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Physical map of the bovine IgH gene locus. Open boxes indicate the IGHV pseudogenes. H, hinge region-encoding exon; IGHDP1, IGHDP2, IGHDP3, the pseudo-Ig C region H chain d genes; M, membrane regionencoding exon.
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It has been suspected for many years that cattle possess two functional IgH gene loci, located onBos taurusautosome (BTA) 21 and BTA11, respectively. In this study, based on fluorescence in situ hybridization and additional experiments, we showed that all functional bovine IgH genes were located on BTA21, and only a truncated μCH2 exon was present...
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... that previous investigations were incomplete. To map the entire IGHC locus, we selected seven IgH gene-positive BAC clones (RP42-195P14, 49A20, 498B11, 567N23, 90B11, 4E14, and INRA-944D11) for sequencing, which generated a 678-kb contiguous genomic sequence covering part of the IGHV region and IGHD, IGHJ, and IGHC genes in their entirety (Fig. ...
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... expression, we generated specific nested PCR primers for the amplification of the recombined Sm1-Sm2, Sm1-Sg1, and Sm2-Sg1 fragments (Fig. 4A). Using spleen genomic DNA as a template, we amplified the desired recombination products with these primers. A total of 13 unique Sm1-Sm2, 12 Sm1-Sg1, and 20 Sm2-Sg1 recombined fragments were sequenced (Fig. 4B, Supplemental Fig. 2), which clearly demonstrated that the bovine IGHM2 gene could also be expressed through a CSR ...
Citations
... Pairing between HCs and LCs from different germline segments creates additional variation in antibody repertoires (Jayaram et al., 2012). In cows, there are 12 IGHV, 16 IGHD, and 4 IGHJ functional segments (Deiss et al., 2019;Ma et al., 2016). Interestingly, a specific combination of V, D, and J segments (IGHV1-7, IGHD8-2, IGHJ2-4) is utilized in the HCs of antibodies with ultra-long CDRs (Deiss et al., 2019;Li et al., 2023;Stanfield et al., 2016). ...
The antigen‐binding sites in conventional antibodies are formed by hypervariable complementarity‐determining regions (CDRs) from both heavy chains (HCs) and light chains (LCs). A deviation from this paradigm is found in a subset of bovine antibodies that bind antigens via an ultra‐long CDR. The HCs bearing ultra‐long CDRs pair with a restricted set of highly conserved LCs that convey stability to the antibody. Despite the importance of these LCs, their specific features remained unknown. Here, we show that the conserved bovine LC found in antibodies with ultra‐long CDRs exhibits a distinct combination of favorable physicochemical properties such as good secretion from mammalian cells, strong dimerization, high stability, and resistance to aggregation. These physicochemical traits of the LCs arise from a combination of the specific sequences in the germline CDRs and a lambda LC framework. In addition to understanding the molecular architecture of antibodies with ultra‐long CDRs, our findings reveal fundamental insights into LC characteristics that can guide the design of antibodies with improved properties.
... In addition to inter-individual haplotype variation, the IG loci across species are enriched with segmental duplications (SDs) [33,[38][39][40]. SD sequences are known blind-spots when mapping and analyzing short-read sequencing data [41,42] due to poor mappability. ...
The expressed antibody repertoire is a critical determinant of immune-related phenotypes. Antibody-encoding transcripts are distinct from other expressed genes because they are transcribed from somatically rearranged gene segments. Human antibodies are composed of two identical heavy and light chain polypeptides derived from genes in the immunoglobulin heavy chain (IGH) locus and one of two light chain loci. The combinatorial diversity that results from antibody gene rearrangement and the pairing of different heavy and light chains contributes to the immense diversity of the baseline antibody repertoire. During rearrangement, antibody gene selection is mediated by factors that influence chromatin architecture, promoter/enhancer activity, and V(D)J recombination. Interindividual variation in the composition of the antibody repertoire associates with germline variation in IGH, implicating polymorphism in antibody gene regulation. Determining how IGH variants directly mediate gene regulation will require integration of these variants with other functional genomic datasets. Here, we argue that standard approaches using short reads have limited utility for characterizing regulatory regions in IGH at haplotype-resolution. Using simulated and ChIP-seq reads, we define features of IGH that limit use of short reads and a single reference genome, namely 1) the highly duplicated nature of DNA sequence in IGH and 2) structural polymorphisms that are frequent in the population. We demonstrate that personalized diploid references enhance performance of short-read data for characterizing mappable portions of the locus, while also showing that long-read profiling tools will ultimately be needed to fully resolve functional impacts of IGH germline variation on expressed antibody repertoires.
... As one of the most important livestock, cattle have made important historical contributions and are continuing to contribute to our understanding of the basic and applied immunology [15][16][17][18] . Recent studies on the gene structure of bovine immunogenomic loci have significantly advanced our understanding of the bovine immune-genome, such as MHC 19,20 , IG 21,22 and TR [23][24][25][26] . Interestingly, the long third heavy chain complementary determining regions (CDRH3) in cattle are capable of rapidly generating broad-neutralizing antibodies against human immunodeficiency virus (HIV) 27 . ...
... In NCBA_BosT1.0, the IGH locus contained 48 IGHV genes (11 functional) belonging to 3 IGHV subgroups as well as 17 IGHD (all functional), 12 IGHJ (3 functional) and 10 IGHC (8 functional) genes (Fig. 2b). A previous study assembled the IGH locus by sequencing seven BAC clones and generated an IGH gene structure containing three tandem [IGHDP-IGHV3-(IGHDv) 5/6 ] repeats 21 . In contrast, we observed only two tandem repeats [IGHDP-IGHV3-(IGHDv) 5/6 ] in the IGH locus of NCBA_BosT1.0, and the repeat regions as well as the adjacent gene loci were fully covered with multiple ultra-long reads greater than 100 Kb (Supplementary Fig. 9b, and Supplementary Fig. 10). ...
Immunogenomic loci remain poorly understood because of their genetic complexity and size. Here, we report the de novo assembly of a cattle genome and provide a detailed annotation of the immunogenomic loci. The assembled genome contains 143 contigs (N50 ~ 74.0 Mb). In contrast to the current reference genome (ARS-UCD1.2), 156 gaps are closed and 467 scaffolds are located in our assembly. Importantly, the immunogenomic regions, including three immunoglobulin (IG) loci, four T-cell receptor (TR) loci, and the major histocompatibility complex (MHC) locus, are seamlessly assembled and precisely annotated. With the characterization of 258 IG genes and 657 TR genes distributed across seven genomic loci, we present a detailed depiction of immune gene diversity in cattle. Moreover, the MHC gene structures are integrally revealed with properly phased haplotypes. Together, our work describes a more complete cattle genome, and provides a comprehensive view of its complex immune-genome.
... Moreover, multiple IgM subclasses have been discovered in some tetrapods; for instance, crocodilians, including Crocodylus siamensis and Alligator sinensis, have three IgM subclasses, named IgM1, IgM2, and IgM3, among which IgM2 and IgM3 H chain genes (Igl2 and Igl3) are expressed through class-switch recombination (20). Cattle (Bos taurus) have two functional IgM subclasses, IgM1 and IgM2; Igl1 and Igl2 can be expressed via independent VDJC recombination, whereas Igl2 can also be expressed through classswitch recombination (21). Unlike tetrapods, which possess at least four Ig classes, teleost fish have only three Ig classes, IgM, IgD, and IgT, among which IgM plays the dominant role in the systemic immune responses (5). ...
... The duplication and diversification of Ig classes or subclasses in vertebrates should have occurred in a long evolutionary period under environmental selection pressures, thus conferring survival advantages (53). As the most ancient and evolutionarily conserved Ig class, IgM has subclasses in some vertebrates ranging from mammals to fish, such as cattle (21), bird (Struthio camelus) (54), crocodilians (20), lungfish (Protopterus dolloi and Protopterus annectens) (23), and Atlantic salmon (55). In this study, we found that grass carp contains three IgMs, and a large frequency (>33%) of IgM 1 B cells coexpressed multiple IgM subclass genes, in a manner similar to that reported in rainbow trout IgT1, IgT2, and/or IgT3 1 B cells (19). ...
Teleost B cells are primitive lymphocytes with both innate and adaptive immune functions. However, the heterogeneity and differentiation trajectory of teleost B cells remain largely unknown. In this study, the landscape of grass carp IgM+ (gcIgM+) B cells was revealed by single-cell RNA sequencing. The results showed that gcIgM+ B cells mainly comprise six populations: (im)mature B cells, innate B cells, proliferating B cells, plasma cells, CD22+ cells, and CD34+ cells, among which innate B cells and proliferating B cells were uncommon B cell subsets with, to our knowledge, new characteristics. Remarkably, three functional IgMs were discovered in grass carp, and a significant percentage of gcIgM+ B cells, especially plasma cells, expressed multiple Igμ genes (Igμ1, Igμ2, and/or Igμ3). More importantly, through single-cell sorting combined with Sanger sequencing, we found that distinct VHDJH recombination patterns of Igμ genes were present in single IgM+ B cells, indicating that individual teleost B cells might produce multiple Abs by coexpressing rearranged IgM subclass genes. Moreover, the percentage of IgM1highIgM2highIgM3high plasma cells increased significantly after bacterial infection, suggesting that individual plasma cells might tend to produce multiple IgMs to resist the infection in teleost fish. In summary, to our knowledge, this study not only helps to uncover the unique heterogeneity of B cells in early vertebrates but also provided significant new evidence supporting the recently proposed "one cell-multiple Abs" paradigm, challenging the classical rule of "one cell-one Ab."
... Cattle antibodies have several unusual characteristics compared to most other species. They have relatively few functional V H gene segments (12) that are over 90% identical to one another [13]. The diversity of the circulating repertoire is driven through the combinatorial and imprecise assembly of V(D)J gene segments followed by extensive antigen-independent somatic hypermutation of the variable region to generate a virtually infinite diversity of the cattle antibody repertoire [14]. ...
... These percentages were lower than the previously described 5-10% [89,90], which could be attributed to (i) PCR bias against ultralong antibodies, (ii) NGS sequencing bias against longer amplicons, or (iii) variation in ultralong heavy-chain proportions depending on individuals. Given that cattle VH gene segments share an average of 90% identity [13], the genomic origin of a single antibody sequence is often ambiguous depending on the level of somatic mutation. Therefore, the most probable V gene segment used was assigned to each sequence, and when ambiguous alternative V gene segments were added (Table S4A-C and Figure S3). ...
Studying the antibody response to infection or vaccination is essential for developing more effective vaccines and therapeutics. Advances in high-throughput antibody sequencing technologies and immunoinformatic tools now allow the fast and comprehensive analysis of antibody repertoires at high resolution in any species. Here, we detail a flexible and customizable suite of methods from flow cytometry, single cell sorting, heavy and light chain amplification to antibody sequencing in cattle. These methods were used successfully, including adaptation to the 10x Genomics platform, to isolate native heavy–light chain pairs. When combined with the Ig-Sequence Multi-Species Annotation Tool, this suite represents a powerful toolkit for studying the cattle antibody response with high resolution and precision. Using three workflows, we processed 84, 96, and 8313 cattle B cells from which we sequenced 24, 31, and 4756 antibody heavy–light chain pairs, respectively. Each method has strengths and limitations in terms of the throughput, timeline, specialist equipment, and cost that are each discussed. Moreover, the principles outlined here can be applied to study antibody responses in other mammalian species.
... In contrast to mice and humans, the genome of cattle (Bos taurus) and some other species (i.e., rabbits, Oryctolagus cuniculus; pigs, Sus scrofa; and sheep, Ovis aries) contain relatively few functional IGHV gene segments, thereby constraining the combinatorial diversity typically generated through VDJ recombination (Dufour et al. 1996;Knight and Becker 1990;Ma et al. 2016;Saini et al. 1997). Furthermore, the twelve functional IGHV genes (of 47 total) in the cattle genome all belong to the same IGHV1 subgroup within clan II (Berens et al. 1997;Ma et al. 2016;Niku et al. 2012), and there is evidence that VDJ recombination biases expression towards a single IGHV sequence (IGHV1-10) in conventional antibodies . ...
... In contrast to mice and humans, the genome of cattle (Bos taurus) and some other species (i.e., rabbits, Oryctolagus cuniculus; pigs, Sus scrofa; and sheep, Ovis aries) contain relatively few functional IGHV gene segments, thereby constraining the combinatorial diversity typically generated through VDJ recombination (Dufour et al. 1996;Knight and Becker 1990;Ma et al. 2016;Saini et al. 1997). Furthermore, the twelve functional IGHV genes (of 47 total) in the cattle genome all belong to the same IGHV1 subgroup within clan II (Berens et al. 1997;Ma et al. 2016;Niku et al. 2012), and there is evidence that VDJ recombination biases expression towards a single IGHV sequence (IGHV1-10) in conventional antibodies . Additionally, while some IGHD gene segments in mice and humans can be used in all three reading frames within functional IgH rearrangements (Jackson 2019;Larimore et al. 2012;Schroeder et al. 2010), all functional cattle antibodies discovered to date incorporate IGHD in only one reading frame. ...
... In the utilized reading frame, IGHD segments contain a repeating GYG motif (even for shorter IGHD), while alternate reading frames are severely hydrophobic or contain stop codons (see Wang et al. (2013)). Thus, cattle and other species with limited recombination potential must employ innovative approaches for augmenting receptor diversity (Berens et al. 1997;Deiss et al. 2019;Lopez et al. 1998;Ma et al. 2016;Wang et al. 2013). One mechanism used by cattle (and other ruminants) is the use of AID-catalyzed SHM in the periphery, extensively mutating CDR H3 of the naïve repertoire within the illeal Peyer's patch and spleen (Liljavirta et al. 2013(Liljavirta et al. , 2014Zhao et al. 2006). ...
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.
... The genetic origins of the ulCDRs are fascinating (Burke et al., 2020;Haakenson et al., 2018). Bovines have only a limited number of functional V, D and J genes (12 IGHV, 16 IGHD and 4 IGHJ) (Ma et al., 2016;Stanfield et al., 2018) compared to 57 IGHV, 23 IGHD and 6 IGHJ gene segments in humans (Mikocziova et al., 2021). Due to this limited germline diversity, cows exploit other mechanisms to expand their antibody repertoire. ...
... Cattle also rely heavily on SHM, which already starts before antigen exposure, while in humans SHM is extensive only after contact with the antigen (Stanfield et al., 2018;Deiss et al., 2019;Kurosawa and Tonegawa, 1982;Tonegawa, 1983;Wang et al., 2013). Remarkably, the SHM in the ulCDR-H3 leads to unusually high usage of cysteines that can form disulfide bonds contributing further to paratope diversification (Ma et al., 2016;Prabakaran and Chowdhury, 2020;Haakenson et al., 2019). The bias towards cysteine usage in the ulCDR-H3 is programmed in the bovine germlineabout 80 % of the residues in the ultra-long DH segment can be changed to cysteine via a single nucleotide mutation (Wang et al., 2013). ...
In contrast to other species, cattle possess exceptional antibodies with ultra-long complementarity-determining regions (ulCDRs) that can consist of 40-70 amino acids. The bovine ulCDR is folded into a stalk and a disulfide-rich knob domain. The binding to the antigen is via the 3-6 kDa knob. There exists an immense sequence and structural diversity in the knob that enables binding to different antigens. Here we summarize the current knowledge of the ulCDR structure and provide an overview of the approaches to discover ulCDRs against novel antigens. Furthermore, we outline protein engineering approaches inspired by the natural ulCDRs. Finally, we discuss the enormous potential of using isolated bovine knobs, also named picobodies, as the smallest antigen-binding domains derived from natural antibodies.
... Compared with other species, the bovine Ab repertoire is limited in terms of the number of germline genetic components and thus has a lower potential for combinatorial diversity (5,15,2123). The functional gene segments of the bovine Ab repertoire appear to include only 12 V H , 23 D H , and 2 J H at the H chain locus; 25 V l and 3 J l at the l locus; and 8 V k and 3 J k at the k locus (5,21,24,25). There is even less potential for combinatorial diversity in bovine H chains with ultralong CDR H3s, because all Abs in this subset appear to use the same germline V H , D H , and J H gene segments: IGHV1-7, IGHD8-2, and IGHJ2-4 (15,16,2124). ...
Ab "ultralong" third H chain complementarity-determining regions (CDR H3) appear unique to bovine Abs and may enable binding to difficult epitopes that shorter CDR H3 regions cannot easily access. Diversity is concentrated in the "knob" domain of the CDR H3, which is encoded by the DH gene segment and sits atop a β-ribbon "stalk" that protrudes far from the Ab surface. Knob region cysteine content is quite diverse in terms of total number of cysteines, sequence position, and disulfide bond pattern formation. We investigated the role of germline cysteines in production of a diverse CDR H3 structural repertoire. The relationship between DH polymorphisms and deletions relative to germline at the nucleotide level, as well as diversity in cysteine and disulfide bond content at the structural level, was ascertained. Structural diversity is formed through (1) DH polymorphisms with altered cysteine positions, (2) DH deletions, and (3) new cysteines that arise through somatic hypermutation that form new, unique disulfide bonds to alter the knob structure. Thus, a combination of mechanisms at both the germline and somatic immunogenetic levels results in diversity in knob region cysteine content, contributing to remarkable complexity in knob region disulfide patterns, loops, and Ag binding surface.
... Although many IG loci have been assembled in the last two years, their automated annotation remains an open problem. Previous studies of IG genes combined a time-consuming experimental approach with semimanual computational analysis, such as in the studies of the platypus (Gambón-Deza et al. 2009), the cow (Ma et al. 2016), and the ferret (Wong et al. 2020). These studies used the human IG genes for a similarity-based detection of IG genes in the novel species and may have missed diverged IG genes. ...
The V(D)J recombination process rearranges the variable (V), diversity (D), and joining (J) genes in the immunoglobulin loci to generate antibody repertoires. Annotation of these loci across various species and predicting the V, D, and J genes (IG genes) is critical for studies of the adaptive immune system. However, since the standard gene finding algorithms are not suitable for predicting IG genes, they have been semi-manually annotated in very few species. We developed the IGDetective algorithm for predicting IG genes and applied it to species with the assembled IG loci. IGDetective generated the first large collection of IG genes across many species and enabled their evolutionary analysis, including the analysis of the "bat IG diversity" hypothesis. This analysis revealed extremely conserved V genes in evolutionary distant species indicating that these genes may be subjected to the same selective pressure, e.g., pressure driven by common pathogens. IGDetective also revealed extremely diverged V genes and a new family of evolutionary conserved V genes in bats with unusual noncanonical cysteines. Moreover, in difference from all other previously reported antibodies, these cysteines are located within complementarity-determining regions. Since cysteines form disulfide bonds, we hypothesize that these cysteine-rich V genes might generate antibodies with noncanonical conformations and could potentially form a unique part of the immune repertoire in bats. We also analyzed the diversity landscape of the recombination signal sequences and revealed their features that trigger the high/low usage of the IG genes.
... The IgH consists of 43 VH genes, 23 DH genes and 12 JH genes. The bovine IgH had an ultra-long CDR3H not found in other species, encoding up to 65 amino acids (AA) (10,11). Ultra-long CDR3H had previously been found in IgNAR of sharks and was thought to exist only in cartilaginous fish. ...
... It is critical to understand the natural immune mechanisms that fight infection and how vaccination, biosafety, nutrition, livestock and management practices could be used to maintain and enhance immune protection (43). The sequence and structure of IgH constant genes in yak were similar to those in bovids (11). Interestingly, two m genes with high similarity were found in the genome of cattle, but only one m gene had been found in the genome of other bovids (yak, sheep and goat) (11,24,44,45). ...
... The sequence and structure of IgH constant genes in yak were similar to those in bovids (11). Interestingly, two m genes with high similarity were found in the genome of cattle, but only one m gene had been found in the genome of other bovids (yak, sheep and goat) (11,24,44,45). Three JH genes were found in the yak genome, but none of them were transcribed. ...
As important livestock in Qinghai-Tibet Plateau, yak provides meat and other necessities for Tibetans living. Plateau yak has resistance to diseases and stress, yet is nearly unknown in the structure and expression mechanism of yak immunoglobulin loci. Based on the published immunoglobulin genes of bovids (cattle, sheep and goat), the genomic organization of the yak immunoglobulin heavy chain (IgH) and immunoglobulin light chain (IgL) were described. The assemblage diversity of IgH, Igλ and Igκ in yak was similar to that in bovids, and contributes little to the antibody lineage compared with that in humans and mice. Somatic hypermutation (SHM) had a greater effect on immunoglobulin diversity in yak than in goat and sheep, and in addition to the complementarity-determining region (CDR), some loci in the framework region (FR) also showed high frequency mutations. CDR3 diversity showed that immunological lineages in yak were overwhelmingly generated through linkage diversity in IgH rearrangements. The emergence of new high-throughput sequencing technologies and the yak whole genome (2019) publication have greatly improved our understanding of the immune response in yaks. We had a more comprehensive analysis of yak immunoglobulin expression diversity by PE300, which avoided the disadvantage of missing low-frequency recombination in traditional Sanger sequencing. In summary, we described the schematic structure of the genomic organization of yak IgH loci and IgL loci. The analysis of immunoglobulin expression diversity showed that yak made up for the deficiency of V(D)J recombinant diversity by junctional diversity and CDR3 diversity. In addition, yak, like cattle, also had the same ultra-long IgH CDR3 (CDR3H), which provided more contribution to the diverse expression of yak immunoglobulin. These findings might provide a theoretical basis for disease resistance breeding and vaccine development in yak.
