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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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Enrichment of VH replacement products in IgH genes derived from different strains of autoimmune prone mice and IgH genes encoding autoantibodies.The frequencies of VH replacement products in IgH genes derived from different strains of mice were analyzed using the VHRFA program based on the keyword linked to each IgH gene in the NCBI database. VH replacement products were assigned based on the identification of (A) 5-mer VH replacement footprints, (B) 4-mer VH replacement footprints, or (C) 3-mer VH replacement footprints within the VH-DH junctions (N1 regions). The frequencies of VH replacement products in different subcategories were compared with that in the BALB/c mice. n, number of IgH sequences in each subcategory. Statistical significance was determined using a two-tailed Chi square test with Yate’s correction. p<0.05 (*) is considered significant and p<0.0001 (**) is considered extremely significant. The detailed sequence analysis and the identified VH replacement products with 5-mer VH replacement footprints correlating with keywords are included in Table S6.
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pone-0057877-g002: Enrichment of VH replacement products in IgH genes derived from different strains of autoimmune prone mice and IgH genes encoding autoantibodies.The frequencies of VH replacement products in IgH genes derived from different strains of mice were analyzed using the VHRFA program based on the keyword linked to each IgH gene in the NCBI database. VH replacement products were assigned based on the identification of (A) 5-mer VH replacement footprints, (B) 4-mer VH replacement footprints, or (C) 3-mer VH replacement footprints within the VH-DH junctions (N1 regions). The frequencies of VH replacement products in different subcategories were compared with that in the BALB/c mice. n, number of IgH sequences in each subcategory. Statistical significance was determined using a two-tailed Chi square test with Yate’s correction. p<0.05 (*) is considered significant and p<0.0001 (**) is considered extremely significant. The detailed sequence analysis and the identified VH replacement products with 5-mer VH replacement footprints correlating with keywords are included in Table S6.

Mentions: To explore the biological significance of VH replacement in mouse, we analyzed the distribution of VH replacement products in IgH genes correlating with different keywords in the NCBI database. Based on the identification of 5-mer VH replacement footprints within the N1 regions, the frequencies of VH replacement products in IgH genes derived from C57BL/6 and BALB/c strains of mice are 3.17% and 5%, respectively (Fig. 2A and Table S6). Such numbers may serve as the basal levels of VH replacement products in these mice. Comparing IgH genes derived from several strains of mice, the frequencies of VH replacement products are highly elevated in IgH genes derived from different strains of autoimmune prone mice (Fig. 2A). In particular, the frequencies of VH replacement product are elevated in IgH genes derived from lupus prone NZB/NZW F1, NZM2410, MRL/lpr, and SLE1/SLE3 mice. In IgH genes derived from mice carrying the spontaneous Faslpr mutation (MRL/MpJ-Lpr/Lpr), the frequency of VH replacement products is 15.38%. In IgH genes from the Sle1/Sle3 mice, the frequency of VH replacement products is 30%. These frequencies are significantly higher than that in the BALB/c or C57BL/6 mice (p<0.05, two tailed Chi-square test) (Fig. 2A). The elevated levels of VH replacement products in autoimmune prone mice suggest that VH replacement products contribute to the generation of autoantibodies. Indeed, further analyses of the IgH genes encoding different antibodies showed that the frequencies of VH replacement products are 12.1% in IgH genes encoding ANA antibody and 9.34% in IgH genes encoding anti-DNA antibodies. These levels are significantly higher than those in the BALB/c or C57BL/6 mice. As a negative control, the frequency of VH replacement products in IgH genes obtained from mice immunized with NP is 3.66%, which is similar to that in the C57BL/6 mice. Taken together, these results provide the first information that VH replacement products are highly enriched in IgH genes derived from different strains of autoimmune prone mice and in IgH genes encoding anti-DNA and ANA autoantibodies.


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)

Enrichment of VH replacement products in IgH genes derived from different strains of autoimmune prone mice and IgH genes encoding autoantibodies.The frequencies of VH replacement products in IgH genes derived from different strains of mice were analyzed using the VHRFA program based on the keyword linked to each IgH gene in the NCBI database. VH replacement products were assigned based on the identification of (A) 5-mer VH replacement footprints, (B) 4-mer VH replacement footprints, or (C) 3-mer VH replacement footprints within the VH-DH junctions (N1 regions). The frequencies of VH replacement products in different subcategories were compared with that in the BALB/c mice. n, number of IgH sequences in each subcategory. Statistical significance was determined using a two-tailed Chi square test with Yate’s correction. p<0.05 (*) is considered significant and p<0.0001 (**) is considered extremely significant. The detailed sequence analysis and the identified VH replacement products with 5-mer VH replacement footprints correlating with keywords are included in Table S6.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585286&req=5

pone-0057877-g002: Enrichment of VH replacement products in IgH genes derived from different strains of autoimmune prone mice and IgH genes encoding autoantibodies.The frequencies of VH replacement products in IgH genes derived from different strains of mice were analyzed using the VHRFA program based on the keyword linked to each IgH gene in the NCBI database. VH replacement products were assigned based on the identification of (A) 5-mer VH replacement footprints, (B) 4-mer VH replacement footprints, or (C) 3-mer VH replacement footprints within the VH-DH junctions (N1 regions). The frequencies of VH replacement products in different subcategories were compared with that in the BALB/c mice. n, number of IgH sequences in each subcategory. Statistical significance was determined using a two-tailed Chi square test with Yate’s correction. p<0.05 (*) is considered significant and p<0.0001 (**) is considered extremely significant. The detailed sequence analysis and the identified VH replacement products with 5-mer VH replacement footprints correlating with keywords are included in Table S6.
Mentions: To explore the biological significance of VH replacement in mouse, we analyzed the distribution of VH replacement products in IgH genes correlating with different keywords in the NCBI database. Based on the identification of 5-mer VH replacement footprints within the N1 regions, the frequencies of VH replacement products in IgH genes derived from C57BL/6 and BALB/c strains of mice are 3.17% and 5%, respectively (Fig. 2A and Table S6). Such numbers may serve as the basal levels of VH replacement products in these mice. Comparing IgH genes derived from several strains of mice, the frequencies of VH replacement products are highly elevated in IgH genes derived from different strains of autoimmune prone mice (Fig. 2A). In particular, the frequencies of VH replacement product are elevated in IgH genes derived from lupus prone NZB/NZW F1, NZM2410, MRL/lpr, and SLE1/SLE3 mice. In IgH genes derived from mice carrying the spontaneous Faslpr mutation (MRL/MpJ-Lpr/Lpr), the frequency of VH replacement products is 15.38%. In IgH genes from the Sle1/Sle3 mice, the frequency of VH replacement products is 30%. These frequencies are significantly higher than that in the BALB/c or C57BL/6 mice (p<0.05, two tailed Chi-square test) (Fig. 2A). The elevated levels of VH replacement products in autoimmune prone mice suggest that VH replacement products contribute to the generation of autoantibodies. Indeed, further analyses of the IgH genes encoding different antibodies showed that the frequencies of VH replacement products are 12.1% in IgH genes encoding ANA antibody and 9.34% in IgH genes encoding anti-DNA antibodies. These levels are significantly higher than those in the BALB/c or C57BL/6 mice. As a negative control, the frequency of VH replacement products in IgH genes obtained from mice immunized with NP is 3.66%, which is similar to that in the C57BL/6 mice. Taken together, these results provide the first information that VH replacement products are highly enriched in IgH genes derived from different strains of autoimmune prone mice and in IgH genes encoding anti-DNA and ANA autoantibodies.

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