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Antibody Structure
The document discusses the conservation of residues in the variable region of antibodies, focusing on somatic hypermutation and affinity maturation in the immune response. It presents estimates that approximately half of the replacement mutations in framework regions are lethal, categorizing mutations into invariant, conservative, and non-conservative types. Future directions include the need for more sequence data and better alignments to further analyze and understand antibody diversity.
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Antibody Structure
- 1. CBB Spring RotationwithSteven KleinsteinConserved Residues ofan Antibody’s V RegionDaniel Gadala-Maria7 June 2010
- 2. 1° immune response:mostlygermline antibodies Somatic Hypermutation& Affinity Maturation2 ° immune response: sequences with point mutationsAgAgMutations improve antibody affinityD F Gadala-Maria 7 June 2010
- 3. Antibodies have severaldomains4 protein chains2 Light chains1 Variable domain (VL) each1 Constant domain (CL) each2 Heavy chains1 Variable domain (VH) each3 Constant domains (CH) eachVHVHVLVLCHCHCLCLCHCHCHCHD F Gadala-Maria 7 June 2010
- 4. VH & VLare divided into 2 partsComplementarityDetermining Regions (CDRs)Mutated to improvebinding to antigen (Ag)Positive selection of certain mutantsFramework Regions (FWs)Provide structure and supportFraction of mutations are not tolerableAgVHVHVLVLD F Gadala-Maria 7 June 2010
- 5. Estimate that ½of replacement mutations are lethal in FWShlomchik et al (1989)1, using two methods:Use replacement:silent mutation ratioExpect R:S of ≈2.9 if no selectionObserve R:S of 155/107 ≈ 1.45 in FWEstimate ≈ ½ R Mutations lethal in FWCategorize and count replacement mutations79 positions categorized as into:21 invariant, 27 conservative, 31 non-conservativeEstimate of 0.48 R Mutations lethal in FW1Shlomchik 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 linethem up and count, if we had a good alignment…Many more sequences available!IMGT (the international ImMunoGeneTics information system) http://www.imgt.orgLefrancet 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 conservationD F Gadala-Maria 7 June 2010
- 8. Can we categorizepositions?20/ 56 = 0.357136/ 56D F Gadala-Maria 7 June 2010
- 9. Loose ends &future directionsCould use more sequences (readily available),especiallynon-functional (not so common)Correct bad alignments for FW1Looked at just V, but could do V(D)J Other methods for assessing % lethalPlot conserved positions onto crystal structureD F Gadala-Maria 7 June 2010
- 10. Thank you forlistening!Special thanks to:Steve KleinsteinMohamed UdumanChris BolenUri HershbergD F Gadala-Maria 7 June 2010
Editor's Notes
- #2 Lethal FR mutations hinder detecting selection
- #3 Likely to re-encounted the same antigens.These mutated sequences help provide an efficient secondary response.
- #5 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)
- #6 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
- #7 Talk a little about where the data came from.Next move onto numbering
- #8 Log2(20) ~ 4.32Lead in to dividing into two groups: conserved and not…
- #9 Log2(20) ~ 4.32Lead into non-functional (expect conservation to not matter, good control)
- #10 variable (V), diversity (D) and joining (J) segmentsCould analyze which of the 9 reachable-in-one-mutation amino acids are found