Evolution of immunogenetic components encoding ultralong CDR H3

Abstract

The genomes of most vertebrates contain many V, D, and J gene segments within their Ig loci to construct highly variable CDR3 sequences through combinatorial diversity. This nucleotide variability translates into an antibody population containing extensive paratope diversity. Cattle have relatively few functional VDJ gene segments, requiring innovative approaches for generating diversity like the use of ultralong-encoding IGHV and IGHD gene segments that yield dramatically elongated CDR H3. Unique knob and stalk microdomains create protracted paratopes, where the antigen-binding knob sits atop a long stalk, allowing the antibody to bind both surface and recessed antigen epitopes. We examined genomes of twelve species of Bovidae to determine when ultralong-encoding IGHV and IGHD gene segments evolved. We located the 8-bp duplication encoding the unique TTVHQ motif in ultralong IGHV segments in six Bovid species (cattle, zebu, wild yak, domestic yak, American bison, and domestic gayal), but we did not find evidence of the duplication in species beyond the Bos and Bison genera. Additionally, we analyzed mRNA from bison spleen and identified a rich repertoire of expressed ultralong CDR H3 antibody mRNA, suggesting that bison use ultralong IGHV transcripts in their host defense. We found ultralong-encoding IGHD gene segments in all the same species except domestic yak, but again not beyond the Bos and Bison clade. Thus, the duplication event leading to this ultralong-encoding IGHV gene segment and the emergence of the ultralong-encoding IGHD gene segment appears to have evolved in a common ancestor of the Bos and Bison genera 5-10 million years ago.

Keywords: Antibody; Cattle; Diversity segment; Evolution; IgH locus; Ultralong CDR H3.

Fig. 1

Structural model illustrating the stalk and knob structure characteristic of cattle ultralong antibodies. a The linear form of an ultralong antibody heavy chain variable region with the gene segments color coded (variable, V: green, diversity, D: yellow; joining, J: blue). The portions of the stalk and knob that correspond to these segments are denoted above the segments. b A crystal structure of the bovine ultralong antibody (PDB:6E9H; Dong et al. 2019) with the V, D, and J segments shaded as in a. c The same crystal structure illustrating the heavy and light chains (heavy chain: cyan; light chain: magenta), with the knob and stalk domains annotated

Fig. 2

Germline IGHV gene segments are highly conserved in bison. a Amino acid alignment of bison (Bison bison) immunoglobulin heavy chain variable (IGHV) gene segments illustrating extensive similarity between functional bison sequences. We compare bison ultralong IGHV1-07 and a typical IGHV1-10 to similar sequences from cattle (Bos taurus). The V-segment ultralong motif (TTVHQ) is boxed in green. Shading within the alignment indicates amino acid conservation based on a Blosum62 scoring matrix (threshold = 1, gaps ignored; highlights indicate similarity: black = 100% similar; dark gray = 80–100% similar; light gray = 60–80% similar; white =  < 60% similar). Values to the right of the alignment show the percent nucleotide identity to the first sequence. Highlighting within the scale indicates leader peptides (part 1—light gray; part 2—dark gray), framework regions (FR, blue), and complementarity-determining regions (CDR, red). Gaps within a sequence are for alignment purposes only. b Nucleotide alignments of ultralong IGHV1-07 and typical IGHV1-10 demonstrate similarity between sequences and hotspot locations for activation-induced cytidine deaminase (AID), which mediates somatic hypermutation of B cell receptors during affinity maturation. Values to the right of the alignments show the percent nucleotide identity of the bison sequence to the cattle sequence. Nonsynonymous nucleotide differences between sequences are highlighted in black, and synonymous nucleotide differences are underlined. Shading within the alignments represents locations of the mutable base (G or C nucleotides) within AID-preferred hotspot motifs (DGYW/WRCH: G:C is the mutable position; D = A/G/T, Y = C/T, W = A/T, R = A/G, and H = T/C/A). Highlighting within the scale is as for a

Fig. 3

Germline IGHD gene segments in bison vary greatly in length. a Amino acid and nucleotide sequence alignments of bison (Bison bison) immunoglobulin heavy chain diversity (IGHD/ IGHD) gene segments. We compare the bison and cattle (Bos taurus) ultralong IGHD8-2 gene segments. Values to the right of the alignments show the number of amino acids (top) or nucleotides (bottom) in each gene segment. Differences between cattle and bison ultralong IGHD sequences are highlighted in yellow. IGHD2, 3, 5, and 9 contain multiple, identical germline sequences and are represented by a single consensus of each segment with the number of sequences in parentheses. b Nucleotide and amino acid sequences of germline ultralong IGHD8-2 gene segments from cattle (top) and bison (bottom). Nucleotides that can be altered with a single base change to a codon that encodes a cysteine (C) residue are colored red. Boxed areas indicate locations of AID-preferred hotspot motifs DGYW or WRCH (G:C is the mutable position; D = A/G/T, Y = C/T, W = A/T, R = A/G, and H = T/C/A). Canonical cysteine residues are highlighted in yellow. Red amino acids illustrate the large fraction of codons that can be mutated to cysteine. (b is modified from Stanfield et al. 2018)

Fig. 4

Germline IGHV segment genes are highly conserved within Bovidae. a We obtained Bovidae IGHV germline gene segments (exon 2) from eleven extant species representing eight Bovidae genera that are most identical to the ultralong IGHV of Bos taurus (cattle). Sequences were ordered phylogenetically (see also Fig. 5) and aligned to the cattle ultralong IGHV (IGHV1-07). We also found ultralong IGHV from auroch (Bos primigenius), the extinct ancestor of Bos taurus and Bos indicus. The cattle amino acid sequence is shown above the alignment for orientation. Dots indicate nucleotide identity to the cattle sequence, while letters indicate disagreements (nonsynonymous base changes are highlighted in black, and synonymous changes are underlined). The germline sequence that is duplicated in ultralong IGHV is outlined by a red box, and the 8-bp duplication within the CTTVHQ motif is highlighted in yellow. Gaps within a sequence are for alignment purposes only. Percent nucleotide identity to the cattle ultralong sequence is shown to the right of the alignment. A model cladogram is provided for reference. b Amino acid sequence alignment of the same twelve IGHV germline gene segments reveals tremendous amino acid conservation. Shading within the alignment indicates amino acid conservation based on a Blosum62 scoring matrix (threshold = 1, gaps ignored; highlights indicate similarity: black = 100% similar; dark gray = 80–100% similar; light gray = 60–80% similar; white =  < 60% similar). Values to the right of the alignment show the percent amino acid identity to the cattle ultralong IGHV gene segment. In both alignments, highlighting within the scale indicates peptides within the leader (pink), framework regions (FR, blue), or complementarity-determining regions (CDR, green) of the immunoglobulin variable region. Accession numbers and/or genomic locations can be found in Supplemental Table 1

Fig. 5

The evolutionary event leading to the ultralong-encoding IGHV and IGHD gene segments occurred in a common ancestor of the Bos and Bison group. A phylogenetic tree of the major clades within the Bovidae family represents eight genera. The 8-bp duplication event that led to the evolution of the ultralong IGHV gene segment arose at the base of the Bos and Bison group, indicated by the circle and clade colored purple. The amino acid length of the longest known IGHD (which forms the knob structure in cattle ultralong antibodies) is shown to the right of each branch next to a line drawing of each species (see Supplementary Table 1). The origin of ultralong-encoding IGHD gene segments (colored red) likely coincides with the origin of the ultralong-encoding IGHV. We did not find elongated IGHD segments in domestic yak (Bos grunniens), and we could not locate any IGHD segments in Barbary sheep (Ammotragus lervia), though incomplete genome assemblies limited our exploration. An approximate evolutionary timeline is provided below the tree (MYA, million years ago)

Fig. 6

Maximum length of germline IGHD gene segment is highly variable. a Nucleotide alignment of the longest known diversity (IGHD) gene segment from nine species of the Bovidae family. Germline sequences were ordered by maximum IGHD length and aligned to the Bos taurus (cattle) ultralong IGHD (IGHD1-08) sequence. The cattle amino acid sequence is shown above the alignment for orientation. Recombination signal sequences (RSS) are highlighted (dark grey: nonamer; light grey: heptamer; with a 12-bp spacer in between). Dots indicate nucleotide identity to the cattle sequence, while letters indicate disagreements. Gaps within a sequence are for alignment purposes only. Percent nucleotide identity to the cattle ultralong sequence is shown to the right of the alignment. A model cladogram is provided for reference. b Amino acid alignment of the same nine IGHD germline gene segments illustrates the variability in maximum length between species. Canonical cysteine residues are highlighted in yellow. Values to the right of the alignment show the percent amino acid identity to the cattle ultralong IGHD gene segment (%) and the amino acid length of the sequence (length), and values colored red indicate ultralong-encoding IGHD

Fig. 7

Modeled structures of bison ultralong IgH antibodies reflect the same knob diversity as seen in cattle. a Mutation within bison (Bison bison) IGHD usually results in an even number of cysteine (C) residues within the IGHD-encoded knob structure, permitting disulfide (C = C) bonds to form. b Modeled bison amplicon sequences aligned with cattle template sequences for each model [1–4]. Conserved cysteine (C) and tryptophan (W) residues at IMGT positions 104 and 118, respectively (highlighted in gray), delineate the boundaries of the CDR H3. Canonical cysteine residues within the CDR H3 are highlighted yellow. The scale is colored as follows: V segment to the cysteine at position 104 of the YYC motif (cyan), TTVHQ motif (red), D segment (green), J segment up to the tryptophan at position 118 of the WGxG motif (magenta), remaining J segment (blue), constant region (gray). c Bison ultralong IgG amplicons modeled against cattle templates illustrate the diversity of knob regions with decreasing numbers of cysteines (1: eight Cs; 2: six Cs; 3: four Cs; 4: two Cs). For each pair of structures, both the cattle template (left) and the bison amplicon sequence (right) contain the same number of cysteines within the IGHD-encoded knob. Disulfide bonds and the paired cysteine residues are colored yellow. Either the PDB entry ID# (cattle templates; protein data base, rcsb.org) or amplicon number (bison IgG sequence) are shown next to each model. Amplicon sequences are colored according to the scale (see b) to indicate gene segment and residue position. Values to the right of the sequences indicate the total number of cysteines within CDR H3. For a more detailed summary of templates, see results