B cell Epit opes - CBS · 2008. 6. 10. · B cell Epit opes. Technical University of Denmark - DTU...
Transcript of B cell Epit opes - CBS · 2008. 6. 10. · B cell Epit opes. Technical University of Denmark - DTU...
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B cell Epitopes
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Antibody Effect
Virus or Toxin Antibodies
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Antibody Effect
Virus or Toxin Antibodies
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Antibodies
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Antibodies
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Antibody - Antigen interactionAntigen
Antibody
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Antibody - Antigen interaction
Fab
Antigen
Antibody
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Antibody - Antigen interaction
Fab
Antigen
Epitope
Antibody
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Antibody - Antigen interaction
Fab
Antigen
Paratope
Antibody
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B-Cells
B-lymphocytes each displaying a unique B-cell receptor
Stem Cell
Precurser B-lymphocytes
Gene rearrangements
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Figure 4-2
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Figure 4-3
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Figure 4-4
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Figure 4-5
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Figure 4-6
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P-nucleotide introduction
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N-nucleotide introduction
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Somatic Hypermutations
© 2001 by Garland Publishing
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B-Cell ActivationNo AffinityLow AffinityNo Affinity
High Affinity
Somatic Hypermutations
Memory B-cells
Plasma cells
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B-Cell Activation
B Cell
T Helper Cell
Class II MHC
Bound Peptide
TCR
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Cartoon by Eric Reits
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Monoclonal Abs
•Hybridomas
•Phage Display
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Hybridomas
http://www.britannica.com/
http://www.britannica.comhttp://www.britannica.com
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Phage Display
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Phage Display
http://www.bio.anl.gov/highthroughputbiology/images/phage_display1.jpg
http://www.bio.anl.gov/highthroughputbiology/images/phage_display1.jpghttp://www.bio.anl.gov/highthroughputbiology/images/phage_display1.jpg
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Structural Epitopes
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Discontinuous Epitopes
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Discontinuous Epitopes
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Discontinuous Paratopes
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Variable regionsAlpha-carbon trace of the structure of the heavy chain and
light chain variable regions of a typical antibody. The framework regions of both chains are shown in grey whilst
the complementarity determining regions (CDRs) are coloured individually, i.e.
Heavy chainCDR 1 = Light blue
CDR 2 = CeriseCDR 3 = Yellow
Light ChainCDR 1 = Red
CDR 2 = GreenCDR 3 = Blue
CDR RegionsCDR = complementarity determining region
http://212.219.234.139/html/anti_alpha.html
http://212.219.234.139/html/anti_alpha.htmlhttp://212.219.234.139/html/anti_alpha.html
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Identification of germ-line genes
Why?
As bookkeeping and reporting tool
For detection of potentially important residues in binding affinity.
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Joinsolver
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Vbase
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Vquest
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VDJsolver
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Questions to be addressed
•Can multiple D genes be inserted?
•Violation of 12/23 rule
•Can D genes be inserted backwards?
•Is there a D gene preference?
•Is there a reading frame preference for D genes?
•If yes, is it part of the gene rearrangement?
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Data sets
•6329 clonally unrelated rearrangements.
•1968 un-mutated functional
•3707 mutated functional
•274 un-mutated non-functional
•380 mutated non-functional
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Sequences Permutated sequences
Distance from heptamer to gene end
No. ofseq
No. withP % with P
No. ofseq
No. with P % with P p-value
VH gene
1 1448 474 32.7 1635 103 6.3
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Sequences Permutated sequences
Distance from heptamer to gene end
No. ofseq
No. withP % with P
No. ofseq
No. with P % with P p-value
VH gene
1 1448 474 32.7 1635 103 6.3
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Sequences Permutated sequences
Distance from heptamer to gene end
No. ofseq
No. withP % with P
No. ofseq
No. with P % with P p-value
VH gene
1 1448 474 32.7 1635 103 6.3
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How many types of D genes?
•Conventional D genes
• Identified in 81% of sequences unmutated sequences, 64% of mutated sequences
•Inverted D genes
•Long inverted D genes can not be excluded
•Two or more D genes
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D gene usage27 conventional D genes, 34 known alleles
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D-gene usage and JH gene
•JH proximal D genes more often recombined to JH4 than JH6 and JH distal D genes more often to JH6
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Inverted (palindrom) D genes
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Inverted (palindrom) D genes
Inverted D genes are not used!(or used extremely infrequent)
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Multiple D genes
•65 sequences with two D genes
•Average length of shortest D genes: 11.6bp
•Average length of longest D genes: 18.8bp
•Average length of D genes in permuted sequences: 11.3bp
•Average length of D genes in normal sequences: 17.8bp
• => multiple D genes are not present!!!
Longest-D Shortest-DV-gene J-gene
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D gene reading frames
• The recombination mechanism utilizes each D gene reading frame at same frequency
Reading Frame Stop Hydrophilic Hydrophobic
Gene P NP P NP P NP
D2-2*01 RIL**YQLLC (1) 6.5 34.7 GYCSSTSCYA (2) 61.2 32.6 DIVVVPAAM (3) 32.2 32.6
D2-2*02 RIL**YQLLY (1) 11.3 46.7 GYCSSTSCYT (2) 55.0 20.0 DIVVVPAAI (3) 33.8 33.3
D2-2*03 WIL**YQLLC (1) 0.0 50.0 GYCSSTSCYA (2) 66.7 50.0 DIVVVPAAM (3) 33.3 0.0
D2-8*01 RILY*WCMLY (1) 2.4 42.9 GYCTNGVCYT (2) 68.3 28.6 DIVLMVYAI (3) 29.3 28.6
D2-8*02 RILYWWCMLY (1) 0.0 0.0 GYCTGGVCYT (2) 88.9 0.0 DIVLVVYAI (3) 11.1 100
D2-15*01 RIL*WW*LLL (1) 1.5 32.5 GYCSGGSCYS (2) 70.8 37.5 DIVVVVAAT (3) 27.7 30.0
D2-21*01 SILWW*LLF (1) 8.3 50.0 AYCGGDCYS (2) 58.3 25.0 HIVVVIAI (3) 33.3 25.0
D2-21*02 SILWW*LLF (1) 0.0 54.5 AYCGGDCYS (2) 78.0 18.2 HIVVVTAI (3) 22.0 27.3
Total - 10.8 33.6 - 62.2 32.4 - 26.9 34.0
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D gene reading frames
• The recombination mechanism utilizes each D gene reading frame at same frequency
Reading Frame Stop Hydrophilic Hydrophobic
Gene P NP P NP P NP
D2-2*01 RIL**YQLLC (1) 6.5 34.7 GYCSSTSCYA (2) 61.2 32.6 DIVVVPAAM (3) 32.2 32.6
D2-2*02 RIL**YQLLY (1) 11.3 46.7 GYCSSTSCYT (2) 55.0 20.0 DIVVVPAAI (3) 33.8 33.3
D2-2*03 WIL**YQLLC (1) 0.0 50.0 GYCSSTSCYA (2) 66.7 50.0 DIVVVPAAM (3) 33.3 0.0
D2-8*01 RILY*WCMLY (1) 2.4 42.9 GYCTNGVCYT (2) 68.3 28.6 DIVLMVYAI (3) 29.3 28.6
D2-8*02 RILYWWCMLY (1) 0.0 0.0 GYCTGGVCYT (2) 88.9 0.0 DIVLVVYAI (3) 11.1 100
D2-15*01 RIL*WW*LLL (1) 1.5 32.5 GYCSGGSCYS (2) 70.8 37.5 DIVVVVAAT (3) 27.7 30.0
D2-21*01 SILWW*LLF (1) 8.3 50.0 AYCGGDCYS (2) 58.3 25.0 HIVVVIAI (3) 33.3 25.0
D2-21*02 SILWW*LLF (1) 0.0 54.5 AYCGGDCYS (2) 78.0 18.2 HIVVVTAI (3) 22.0 27.3
Total - 10.8 33.6 - 62.2 32.4 - 26.9 34.0
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VDJsolver performanceUnmutated sequences
Mutated sequences
#: p
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Results regarding recombination and diversity and open questions
•DIR, OR15, multiple D genes and VH replacements are not used at a significant rate
•Inverted D genes are used rarely
•All D genes not used at same frequencyWhat determines if a D genes is used?
•D gene usage somewhat dependent on JH gene Does multiple D-J recombination steps take place?
•All D gene reading frames used at equal rate at the recombination step
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Results regarding recombination and diversity and open questions (cont.)
•N addition not random but dependent on end nucleotide
Does nucleotide availability or the specificity of TdT determine the N addition?
•Trimming not random but dependent on gene and sequence
What enzyme(s) is responsible for the trimming?
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Numbering Schemes
The Kabat numbering scheme is a widely adopted standard for numbering the residues in an antibody in a consistent manner. However the scheme has problems!
The Chothia numbering scheme is identical to the Kabat scheme, but places the insertions in CDR-L1 and CDR-H1 at the structurally correct positions. This means that topologically equivalent residues in these loops do get the same label (unlike the Kabat scheme).
The IMGT unique numbering for all IG and TR V-REGIONs of all species relies on the high conservation of the structure of the variable region. This numbering, set up after aligning more than 5 000 sequences, takes into account and combines the definition of the framework (FR) and complementarity determining regions (CDR), structural data from X-ray diffraction studies, and the characterization of the hypervariable loops.
http://www.bioinf.org.uk/abs/#kabatnum http://imgt.cines.fr/
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Identification of CDR regionsCDR-L1Start Approx residue 24Residue before is always CResidue after is always W. Typically WYQ, but also, WLQ, WFQ, WYLLength 10 to 17 residues
CDR-L2Start always 16 residues after the end of CDR-L1Residues beforegenerally IY, but also, VY, IK, IFLength always 7 residues
CDR-L3Start always 33 residues after end of CDR-L2Residue before is always CResidues after always FGXGLength 7 to 11 residues
CDR-H1Start Approximately residue 31 (always 9 after a C) (Chothia/AbM defintion starts 5 residues earlier)Residues beforealways CXXXXXXXXResidues after always W. Typically WV, but also WI, WALength 5 to 7 residues (Kabat definition); 7 to 9 residues (Chothia definition); 10 to 12 residues (AbM definition)
CDR-H2Start always 15 residues after the end of Kabat/AbM definition of CDR-H1Residues beforetypically LEWIG, but a number of variationsResidues after K[RL]IVFT[AT]SIA (where residues in square brackets are alternatives at that position)Length Kabat definition 16 to 19 residues (AbM definition and most recent Chothia definition ends 7 residues earlier; earlier Chothia definition starts 2 residues later and ends 9 earlier)
CDR-H3Start always 33 residues after end of CDR-H2 (always 3 after a C)Residues beforealways CXX (typically CAR)