CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark Creation of a functional B cell receptor/Antibody

CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark Germ line gene organization © 2001 by Garland Publishing

CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark Gene organization © 2001 by Garland Publishing

CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark The 12/23 rule of recombination recombination signal sequence (RSS) { Only combined 12 RSS to 23 RSS

CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark Mechanism of gene rearrangement

CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark Addition of P and N nucleotides TdT: terminal deoxynucleotidyl transferase

CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark 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? Who is doing the end trimming?

CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark P nucleotides SequencesPermutated sequences Distance from heptamer to gene end No. of seq No. with P % with PNo. of seq No. with P % with Pp-value VH gene < JH gene < ’ end of D gene < ’ end of D gene Table 1. Manuscript 2.

CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark How many types of D genes? Conventional D genes – Identified in 81% of sequences unmutated sequences, 64% of mutated sequences D genes with irregular RSS (DIR) Chromosome 15 OR Two D genes Inverted D genes –Long inverted D genes can not be excluded

CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark Inverted (palindrom) D genes

CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark D genes with irregular RSS (DIR) Very long, >180 bp Contain a family 1 D gene Found in 1% of sequences, inverted in 1.2% Some explained as family 1 gene plus N additions Median length of remaining not different from in permutated sequences

CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark Two D genes 2 D genes found in 1% of sequences Frequency not different from permutated sequences Some explained as one long D genes with deletion Some not possible due to D genes location Median lengths of longest gene resembles normal D genes, shortest resembles permutated sequences

CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark Chromosome 15 OR 10 OR resembling D genes on chromosome 15 High homology to conventional D genes Very few OR15 in un-mutated sequences Median length not different from hits in permutated sequences

CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark D gene usage 27 conventional D genes, 34 known alleles

CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark 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

CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark D gene reading frames The recombination mechanism utilises each D gene reading frame at same frequency Reading Frame StopHydrophilicHydrophobic GenePNPP P D2-2*01RIL**YQLLC (1) GYCSSTSCYA (2) DIVVVPAAM (3) D2-2*02RIL**YQLLY (1) GYCSSTSCYT (2) DIVVVPAAI (3) D2-2*03WIL**YQLLC (1) GYCSSTSCYA (2) DIVVVPAAM (3) D2-8*01RILY*WCMLY (1) GYCTNGVCYT (2) DIVLMVYAI (3) D2-8*02RILYWWCMLY (1)0.0 GYCTGGVCYT (2) DIVLVVYAI (3) D2-15*01RIL*WW*LLL (1) GYCSGGSCYS (2) DIVVVVAAT (3) D2-21*01SILWW*LLF (1) AYCGGDCYS (2) HIVVVIAI (3) D2-21*02SILWW*LLF (1) AYCGGDCYS (2) HIVVVTAI (3) Total Tabel 4. Manuscript 2.

CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark N nucleotide dependence on end nucleotide Position X+1 Position XATGCP-value A T G C < Expected N addition is not random but dependent on end nucleotide

CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark Trimming of gene ends  Trimming depends on the gene end and can not only be described by a simple removal of one nucleotide at a time Avg. 3.8 bp

CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark 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 frequency  What 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  At what step in the development is the selection for the hydrophilic reading frame

CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark 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?

CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark The translated functional rearrangement

CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark Numbering Schemes

CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark 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 [1-6]. This numbering, set up after aligning more than sequences, takes into account and combines the definition of the framework (FR) and complementarity determining regions (CDR) [8], structural data from X-ray diffraction studies [9], and the characterization of the hypervariable loops [10].

CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS BiC BioCentrum-DTU Technical University of Denmark Identification of CDR regions Indentifying the CDRs CDR-L1 StartApprox residue 24 Residue beforeis always C Residue afteris always W. Typically WYQ, but also, WLQ, WFQ, WYL Length10 to 17 residues CDR-L2 Startalways 16 residues after the end of CDR-L1 Residues beforegenerally IY, but also, VY, IK, IF Lengthalways 7 residues CDR-L3 Startalways 33 residues after end of CDR-L2 Residue beforeis always C Residues afteralways FGXG Length7 to 11 residues CDR-H1 StartApproximately residue 31 (always 9 after a C) (Chothia/AbM defintion starts 5 residues earlier) Residues beforealways CXXXXXXXX Residues afteralways W. Typically WV, but also WI, WA Length5 to 7 residues (Kabat definition); 7 to 9 residues (Chothia definition); 10 to 12 residues (AbM definition) CDR-H2 Startalways 15 residues after the end of Kabat/AbM definition of CDR-H1 Residues beforetypically LEWIG, but a number of variations Residues afterK[RL]IVFT[AT]SIA (where residues in square brackets are alternatives at that position) LengthKabat 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-H3 Startalways 33 residues after end of CDR-H2 (always 3 after a C) Residues beforealways CXX (typically CAR)