Revisiting the Pig IGHC Gene Locus in Different Breeds Uncovers Nine Distinct IGHG Genes

Ming Zhang, Zhenrong Li, Jingying Li, Tian Huang, Gaochuang Peng, Wenda Tang, Guoqiang Yi, Lifan Zhang, Yu Song, Tianran Liu, Xiaoxiang Hu, Liming Ren, Honglin Liu, John E. Butler, Haitang Han and Yaofeng Zhao
J Immunol October 15, 2020, 205 (8) 2137-2145; DOI: https://doi.org/10.4049/jimmunol.1901483

Key Points

  • Pig IGHG genes can be classified into nine subclasses.

  • An evolutionary model for pig IGHG genes was proposed.

  • A mouse mAb 4A4 specific to the pig IgG3 was developed.

Abstract

IgG subclass diversification is common in placental mammals. It has been well documented in humans and mice that different IgG subclasses, with diversified functions, synergistically regulate humoral immunity. However, our knowledge on the genomic and functional diversification of IgG subclasses in the pig, a mammalian species with high agricultural and biomedical importance, is incomplete. Using bacterial artificial chromosome sequencing and newly assembled genomes generated by the PacBio sequencing approach, we characterized and mapped the IgH C region gene locus in three indigenous Chinese breeds (Erhualian, Xiang, and Luchuan) and compared them to that of Duroc. Our data revealed that IGHG genes in Chinese pigs differ from the Duroc, whereas the IGHM, IGHD, IGHA, and IGHE genes were all single copy and highly conserved in the pig breeds examined. Most striking were differences in numbers of IGHG genes: there are seven genes in Erhualian pigs, six in the Duroc, but only five in Xiang pigs. Phylogenetic analysis suggested that all reported porcine IGHG genes could be classified into nine subclasses: IGHG1, IGHG2a, IGHG2b, IGHG2c, IGHG3, IGHG4, IGHG5a, IGHG5b, and IGHG5c. Using sequence information, we developed a mouse mAb specific for IgG3. This study offers a starting point to investigate the structure-function relationship of IgG subclasses in pigs.

Introduction

Immunoglobulins play a major role in the adaptive immunity of jawed vertebrates (1). In mammals, there are five Ig isotypes, including IgM, IgD, IgG, IgE, and IgA, which are encoded by the IGHM, IGHD, IGHG, IGHE, and IGHA genes, respectively (2). Among these Ig isotypes, IgG is one of the most abundant proteins in the serum and is critical for humoral immunity, as demonstrated in animals with IgG deficiencies (3–6). Perhaps because of the functional importance of IgG in mammals, the genes encoding IgG (IGHG) have experienced gene duplications in most placental mammals (7–9). These encode different IgG subclasses, which differ subtly in sequences in structures and somewhat in functions. For example, there are four IgG subclasses in humans (IgG1, IgG2, IgG3, and IgG4) (10, 11). IgG4 is the only subclass that fails to fix complement, and the affinity for C1q among the remaining three follows the order: IgG3 > IgG1 > IgG2. IgG1 and IgG3 bind to all three FcγR classes, whereas IgG4 binds only FcγRII and FcγRIII, and IgG2 binds only to FcγRII (12). IgG subclasses are also differently distributed locally and systemically. In cattle and other ruminants, IgG1 is selectively transported by the alveolar epithelial cells in the mammary gland and other sites and can lead to levels exceeding 100 mg/ml IgG1 in colostrum compared with <5 mg/ml IgG2 (13). Also, the response profile to different antigenic stimuli differs among subclasses (14–18). During early HIV-1 infection, functional Ab responses decline drastically with HIV disease progression, which is correlated with a selective waning IgG3 concentration (19–21).

The diversification of IGHG genes is also prevalent in domestic animals, as three IGHG genes in cattle, two in sheep, and seven in horses have previously been identified (8, 9, 22, 23). In Sus scrofa (pig), nearly all work has been done with breeds of European origin: Yorkshire, various Landrace, Duroc, crosses among these, and with various mini-pigs. The earliest reports, based on immunoelectrophoresis and chromatography, identified two to four candidate subclasses, two of which were designated and eventually used to generate mAbs that have been marketed for >20 y (14–17, 24). When first examined in cDNAs, five IGHG genes were identified (25). The subclass originally designated IgG1 by this group was later renamed IgG3 (26). This subclass is most different because of its extended hinge and its early ontogeny in the ileal Peyer patches. In a more-extensive study, six IGHG genes were recovered from Landrace-Yorkshire crosses in which allotypic variants of five of these were found (27). However, when the IGHG genes from a Landrace in Japan were mapped (28), the number of IGHG genes was the same, but IGHG genes reported as allotypic variants in the Landrace-Yorkshire crosses were shown to be separate genes in the Japanese pig. This observation/discrepancy together with the fact some Landrace-Yorkshire crosses expressed only four IGHG genes raised a question with regard to the authentic number of IGHG genes and how the porcine IGHG genes are related and how they evolved.

Worldwide, there are perhaps hundreds of distinct pig breeds, but only a number of them have extensively been used in agriculture and biomedical studies (29–32). Several commercial pig breeds, such as Landrace, Duroc, and Large White/Yorkshire, contribute considerably to the world pork production, whereas other lines, such as Göttingen and Chinese Xiang, which differ significantly from their commercial counterparts in body size, have increasingly been used as model animals in studies for biomedicine (33, 34). Most notable are the Göttingen mini-pigs especially suitable for gnotobiotic studies (18, 35) and the inbred National Institutes of Health mini-pig developed for use in transplantation biology (36). Considering the importance of pigs in both aspects, more effort is needed to characterize the structure-function relationship among the multiple IgG subclasses.

Annually, China alone produces ∼700 million commercial pigs, roughly half of the number produced globally for the pork supply (37). However, infectious diseases such as African swine fever and porcine reproductive and respiratory syndrome (PRRS) are currently the major threat to pig production, causing tremendous economic loss worldwide (38–40). Because of the important role played by this species in the world food supply and its value as a model in medical research, including immunology, we believed it was time to resolve the confusion and uncertainty regarding the diversification of the IGHG genes by carefully examining IGHG genes recovered from bacterial artificial chromosome (BAC) libraries of different breeds, especially those of non-European origin.

It is now generally accepted that European and Chinese domestic pigs were domesticated from local wild boars independently some 9000–10,000 y ago, and European wild boars diverged from their Chinese counterparts ∼1 million years ago (41, 42). This basically means that European domestic pigs (including major commercial breeds) are genetically quite different from the Chinese domestic pigs despite some genetic exchanges between them very recently (41, 43). Thus, a more-comprehensive analysis of IGHG genes in Chinese domestic pigs may provide significant insights into the IGHG gene evolution in the pig species. The information gained from this study and the methodology developed in the process will also make it possible to generate mAbs that can then be used to study structure-function relationships among the various IgG subclasses. Such information could prove valuable in the development of vaccines against emerging diseases that threaten this economically important species.

Materials and Methods

Sample collection

Blood and tissue samples of Erhualian pigs were collected from Changzhou Erhualian Production Cooperation (Jiangsu, China). Tissue samples of Xiang, Landrace, and Duroc pigs were collected from the Experimental Pig Base, Zhuozhou, China Agricultural University. These studies were approved by the Animal Care and Use Committee of China Agricultural University.

Construction of Erhualian pig and Xiang pig BAC library

The Erhualian pig and Xiang pig BAC libraries were constructed by Wuhan Bacgene Technology (http://www.bacgene.com). Genomic DNA was extracted from PBLs of Erhualian pigs and from fibroblasts of Xiang pigs, and was then digested with HindIII and subcloned into BAC vector pIndigoBAC5-536S and transformed into DH10B Escherichia coli. Individual white clones were transferred to wells of 384-well plates containing Luria-Bertani medium with 7.5% glycerol, and the plates were incubated overnight at 37°C and then stored at −80°C (44–46).

BAC library pooling and screening

We prepared the super pools, plate pools, and row pools, in which each super pool was made up of 10 384-well plates. A total of 480 plates of the Erhualian pig BAC library were pooled into 48 super pools, and 492 plates of the Xiang pig BAC library were pooled into 49 super pools. The last super pool contained 12 plates (481–492 plates). Each plate pool was made up of one 384-well plate, and each row pool was made up of one of the rows of a 384-well plate. IGHM-, IGHG-, and IGHA-positive BAC clones were screened by PCR, as previously described (47). The screening primers were as follows: IGHM-F: 5′-TTTCCTGCCTGGTCACGG-3′, IGHM-R: 5′-GCTTGGAGACGCTCTGCTT-3′, IGHG-F: 5′-GCCCTCGGTCTTCATCTTC-3′, IGHG-R: 5′-CTGGGAGGTCTTCGTTGTTG-3′, IGHA-F: 5′-TCATCGCCTGCCTGGTC-3′, and IGHA-R: 5′-GGACTGGCTGGATTTGGAG-3′.

The Duroc BAC library CHORI-242 filter was purchased from the BACPAC Resources Center (http://bacpac.chori.org/home.htm), Children’s Hospital Oakland Research Institute. IGHM, IGHG, and IGHA gene–specific probes were labeled with digoxigenin-11-dUTP using a PCR digoxigenin probe synthesis kit (Roche, Basel, Switzerland). PCR primers were IGHM-F, IGHM-R, IGHG-F, IGHG-R, IGHA-F, and IGHA-R. Hybridization and detection were performed using the DIG-High Prime DNA Labeling and Detection Starter Kit II following the manufacturer’s instructions.

BAC end and full-length sequencing

IGHM-, IGHG-, and IGHA-positive BAC clones were sequenced by Sanger sequencing to obtain their end sequences. A BLAST search with the Landrace boar IGHC sequences (accession numbers AB699686, AB699687, and AB699688) was performed to determine the BAC clones that overlap with each other. The overlapping DNA in the BAC clones was isolated using the QIAGEN Large-Construct Kit (Qiagen, Valencia, CA) according to the manufacturer’s protocols. Pulse field gel electrophoresis was used to determine BAC insert size. Because the genomic regions encompassing IGHG genes had extremely high similarity to each other, we selected third-generation sequencing technology (PacBio) for BAC full-length sequencing to avoid incorrect assembly. The original sequence data of PacBio was corrected by Canu first (48), then the corrected data were assembled by Overlap-Layout-Consensus (49). Finally, the assembly data were corrected by RACON (50). BAC full-length sequencing and sequence assembly were completed by BioMarker Biotech (www.biomarker.com.cn).

Computational analysis and sequence alignment

Erhualian pig, Xiang pig, Luchuan pig, and Duroc IGHC region genes were retrieved by comparison with GenBank files. The National Center for Biotechnology Information’s GenBank accession numbers of the sequences used were as follows: AB699686, AB699687, and AB699688. The IGHV, IGHD, and IGHJ gene segments were identified using online software (http://www-archbac.u-psud.fr/genomics/patternSearch.html) and by searching for adjacent recombination signal sequences allowing for mismatches (also retrieved by comparing with GenBank files), and the accession number is AB513624. DNA and protein sequence editing, alignments, and comparisons were performed using DNASTAR’s Lasergene software and GeneDoc.

Phylogenetic tree

All published known pig breed IGHG genes were downloaded from the ImMunoGeneTics/Laboratoire d'Immunogenetique Moleculaire database (http://www.imgt.org/ligmdb/). The National Center for Biotechnology Information’s GenBank accession numbers were AB699686, AB699687, U03778, U03779, and U03782. A phylogenetic tree was then constructed with the new IGHC genes obtained from Erhualian pig, Xiang pig, Luchuan pig, and Duroc by MrBayes3.1, MEGA6, and PhyML3.0 (51–53). The phylogenetic tree was viewed or analyzed by Fig Tree.v1.4 (54).

Preparation of anti-IgG mAbs

After alignment of the deduced constant regions, the specific amino acid sites in each IgG isotype were identified. We then selected all appropriate amino acid sites for synthesizing short peptides conjugated with Keyhole limpet hemocyanin (KLH) as Ags to immunize the BALB/c mouse. The short peptides and the mAb prepared were completed by Abmart (Shanghai, China). The specificity of the obtained mAbs was confirmed by Western blot and ELISA for each IgG isotype expressed in 293T cells.

Expression of pig IgG subclasses

We amplified every exon from each IGHG subclass of Landrace boar and fused the exons by two-step fusing PCR to obtain the complete IgG C region. The V region was amplified from a pig mAb against PRRSV-GP5 to be fused with the IgG C region. To obtain complete IgG, the pig Ig λ L chain was also amplified from the same pig mAb against PRRSV-GP5 to bind to the IgG H chain. Then, the IgG H chain and L chain were subcloned into the eukaryotic expression vector pBudCE4.1 and transfected into 293T cells.

Western blot analysis

After transfection for 48 h, the cells were lysed with RIPA lysis buffer (P0013B; Beyotime). The cell lysate was denatured at 100°C for 10 min with or without DTT, subjected to 10% SDS-PAGE, and then transferred to PVDF membranes (Millipore). The membranes were blocked with 5% nonfat milk in TBST for 1 h at room temperature. Then, the membranes were treated with the primary Ab and anti-pig IgG Ab (AAI41; Bio-Rad) for 1 h at room temperature. Next, the membranes were incubated with HRP-conjugated secondary Abs (ZSGB Biotechnology) for 1 h at room temperature. After determining which IgG subclasses were expressed, the membranes were treated with 24 of the mAbs generated by the procedure described above for 1 h at room temperature to identify which mAbs were IgG3-specific.

ELISA analysis

The cell lysates were coated onto the 96-well ELISA plate and incubated at room temperature for 2 h. The plate was subsequently washed, and 4A4 mAb was added, followed by incubation at room temperature for 1 h. Next, the plate was washed, and anti-mouse IgG Ab (HRP-conjugated) was added, followed by incubation at room temperature for 1 h. Then, the plate was washed, and TMB High Sensitivity Substrate (BioLegend) was added. Finally, the reaction was stopped with 0.18 M sulfuric acid. The plate was read at OD450 on a microplate reader (Tecan).

Results

Construction of Erhualian pig and Xiang pig BAC libraries

To analyze the porcine IGHC genes of pigs in different breeds, we generated two BAC genomic libraries using peripheral blood leukocytes isolated from an Erhualian pig and fibroblasts isolated from a Xiang pig. The Erhualian pig library consisted of 184,320 clones with an average insert size of >120 kb (data not shown), representing <5% of the noninsert clones and ∼10× coverage of the pig genome (∼2800 Mb). The Xiang pig library consisted of 188,928 clones with an average insert size of >120 kb (data not shown), also representing <5% of the noninsert clones and ∼10× coverage of the pig genome (Table I).

View this table:
Table I. The BAC library information

The IGHC locus in Erhualian, Xiang, Duroc, and Luchuan pigs

Three IGHC gene–positive clones (EHL131F13, EHL255N17, and EHL342J23) for Erhualian and two for Xiang pigs (XP159B23 and XP83P15) were identified from the above constructed BAC libraries using a PCR-based approach. Identified with a Southern blot on a filter membrane containing BAC DNA of an entire BAC library, the BAC clone (CH242-418N9) for Duroc was purchased from the BACPAC Resources Center. These BAC clones were sequenced by third-generation sequencing technology (PacBio) and assembled, and we finally obtained a 347,251-bp genomic sequence covering the Erhualian pig IGHC genes and a 241,547-bp genomic sequence covering the Xiang pig IGHC genes. The sequenced Duroc BAC clone (CH242-418N9) showed a perfect match to the sequences of the IGHC gene locus retrieved from the Duroc genome, which was a recent de novo assembly generated by PacBio sequencing. Thus, a 400,000-bp genomic sequence derived from the Duroc genome (NW_018084979) covering the Duroc IGHC genes was used in further analysis. The Luchuan IGHC gene locus was also derived from a de novo genome assembly generated by PacBio sequencing (Y. Yang, J. Lian, B. Xie, M. Chen, Y. Niu, Q. Li, Y. Liu, G. Yi, X. Fan, Y. Tang et al., manuscript posted on bioRxiv, DOI: 10.1101/770958). The IGHC genes of the four breeds are arranged as IGHM-IGHD-IGHGn-IGHE-IGHA as previously reported in Landrace boar (Fig. 1).

FIGURE 1.
FIGURE 1.

Physical map of the pig IgH gene locus. Every darkened rectangle represents a gene, and the name of the gene is marked in the rectangle. IGHG (Cγ) genes were named with 5′-3′ order. IGHV and IGHD genes are not shown. (A) The 347,251-bp contiguous nucleotide sequence was reconstructed by using three BAC clones, EHL131F13, EHL255N17, and EHL342J23. (B) The 400,000-bp contiguous nucleotide sequence was selected from the 5′ end of the Duroc genome sequence (NW_018084979). (C) The 241547-bp contiguous nucleotide sequence was reconstructed by using two BAC clones, XP159B23 and XP83P15. (D) The genome sequence of the Luchuan pig 7 chromosome, which contains the IgH gene locus.

Overall comparisons of the pig IGHC genes in various breeds

As shown in Fig. 1, the IGHM, IGHD, IGHA, and IGHE genes are all present in a single copy in the investigated pig breeds. These genes appear to be highly conserved with regard to their DNA and correspondingly encoded amino acid sequences, as very few nucleotide or amino acid changes were observed for these genes in the five pig breeds (Supplemental Fig. 1).

However, the IGHG genes are very different in different pig breeds both in gene number and sequence. At the Erhualian IGHC gene locus, seven IGHG genes spanned 120,355 bp, according to the location order, which were named IGHG1, IGHG2, IGHG3, IGHG4, IGHG5, IGHG6, and IGHG7, tentatively (Fig. 1A). In the Xiang pig IgH locus, five IGHG genes spanned 85,352 bp, named IGHG1 to IGHG5 (Fig. 1C). Similar to Landrace pigs, both Luchuan and Duroc pigs have six IGHG genes, which were named IGHG1 to IGHG6, according to the location order (Fig. 1B, 1D). Different pig breeds have various numbers of IGHG genes, and these IGHG genes differ from each other to different extents in sequences (Supplemental Fig. 2).

Phylogenetic analysis uncovers nine distinct IGHG genes in pigs

As a large number of IGHG genes were identified in different pig breeds, it seemed appropriate to determine how these would group based on their sequences. Thus, sequences of all IGHG genes obtained in this study, as well as pig IGHG sequences recovered from cDNA libraries and reported by others, were used in our phylogenetic analyses. Considering that the PCR amplification process may introduce artifacts, previously reported IGHG sequences that were generated through PCR amplifications or derived from unknown pig breeds were excluded from the analyses. Three different methods, including MEGA, Phylip (data not shown), and MrBayes (data not shown), were employed to conduct the analyses, which all generated trees with the same topology (Fig. 2), allowing the pig IGHG genes to be placed into five major groups (IGHG1-5). Two groups (IGHG2 and IGHG5) can be further classified into three subgroups based on the presence of distinct IGHG gene sequences recovered from the genomes of Luchuan and Erhualian pigs. Based on these findings, we suggest that the pig IGHG genes should be renamed to avoid inconsistency. As shown in Fig. 3, we systematically named the pig IGHG genes identified in both this study and in previous reports. Our data support that there are nine distinct IGHG genes, including IGHG1, IGHG2a, IGHG2b, IGHG2c, IGHG3, IGHG4, IGHG5a, IGHG5b, and IGHG5c, in pigs. Further comparisons of switch regions (data not shown), intron size, and transmembrane regions of all these IGHG genes also strongly supported the classification (Supplemental Fig. 3, Supplemental Table I).

FIGURE 2.
FIGURE 2.

Phylogenetic tree of pig IGHG coding regions of genes in this study and other publications. Landrace-IGHG1-1, Landrace-IGHG3-1, Landrace-IGHG5-1-01, Landrace-IGHG5-2-01, AB699686; Landrace-IGHG6-1-01, Landrace-IGHG6-1-02, AB699687; Yorkshire-IGHG1-03, U03778; Yorkshire-IGHG2-01, U03779; Yorkshire-IGHG4-02, U03782. Holstein IGHG1 was used as an outgroup. This phylogenetic tree was constructed by MEGA6.

FIGURE 3.
FIGURE 3.

The pig IGHG genes identified in both this study and previous reports were systematically named. Each IGHG subclass was presented by a rectangle in 5′-3′ order. The IGHG genes of five pig breeds are presented. Different IGHG genes were previously named in various studies (24, 28).

For the nine IGHG genes, it is evident that only IGHG1, IGHG3, and IGHG4 are observed in all breeds investigated, whereas the other remaining IGHG genes are dispersedly distributed in different pig breeds. These data suggest that these three IGHG genes are relatively old, and IGHG3 appears to be the oldest in the genesis of pig IGHG genes (Figs. 2, 4).

FIGURE 4.
FIGURE 4.

An evolutionary model for pig IGHG genes. Our phylogenetic analysis and previous studies by others suggest that IGHG3 is the oldest gene in the pig, which should have given rise to IGHG1 and IGHG4 genes via gene duplications during the pig speciation, whereas the IGHG1 and IGHG4 genes duplicated independently later to create other IGHG genes. This figure also suggests that all nine IGHG genes should have been present in the common ancestor of European and Chinese wild boars, and various IGHG gene losses in the domestication process led to the presence of different IGHG genes in different domestic pig breeds. The IA pigs, the common immediate ancestor of European and Chinese wild boars.

Amino acid variations of the nine IgG subclasses

IGHM, IGHD, IGHA, and IGHE all appear to be single-copy genes and are extremely conserved in their amino acid sequences in different breeds. Extensive subclass diversification of the IGHG genes led to not only various copy numbers in different pig lines but also sequence variations either in distinct IGHG genes in one line or in the same IGHG gene in different lines.

For the constant regions encoded by the nine IGHG genes, the most variable part is the hinge region. Six different hinge sequences were identified, in which IGHG3 encodes the longest. Different IGHG genes can have the same hinge coding sequences (such as IGHG5c, IGHG2c, and IGHG4), indicating that genomic gene conversion may have been involved in the genesis of these genes. Interestingly, there are two types of hinges for IgG1 in different pig lines: whereas the Xiang pig and Yorkshire pig share one, the remaining lines share the other.

Like human IgG, all nine IgG subtypes have an N-glycosylation site in CH2, as in human IgG Asn297 (Fig. 5) (55). The glycans at this N-glycosylation site can influence Ab stability, binding to FcγRs and complement activation, consequently modulating effector functions, such as complement-dependent cytotoxicity and Ab-dependent cell cytotoxicity (56–59). In addition, all nine IGHG genes encode a highly conserved transmembrane region with only very few amino acid changes observed.

FIGURE 5.
FIGURE 5.

Comparison of the deduced amino acid sequences of nine pig IgG subclasses. For alignment of sequences, dots indicate the same sequence and dashes indicate deletions. The conserved cysteines and tryptophans are shaded and in bold. Stars are used to indicate amino acids specific to IgG3, whereas an inverted triangle marks the conserved N-linked glycosylation site.

Generation of an mAb specific to the pig IgG3

Because we identified nine IgG subclass encoding genes in different pig breeds, we prepared IgG subclass–specific mAbs. After a thorough examination of the sequences of nine IgG subclasses, we found that IgG3 has some specific sites that are different from the others (Fig. 5). We then designed three polypeptides conjugated with KLH and VLP as Ags to immunize BALB/c mice. Finally, we obtained 24 mAbs.

To confirm whether the 24 clones were specific to IgG3, we expressed five IgG (IgG3, IgG5b, IgG5c, IgG1, and IgG2c) subclasses of Landrace boar in 293T cells, and these five IgG subclasses have different sites in three polypeptides. After 48 h of transfection, we confirmed that all five IgG subclasses were expressed by Western blot using the anti-pig IgG Ab (Fig. 6A).

FIGURE 6.
FIGURE 6.

Detection of the specificity of the 4A4 mAb. (A) Detection of the expression of five IgG subclasses in 293T with anti-pig total IgG Ab. Detection of the specificity of the 4A4 mAb in reduced condition (B) and nonreduced condition (C) by Western blot. (D) Detection of the specificity of 4A4 mAb by ELISA.

Then, we used the cell lysates to detect the specificity of 24 mAb clones by Western blot. We found that only 4A4 was specific to IgG3 in both reduced and nonreduced conditions (Fig. 6B, 6C). Furthermore, 4A4 can also be used in ELISA to detect IgG3, specifically (Fig. 6D).

Discussion

In this study, we compared the IGHC gene locus in three Chinese breeds (Erhualian, Xiang, Luchuan) with that of a Duroc of European stock used in the pig genome project (42 and A. Warr, N. Affara, B. Aken, H. Beiki, D. M. Bickhart, K. Billis, W. Chow, L. Eory, H. A. Finlayson, P. Flicek et al., manuscript posted on bioRxiv, DOI: 10.1101/668921). We found that in all pigs, the IGHC gene locus is organized as IGHM-IGHD-IGHGn-IGHE-IGHA. Whereas the IGHM, IGHD, IGHE, and IGHA genes are highly conserved, the IGHG sublocus exhibits extensive variations in copy number and sequence in different pig breeds. We further examined the phylogenetic relationships of IGHG genes identified in both the present and previous studies of swine and found that pig IGHG genes could be classified into nine distinct subclasses. Based on this analysis, we renamed all pig IGHG genes in a manner that, if accepted, could avoid confusion among investigators undertaking future studies.

Four pig breeds, including three indigenous Chinese pig breeds and one European pig breed, were used in this study. Despite their different geographical origins, these pig breeds retained highly conserved genes for IGHM, IGHD, IGHA, and IGHE genes. In the four pig breeds investigated in this study and Landrace, both IGHM and IGHE showed no amino acid changes in their encoded IgM and IgE H chain constant regions, whereas only one amino acid change in Erhualian pig for IgA and one for IgD in Xiang pig were observed. However, genetic variants of porcine IgA are well known among European breeds (60, 61). The high degree of conservation in genes encoding IgM and IgD contrasts with the extremely short time in which evolutionary divergence of the IGHG sublocus occurred. The conservation of genes encoding IgM and IgG probably reflects the functional importance of these genes in B cell development in all mammals.

In contrast to the IGHM, IGHD, IGHA, and IGHE genes, extensive subclass diversification and copy number variations were much greater among IGHG genes. A systematic phylogenetic analysis of all reported IGHG genes in pigs indicated they could be defined as nine distinct IGHG genes. We renamed these as IGHG1, IGHG2a, IGHG2b, IgHG2c, IGHG3, IGHG4, IGHG5a, IGHG5b, and IGHG5c. In pig breeds so far investigated, the Chinese indigenous Erhualian pig, which closely related to the Chinese Meishan pig, and the Taihu lake pig, (distributed in the Taihu Basin of Jiangsu Province) were shown to have seven IGHG genes, whereas another indigenous Chinese mini-pig breed, the Xiang pig, only has five genes in its genome.

According to our data, among the nine IGHG genes, only IGHG1, IGHG3, and IGHG4 are present in all pig breeds, indicating that these three IGHG genes are evolutionarily more ancient than the others. Consistent with previous studies (28, 62), our phylogenetic tree also clearly suggested that IGHG3 was the most ancient IGHG gene in pigs. Based on these analyses, we hypothesize that during the evolutionary process of pig IGHC gene locus, the IGHG3 gave rise to IGHG1 and IGHG4 genes via gene duplications initially, and later, IGHG1 and IGHG4 duplicated independently to create other IGHG genes (Fig. 4). Our data strongly suggest that all the nine IGHG genes should have been present in the common ancestor of European and Chinese wild boars, and various IGHG gene losses in the domesticating process led to presence of different IGHG genes in different domestic pig breeds (Fig. 4).

IgG is one of the most abundant proteins in the serum of mammals, accounts for >85% of all Abs in serum, and plays significant roles in humoral immunity. The extensive diversification of the genes encoding IgG among mammals raises the question as to why this occurred. One reason for diversification could have been evolutionary pressure to generate specialized variants, each of which may possess specialized effector capabilities to best counter the diverse array of pathogens that can threaten mammals. Differences in the ability to activate complement and bind with different affinity to FcγRs, including transcellular transport, are well documented. It is noteworthy that in the rabbit family, which shows no diversification in the IGHG sublocus, there might be as many as 13 IGHA variants (63). In horses, there are also multiple variants of IGHE (64). This might suggest that IgG, IgA and IgE, which are associated with secondary and memory responses, have been targets of diversification, whereas IgM and IgD are largely restricted to the primary immune response.

Although the role of the IgG subclasses are best known from studies in humans, mice, and cattle, nothing is known about structure-function relationships for the IgG subclasses in pigs. A major obstacle has been the unavailability of mAbs that can recognize the major subclasses and therefore allow investigators to follow the role of each in an immune response. This is rather surprising because pigs are the major meat animal worldwide, and for a relatively long time, they have been used as models for transplantation immunology (36) as convenient gnotobiotic mini-pigs (35), melanoma (65), and, more recently, cystic fibrosis (66). Conferences have been regularly held on the use of swine models (67), and treatises have been published on pig genetics, including immunogenetics (68). The major obstacles with studies on subclass function have been that the subclasses in swine cannot be physico-chemically purified as in cattle, and unlike mice and humans, there are no natural or induced plasmacytomas from which to recover a “pure subclass protein.” These difficulties have led to the marketing of mAb reagents that claimed to be IgG1 and IgG2 specific (for >20 y) well before the complex structure of the IGHG sublocus described in this study and by others was investigated. Using such reagents that are not subclass specific (69) has only caused confusion in determining structure-function relationships. Using the technology described in this study and previously (70), it will now be possible to characterize the role of the many swine IgG subclasses. Such information could prove valuable in the design of vaccines for emerging diseases that threaten the pork industry.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Desen Wu (China Railway Eighth Civil Engineering Group Corporation) and Yingpeng Yao, (China Agricultural University) for help in mapping. We also thank Fang Wang (China Agricultural University) for help in bioinformatics analysis.

Footnotes

  • This work was supported by the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (31530070).

  • The sequences presented in this article have been submitted to the National Center for Biotechnology Information’s GenBank (http://www.ncbi.nlm.nih.gov/) under accession numbers MN735459, MN735460, and MN735461.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    BAC
    bacterial artificial chromosome
    PRRS
    porcine reproductive and respiratory syndrome.

  • Received December 16, 2019.
  • Accepted August 13, 2020.

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