B cell Epit opes - CBS · 2008. 6. 10. · B cell Epit opes. Technical University of Denmark - DTU Department of systems biology CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS Antibody E

Technical University of Denmark - DTUDepartment of systems biology

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B cell Epitopes


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Antibody Effect

Virus or Toxin 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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Fab

Antigen

Epitope

Antibody


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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

Figure 4-2

Figure 4-3

Figure 4-4

Figure 4-5

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 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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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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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)

Documents

B cell Epit opes - CBS · 2008. 6. 10. · B cell Epit opes. Technical University of Denmark - DTU Department of systems biology CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS Antibody E