Regulation of Repertoire Development through Genetic Control of DH Reading Frame Preference

Abstract

In jawed vertebrates most expressed Ig H chains use only one of six possible DH reading frames. Reading frame (RF)1, the preferred reading frame, tends to encode tyrosine and glycine, whereas the other five RFs tend to be enriched for either hydrophobic or charged amino acids. Mechanisms proposed to favor use of RF1 include a preference for deletion over inversion that discourages use of inverted RF1, RF2, and RF3; sequence homology between the 5′ terminus of the JH and the 3′ terminus of the DH that promotes rearrangement into RF1; an ATG start site upstream of RF2 that permits production of a truncated Dμ protein; stop codons in RF3; and, following surface expression of IgM, somatic, presumably Ag receptor-based selection favoring B cells expressing Igs with tyrosine- and glycine-enriched CDR-H3s. By creating an IgH allele limited to the use of a single, frameshifted DFL16.1 DH gene segment, we tested the relative contribution of these mechanisms in determining reading frame preference. Dμ-mediated suppression via an allelic exclusion-like mechanism dominated over somatic selection in determining the composition of the CDR-H3 repertoire. Evidence of somatic selection for RF1-encoded tyrosine in CDR-H3 was observed, but only among the minority of recirculating, mature B cells that use DH in RF1. These observations underscore the extent to which the sequence of the DH acts to delimit the diversity of the Ab repertoire.

Conservation and diversity in the ultralong third heavy-chain complementarity-determining region of bovine antibodies

“…These amino acids are also found frequently in human and murine CDR H3 regions (3, 39), but in a survey of the complete UniProtKB/SwissProt sequence database as of February 2016 (http://web.expasy.org/docs/relnotes/relstat.html), frequencies for these particular amino acids in all available protein sequences are 2.9% (Tyr), 7.1% (Gly), and 6.6% (Ser), so that they are indeed over-represented in these ultralong CDR H3s. Synthetic libraries utilizing antibody scaffolds as well as other protein folds have been developed using very small subsets of amino acids as recognition elements, with tyrosine, serine, and glycine proving the best minimal combination (40) for effective antigen recognition.…”

“…The ultralong CDR H3s contain a large, usually even, number of cysteine residues (from 2–12) (1–2). The longest CDR H3 regions found to date in the human germline repertoire contain up to 35 residues (3), while camelid single-chain antibodies have up to 24 residues (4) and shark IgNAR antibodies up to 27 residues (5). While CDR H3 cysteines are rare in humans and mice, they are found more extensively in sharks and camelids, and tether the long CDR H3s to the framework region or to a neighboring CDR.…”

Plasma Cells and Nonplasma B Cells Express Differing IgE Repertoires in Allergic Sensitization

“…Ig sequences were numbered according to the ImMunoGeneTics unique numbering (23). A minimum of six nonmutated nucleotides with at least two nonmutated nucleotides at each end was required to identify a D gene (24). The somatic mutation rate was calculated by dividing the sum of mutations in CDR-H1, framework region (FR)-H2, CDR-H2, and FR-H3 (compared with the germline V H gene) by the total number of nucleotides.…”

Partial versus Productive Immunoglobulin Heavy Locus Rearrangements in Chronic Lymphocytic Leukemia: Implications for B-Cell Receptor Stereotypy

“…The biological mechanism, which determines whether a partial IGHD-IGHJ rearrangement will develop into an IGHV-IGHD-IGHJ rearrangement is still unknown. Studies in the mouse have shown a biased usage of IGHD genes in reading frames (RFs) encoding for hydrophilic amino acids in IGHV-IGHD-IGHJ rearrangements (V-D-J) along with counter-selection of IGHD-IGHJ rearrangements with IGHD genes in RF encoding for hydrophobic amino acids or stop codons (13)(14)(15)(16)(17)(18) . Relevant studies have also shown that IGHD-IGHJ with IGHD genes rearranged in RF encoding for hydrophobic amino acids can be translated into a truncated μ protein, called Dμ (13,19).…”