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Exceptionally long CDR3H are not isotype restricted in bovine immunoglobulins.

ABSTRACT: Exceptionally long third complementarity determining regions of the heavy chain (CDR3H) were previously described as a specificity of bovine IgG and IgM immunoglobulins. In addition, the genomic organization of the immunoglobulin heavy chain locus remains to be elucidated with a special focus on the number of variable segments (IGHV). By analyzing the variable regions according to the isotype-specific PCR using cDNA-PCR, we were able to prove the existence of exceptional long CDR3H in all bovine isotypes. The corresponding sequences of three distinct amplicons were grouped according to the length of the CDR3H. Sequences of CDR3H possessed 5 to 10, 12 to 31 or at least 48 amino acid residues. Long and mid-length CDR3H were composed of mainly hydrophilic amino acid residues, while short CDR3H also contained hydrophobic amino acid residues. All sequences with long CDR3H were related to the germline variable segment 10. Using the current genome assembly, Bos taurus NCBI build 6.1, the genomic organization of the bovine immunoglobulin heavy-chain locus was analyzed. A main locus was investigated on BTA21. Exons coding for variable, diversity, and joining segments, as well as for the constant regions of different isotypes, were also localized on BTA7, BTA8, and BTA20. Together with the information from unplaced contigs, 36 IGHV were detected of which 13 are putatively functional. Phylogenetic analysis revealed two bovine IGHV families (boVH1, boVH2). Thus, the existence of the two bovine families suggested was demonstrated, where boVH1 comprises all functional segments. This study substantially improves the understanding of the generation of immunoglobulin diversity in cattle.

SUBMITTER: Walther S

PROVIDER: S-EPMC3661452 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Exceptionally long CDR3H are not isotype restricted in bovine immunoglobulins.

Walther Stefanie S Czerny Claus-Peter CP Diesterbeck Ulrike S US

PloS one 20130522 5

Exceptionally long third complementarity determining regions of the heavy chain (CDR3H) were previously described as a specificity of bovine IgG and IgM immunoglobulins. In addition, the genomic organization of the immunoglobulin heavy chain locus remains to be elucidated with a special focus on the number of variable segments (IGHV). By analyzing the variable regions according to the isotype-specific PCR using cDNA-PCR, we were able to prove the existence of exceptional long CDR3H in all bovine ...[more]

PMID: 23717573

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Project description:Mass spectrometry (MS) has become an indispensable tool in structural characterization and quality control of monoclonal antibodies (mAbs). Intact-mass analysis is a particularly attractive option that provides a powerful and cost-effective means to not only confirm the structural integrity of the protein, but also probe its interactions with therapeutic targets. To a certain extent, this success can be attributed to relatively modest glycosylation levels exhibited by IgG molecules, which limits their structural heterogeneity and enables straightforward mass measurements at the intact molecule level. The recent surge of interest in expanding the repertoire of mAbs to include other classes of immunoglobulins places a premium on efforts to adapt the IgG-tailored experimental strategies to other classes of antibodies, but their dramatically higher levels of glycosylation may create insurmountable obstacles. The monoclonal murine IgE antibody explored in this work provides a challenging model system, as its glycosylation level exceeds that of conventional IgG mAbs by a factor of nine. The commercial sample, which included various IgE fragments, yields a poorly resolved ionic signal in intact-mass measurements, from which little useful information can be extracted. However, coupling MS measurements with the limited charge reduction of select polycationic species in the gas phase gives rise to well-defined charge ladders, from which both ionic masses and charges can be readily determined. The measurements reveal significant variation of the extent of glycosylation within intact IgE molecules, as well as the presence of low-molecular weight impurities in the commercial IgE sample. Furthermore, incubation of the monoclonal IgE with its antigen (ovalbumin) gives rise to the formation of complexes with varying stoichiometries, which can also be uniquely identified using a combination of native MS, limited charge reduction in the gas phase and data fitting procedures. This work demonstrates that following appropriate modifications, intact-mass analysis measurements can be successfully applied to mAbs beyond the IgG isotype, providing a wealth of information not only on the mass distribution of the intact IgE molecules, but also their large-scale conformational integrity, the integrity of their covalent structure, and their interactions with antigens.

Dataset Information

Exceptionally long CDR3H are not isotype restricted in bovine immunoglobulins.

Publications

Exceptionally long CDR3H are not isotype restricted in bovine immunoglobulins.

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