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Structural analysis of the VH-D-JH segments of human polyreactive IgG mAb. Evidence for somatic selection.

ABSTRACT: Polyreactive (natural) antibodies are primarily IgM and account for a major proportion of circulating Ig in humans. They use various V gene segments, in general, in germ line (unmutated) configuration. To analyze the VH regions of polyreactive antibodies, with particular attention at their somatically mutated status, we generated five IgG (three IgG1 and two IgG3) mAb (using B cells from a healthy subject, a patient with insulin-dependent diabetes mellitus and a patient with SLE), which bound with various efficiencies a number of different self and foreign Ag. Gene cloning experiments showed that the VH region sequences were unique to each IgG mAb. The H chain complementary determining region (CDR3) of two IgG (mAb10 and mAb426.4.2F20) displayed an identical stretch of five amino acids (RFLEW), but the other three IgG mAb CDR3 were divergent in both length and composition. The VH gene sequences of two IgG, mAb426.4.2F20 and mAb410.7.F91, were 99% identical to those of the germ line VH4.11 and VH4.21 genes, respectively. Those of the remaining three IgG mAb displayed a number of differences (93.6 to 95.9% identity) when compared with the germ line VH4.18, VH4.11, and hv1263 gene sequences. These and the VH4.21 gene have been found to encode polyreactive IgM and IgA and, in mutated configuration, monoreactive high affinity autoantibodies and antibodies induced by foreign Ag. When compared with the respective framework region, the CDR of three IgG mAb VH segment sequences displayed a significantly higher: 1) frequency of total nucleotide differences (6.1 x 10(-2) vs 4.5 x 10(-2) difference/base); 2) frequency of putative nucleotide changes yielding amino acid replacements (5.6 x 10(-2) vs 1.4 x 10(-2) replacement change/base); and 3) ratio of overall putative replacement to silent (R:S) mutations (11.0 vs 0.4). Thus, the distribution and nature of the nucleotide differences were consistent with a process of somatic mutation and Ag-dependent clonal selection. This was formally proved in IgG mAb426.12.3F1.4 and IgG mAb10 by differentially targeted polymerase chain reaction amplification and cloning and sequencing of the germ line genes that gave rise to the expressed VH segments, using DNA from polymorphonuclear cells of the same subjects whose B cells were used for the generation of these IgG mAb. Somatic mutations might have been responsible for bringing about polyreactivity in originally monoreactive antibodies or, more likely, they accumulated in originally polyreactive antibodies, which after undergoing a process of Ag selection, retained polyreactivity and may have or may have not acquired a higher affinity for the selecting Ag.

SUBMITTER: Ikematsu H

PROVIDER: S-EPMC4627373 | biostudies-literature | 1993 Oct

REPOSITORIES: biostudies-literature

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Structural analysis of the VH-D-JH segments of human polyreactive IgG mAb. Evidence for somatic selection.

Ikematsu H H Kasaian M T MT Schettino E W EW Casali P P

Journal of immunology (Baltimore, Md. : 1950) 19931001 7

Polyreactive (natural) antibodies are primarily IgM and account for a major proportion of circulating Ig in humans. They use various V gene segments, in general, in germ line (unmutated) configuration. To analyze the VH regions of polyreactive antibodies, with particular attention at their somatically mutated status, we generated five IgG (three IgG1 and two IgG3) mAb (using B cells from a healthy subject, a patient with insulin-dependent diabetes mellitus and a patient with SLE), which bound wi ...[more]

PMID: 8376796

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Project description:Anti-DNA IgA autoantibodies play an important immunopathologic role in SLE patients. To analyze the cellular origin and the VH and VL structure of anti-DNA IgA autoantibodies, we generated five IgA1 mAbs to DNA using B lymphocytes from three SLE patients. Two mAbs bound to ssDNA only and one to both ssDNA and dsDNA (monoreactive antibodies). The remaining two mAbs bound to DNA (one to ssDNA and the other to both ssDNA and dsDNA) and to other self and foreign Ag (polyreactive antibodies). The IgA mAb relative avidity for DNA ranged from 7.5 x 10(-8) to 8.0 x 10(-10) g/microliters. The anti-DNA IgA mAb used VH segments of the VHI(VI-3b), VHII (VH2-MC2), VHIII (WHG16G and VH26c), and VHIV (V71-2) families in conjunction with V kappa I, V kappa IIIb, or V lambda I segments. All IgA mAb VH segments were juxtaposed with JH4b segments. The heavy chain CDR3 sequences were divergent in composition and length. When compared with those of the closest reported germ line genes, the IgA mAb VH and VL gene sequences displayed a number of differences. That these differences represented somatic point mutations was formally proved in both the monoreactive IgA mAb 412.67.F1.3 and the polyreactive IgA mAb 412.66.F1 VH segments by differential PCR amplification and cloning and sequencing of genomic DNA from the mAb-producing cell lines and autologous polymorphonuclear cells. The sequences of the germ line genes that putatively gave rise to the mAb 412.67.F1.3 and mAb 412.66.F1 VH segments were identical with those of the WHG16G and VH26c genes, respectively. In not only the monoreactive mAb 412.67.F1.3 but also the polyreactive mAb 412.66.F1 and mAb 448.9G.F1 VH segments, the higher concentration of replacement (R) mutations and the higher R:S (silent) mutation ratios in the complementarity-determining region (infinity; 19:0) than in the framework region (1.0) (p = 0.00001, chi 2 test) were highly consistent with selection by Ag. In the five IgA mAb VH and VL segments, the putative and verified somatic point mutations yielded 68 amino acid replacements, of which 38 were nonconserved. Twenty of these yielded positively charged or polar residues that play a major role in DNA binding, including seven Arg, five Lys, three Tyr, two Gln, two His, and a Thr. The conserved amino acid changes included seven Asn. These findings suggest that anti-DNA IgA autoantibodies use a broad selection of VH and VL genes and enhance their fit for Ag by undergoing somatic hypermutation and Ag selection.(ABSTRACT TRUNCATED AT 400 WORDS)

Project description:B lymphocytes committed to the production of antibodies binding to antigens on pathogenic bacteria and viruses (natural antibodies) are common components of the normal human B cell repertoire. A major proportion of natural antibodies is capable of binding multiple antigens (polyreactive antibodies). Using B cells from three HIV-1 seronegative healthy subjects, and purified HIV-1 and beta-galactosidase from Escherichia coli as selecting antigen, we generated three natural IgM mAb to HIV-1 and a natural IgM mAb to beta-galactosidase. The three HIV-1-selected antibodies (mAb102, mAb103, and mAb104) were polyreactive. They bound with different affinities (Kd = 10(-6) to 10(-8) M) to the HIV-1 envelope gp160, the p24 core protein, and the p66 reverse transcriptase, but not to the 120 glycosylated env protein. They also bound to beta-galactosidase (Kd approximately 10(-7) M), tetanus toxoid, and various various self antigens. In contrast, the natural mAb selected for binding to beta-galactosidase (mAb207.F1) was monoreactive, in that it bound with a high affinity (Kd < 10(-8) M) to this antigen, but to none of the other antigens tested, including HIV-1. Structural analysis of the VH and VL segments revealed that the natural mAb utilized three segments of the VHIV gene family and one of the VHIII family, in conjunction with VL segments of the V lambda I, V lambda II, V lambda III, or V kappa IV subgroups. In addition, the natural mAb VH and VL segments were in unmutated or virtually unmutated (germline) configuration, including those of the monoreactive mAb207.F1 to beta-galactosidase, and were identical or closely related to those utilized by specific autoantibodies or specific antibodies to viral and/or bacterial pathogens. Thus, the present data show that both polyreactive and monoreactive natural antibodies to foreign antigen can be isolated from the normal human B cell repertoire. They also suggest that the VH and VL segments of not only polyreactive but also monoreactive natural antibodies can be encoded in unmutated or minimally mutated genes, and possibly provide the templates for the specific high affinity antibodies elicited by self or foreign antigens.

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Dataset Information

Structural analysis of the VH-D-JH segments of human polyreactive IgG mAb. Evidence for somatic selection.

Publications

Structural analysis of the VH-D-JH segments of human polyreactive IgG mAb. Evidence for somatic selection.

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