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Contribution of V(H) replacement products in mouse antibody repertoire.

Huang L, Lange MD, Yu Y, Li S, Su K, Zhang Z - PLoS ONE (2013)

Bottom Line: The frequencies of VH 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 VH 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 VH replacement products to the diversification of antibody repertoire and potentially, to the generation of autoantibodies in mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, United States of America.

ABSTRACT
VH replacement occurs through RAG-mediated recombination between the cryptic recombination signal sequence (cRSS) near the 3' end of a rearranged VH gene and the 23-bp RSS from an upstream unrearranged VH gene. Due to the location of the cRSS, VH replacement leaves a short stretch of nucleotides from the previously rearranged VH gene at the newly formed V-D junction, which can be used as a marker to identify VH replacement products. To determine the contribution of VH replacement products to mouse antibody repertoire, we developed a Java-based VH Replacement Footprint Analyzer (VHRFA) program and analyzed 17,179 mouse IgH gene sequences from the NCBI database to identify VH replacement products. The overall frequency of VH replacement products in these IgH genes is 5.29% based on the identification of pentameric VH replacement footprints at their V-D junctions. The identified VH replacement products are distributed similarly in IgH genes using most families of VH genes, although different families of VH genes are used differentially. The frequencies of VH 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 VH 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 VH replacement products to the diversification of antibody repertoire and potentially, to the generation of autoantibodies in mice.

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VH replacement footprints preferentially contribute charged amino acids to the CDR3 regions.(A) The frequencies of charged amino acids encoded by the identified pentameric VH replacement footprints or the N1 regions of non-VH replacement products were compared. Detailed amino acid sequences of the IgH CDR3 regions are listed in Table S6. (B) The frequencies of individual amino acid encoded by the identified VH replacement footprints or the N1 regions of non-VH replacement products were compared. n, amino acids encoded by the identified VH replacement footprints or the N1 regions of non-VH replacement products. (C) The frequencies of individual amino acid encoded by the 3′ end of VH germline genes and DH regions were compared. n, amino acids encoded by the VH gene 3′ ends or DH regions. (D) Usages of different amino acids encoded by the identified VH replacement footprints in functional VH replacement products and non-functional VH replacement products. n, amino acids encoded by the identified VH replacement footprints. Statistical significance was determined using a two-tailed Chi square test with Yate’s correction. n, number of amino acid residues encoded by indicated sequences. p<0.05 (*) is considered significant and p<0.0001 (**) is considered extremely significant.
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pone-0057877-g003: VH replacement footprints preferentially contribute charged amino acids to the CDR3 regions.(A) The frequencies of charged amino acids encoded by the identified pentameric VH replacement footprints or the N1 regions of non-VH replacement products were compared. Detailed amino acid sequences of the IgH CDR3 regions are listed in Table S6. (B) The frequencies of individual amino acid encoded by the identified VH replacement footprints or the N1 regions of non-VH replacement products were compared. n, amino acids encoded by the identified VH replacement footprints or the N1 regions of non-VH replacement products. (C) The frequencies of individual amino acid encoded by the 3′ end of VH germline genes and DH regions were compared. n, amino acids encoded by the VH gene 3′ ends or DH regions. (D) Usages of different amino acids encoded by the identified VH replacement footprints in functional VH replacement products and non-functional VH replacement products. n, amino acids encoded by the identified VH replacement footprints. Statistical significance was determined using a two-tailed Chi square test with Yate’s correction. n, number of amino acid residues encoded by indicated sequences. p<0.05 (*) is considered significant and p<0.0001 (**) is considered extremely significant.

Mentions: Our previous analysis of the identified VH replacement products in human IgH genes showed that the VH replacement footprints preferentially encode charged amino acids into the IgH CDR3 regions [31]. Here, analysis of the identified VH replacement products from mouse IgH genes showed that 64% of the amino acids encoded by the identified VH replacement footprints contribute charged amino acids, including K, R, D, E, N, and Q. Such frequency is significantly higher than the overall frequency of charged amino acids in the N1 regions (p<0.0001) (Fig. 3A). Moreover, the frequencies of charged amino acids, including E, K, and R, encoded by the identified VH replacement footprints are significantly higher than those encoded by the N1 regions of non-VH replacement products (p<0.0001) (Fig. 3B). The preferential contribution of charged amino acids by the VH replacement footprints seems to be predetermined by the sequences at the 3′ end of VH germline genes following the cRSS sites. The frequencies of charged amino acids encoded by the 3′ ends of VH germline gene, including K, R, D, E, N, and Q, are significantly higher than those encoded by the DH germline genes (p<0.0001) (Fig. 3C). In non-functional IgH genes, the identified VH replacement footprints also preferentially encode charged amino acids, although the usages of different charged residues are slightly different from those in the functional VH replacement products (Fig. 3D). Such results are consistent with previous findings that the VH replacement footprints identified in human or mouse VH replacement products preferentially encoded charged residues [31], [39].


Contribution of V(H) replacement products in mouse antibody repertoire.

Huang L, Lange MD, Yu Y, Li S, Su K, Zhang Z - PLoS ONE (2013)

VH replacement footprints preferentially contribute charged amino acids to the CDR3 regions.(A) The frequencies of charged amino acids encoded by the identified pentameric VH replacement footprints or the N1 regions of non-VH replacement products were compared. Detailed amino acid sequences of the IgH CDR3 regions are listed in Table S6. (B) The frequencies of individual amino acid encoded by the identified VH replacement footprints or the N1 regions of non-VH replacement products were compared. n, amino acids encoded by the identified VH replacement footprints or the N1 regions of non-VH replacement products. (C) The frequencies of individual amino acid encoded by the 3′ end of VH germline genes and DH regions were compared. n, amino acids encoded by the VH gene 3′ ends or DH regions. (D) Usages of different amino acids encoded by the identified VH replacement footprints in functional VH replacement products and non-functional VH replacement products. n, amino acids encoded by the identified VH replacement footprints. Statistical significance was determined using a two-tailed Chi square test with Yate’s correction. n, number of amino acid residues encoded by indicated sequences. p<0.05 (*) is considered significant and p<0.0001 (**) is considered extremely significant.
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pone-0057877-g003: VH replacement footprints preferentially contribute charged amino acids to the CDR3 regions.(A) The frequencies of charged amino acids encoded by the identified pentameric VH replacement footprints or the N1 regions of non-VH replacement products were compared. Detailed amino acid sequences of the IgH CDR3 regions are listed in Table S6. (B) The frequencies of individual amino acid encoded by the identified VH replacement footprints or the N1 regions of non-VH replacement products were compared. n, amino acids encoded by the identified VH replacement footprints or the N1 regions of non-VH replacement products. (C) The frequencies of individual amino acid encoded by the 3′ end of VH germline genes and DH regions were compared. n, amino acids encoded by the VH gene 3′ ends or DH regions. (D) Usages of different amino acids encoded by the identified VH replacement footprints in functional VH replacement products and non-functional VH replacement products. n, amino acids encoded by the identified VH replacement footprints. Statistical significance was determined using a two-tailed Chi square test with Yate’s correction. n, number of amino acid residues encoded by indicated sequences. p<0.05 (*) is considered significant and p<0.0001 (**) is considered extremely significant.
Mentions: Our previous analysis of the identified VH replacement products in human IgH genes showed that the VH replacement footprints preferentially encode charged amino acids into the IgH CDR3 regions [31]. Here, analysis of the identified VH replacement products from mouse IgH genes showed that 64% of the amino acids encoded by the identified VH replacement footprints contribute charged amino acids, including K, R, D, E, N, and Q. Such frequency is significantly higher than the overall frequency of charged amino acids in the N1 regions (p<0.0001) (Fig. 3A). Moreover, the frequencies of charged amino acids, including E, K, and R, encoded by the identified VH replacement footprints are significantly higher than those encoded by the N1 regions of non-VH replacement products (p<0.0001) (Fig. 3B). The preferential contribution of charged amino acids by the VH replacement footprints seems to be predetermined by the sequences at the 3′ end of VH germline genes following the cRSS sites. The frequencies of charged amino acids encoded by the 3′ ends of VH germline gene, including K, R, D, E, N, and Q, are significantly higher than those encoded by the DH germline genes (p<0.0001) (Fig. 3C). In non-functional IgH genes, the identified VH replacement footprints also preferentially encode charged amino acids, although the usages of different charged residues are slightly different from those in the functional VH replacement products (Fig. 3D). Such results are consistent with previous findings that the VH replacement footprints identified in human or mouse VH replacement products preferentially encoded charged residues [31], [39].

Bottom Line: The frequencies of VH 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 VH 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 VH replacement products to the diversification of antibody repertoire and potentially, to the generation of autoantibodies in mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, United States of America.

ABSTRACT
VH replacement occurs through RAG-mediated recombination between the cryptic recombination signal sequence (cRSS) near the 3' end of a rearranged VH gene and the 23-bp RSS from an upstream unrearranged VH gene. Due to the location of the cRSS, VH replacement leaves a short stretch of nucleotides from the previously rearranged VH gene at the newly formed V-D junction, which can be used as a marker to identify VH replacement products. To determine the contribution of VH replacement products to mouse antibody repertoire, we developed a Java-based VH Replacement Footprint Analyzer (VHRFA) program and analyzed 17,179 mouse IgH gene sequences from the NCBI database to identify VH replacement products. The overall frequency of VH replacement products in these IgH genes is 5.29% based on the identification of pentameric VH replacement footprints at their V-D junctions. The identified VH replacement products are distributed similarly in IgH genes using most families of VH genes, although different families of VH genes are used differentially. The frequencies of VH 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 VH 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 VH replacement products to the diversification of antibody repertoire and potentially, to the generation of autoantibodies in mice.

Show MeSH
Related in: MedlinePlus