Immunoglobulin VH, DH, and JH gene usages in the mouse IgH sequence repertoire.

<p>The mouse IgH gene sequence data set containing 17,179 entries was downloaded from NCBI databases. The potential V_H, D_H, and J_H germline gene assignments were performed using the IMGT/V-QUEST program by sending batches of sequences by the V_HRFA program. Clonally redundant IgH sequences were removed if they contain identical CDR3 regions. The usages of different families of V_H germline genes (A), D_H genes (B), and J_H (C) genes in the functional or non-functional unique IgH genes were analyzed.</p

Contribution of V_H Replacement Products in Mouse Antibody Repertoire

<div><p>V_H replacement occurs through RAG-mediated recombination between the cryptic recombination signal sequence (cRSS) near the 3′ end of a rearranged V_H gene and the 23-bp RSS from an upstream unrearranged V_H gene. Due to the location of the cRSS, V_H replacement leaves a short stretch of nucleotides from the previously rearranged V_H gene at the newly formed V-D junction, which can be used as a marker to identify V_H replacement products. To determine the contribution of V_H replacement products to mouse antibody repertoire, we developed a Java-based V_H Replacement Footprint Analyzer (V_HRFA) program and analyzed 17,179 mouse IgH gene sequences from the NCBI database to identify V_H replacement products. The overall frequency of V_H replacement products in these IgH genes is 5.29% based on the identification of pentameric V_H replacement footprints at their V-D junctions. The identified V_H replacement products are distributed similarly in IgH genes using most families of V_H genes, although different families of V_H genes are used differentially. The frequencies of V_H replacement products are significantly elevated in IgH genes derived from several strains of autoimmune prone mice and in IgH genes encoding autoantibodies. Moreover, the identified V_H replacement footprints in IgH genes from autoimmune prone mice or IgH genes encoding autoantibodies preferentially encode positively charged amino acids. These results revealed a significant contribution of V_H replacement products to the diversification of antibody repertoire and potentially, to the generation of autoantibodies in mice.</p> </div

VH replacement footprints preferentially contribute charged amino acids to the CDR3 regions.

<p>(A) The frequencies of charged amino acids encoded by the identified pentameric V_H replacement footprints or the N1 regions of non-V_H replacement products were compared. Detailed amino acid sequences of the IgH CDR3 regions are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057877#pone.0057877.s006" target="_blank">Table S6</a>. (B) The frequencies of individual amino acid encoded by the identified V_H replacement footprints or the N1 regions of non-V_H replacement products were compared. <i>n</i>, amino acids encoded by the identified V_H replacement footprints or the N1 regions of non-V_H replacement products. (C) The frequencies of individual amino acid encoded by the 3′ end of V_H germline genes and D_H regions were compared. n, amino acids encoded by the V_H gene 3′ ends or D_H regions. (D) Usages of different amino acids encoded by the identified V_H replacement footprints in functional V_H replacement products and non-functional V_H replacement products. n, amino acids encoded by the identified V_H replacement footprints. Statistical significance was determined using a two-tailed Chi square test with Yate’s correction. <i>n</i>, number of amino acid residues encoded by indicated sequences. <i>p</i><0.05 (*) is considered significant and <i>p</i><0.0001 (**) is considered extremely significant.</p

The enriched V_H replacement products identified in different strains of autoim<i>m</i>une prone mice or IgH genes encoding aut<i>o</i>antibodies have been positively selected during autoimmune responses.

<p>(A) Analysis of the frequencies of positively charged versus negatively charged amino acids encoded by the 3′ end V_H genes and the identified V_H replacement footprints from different strains of mice or IgH genes encoding autoantibodies. Statistical significance was determined using a two-tailed Chi square test with Yate’s correction. <i>p</i><0.05 (*) is considered significant. (B) Comparison of the amino acids encoded by the identified V_H replacement footprints in <i>MRL/lpr</i> mice. <i>n</i>, numbers of amino acids encoded by the identified V_H replacement footprints. (C) Mutation status analysis of identified V_H replacement products and non-V_H replacement products from different subgroups of IgH genes.</p

List of potential V_H replacement products in the test IgH sequences.

<p>The identified V_H replacement footprints in the N1 regions are <i>underlined</i>.</p>a<p>The relative positions of the potential donors and recipient V_H genes in the identified V_H replacement product were analyzed to determine if the V_H replacement occurred through an upstream V_H gene replacing a downstream V_H gene (Y) or a downstream V_H gene replacing an upstream gene (N). Only functional V_H germline genes were used in this analysis.</p

Comparison of the amino acids encoded by V_H replacement footprints and the IgH CDR3 lengths of V_H replacement products.

<p>(A) The usages of different amino acids encoded by V_H replacement footprints with 5, 4, or 3 nucleotides were compared. <i>n</i>, number of amino acid residues encoded by the identified V_H replacement footprints with different lengths. Statistical significance was determined using a two-tailed Chi square test with Yate’s correction. <i>p</i><0.05 (*) is considered significant and <i>p</i><0.0001 (**) is considered extremely significant. (B) Comparison of the IgH CDR3 lengths of V_H replacement products containing the 5-mer or the 3-mer V_H replacement products with the CDR3 length of the total functional IgH genes. <i>n</i>, number of IgH sequences or V_H replacement products with 3- or 5-mer V_H replacement footprints. Statistical significance was determined using unpaired <i>t</i> test. <i>p</i><0.05 (*) is considered significant and <i>p</i><0.0001 (**) is considered extremely significant.</p

Enrichment of V_H replacement products in IgH genes derived from different strains of autoimmune prone mice and IgH genes encoding autoantibodies.

<p>The frequencies of V_H replacement products in IgH genes derived from different strains of mice were analyzed using the V_HRFA program based on the keyword linked to each IgH gene in the NCBI database. V_H replacement products were assigned based on the identification of (A) 5-mer V_H replacement footprints, (B) 4-mer V_H replacement footprints, or (C) 3-mer V_H replacement footprints within the V_H-D_H junctions (N1 regions). The frequencies of V_H replacement products in different subcategories were compared with that in the <i>BALB/c</i> mice. <i>n</i>, number of IgH sequences in each subcategory. Statistical significance was determined using a two-tailed Chi square test with Yate’s correction. <i>p</i><0.05 (*) is considered significant and <i>p</i><0.0001 (**) is considered extremely significant. The detailed sequence analysis and the identified V_H replacement products with 5-mer V_H replacement footprints correlating with keywords are included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057877#pone.0057877.s006" target="_blank">Table S6</a>.</p

Frequencies of V_H replacement products in IgH genes using different families of mouse V_H genes.

a<p>Number of IgH gene sequences with V_H replacement “footprint” motifs in the N1 regions divided by the total number of IgH gene sequences assigned to a V_H gene family.</p>b<p>Functional IgH genes using the VH5-2/7183.2 gene were analyzed for potential V_H replacement footprints in the N1 regions.</p>C<p>The frequency of V_H replacement products using VH2/Q52 family of V_H genes is significantly higher than the overall frequency of V_H replacement products in mouse IgH genes.</p