transcript

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