Characterization of antibody V segment diversity in the Tasmanian devil (Sarcophilus harrisii)

https://doi.org/10.1016/j.vetimm.2015.08.001Get rights and content

Abstract

The Tasmanian devil (Sarcophilus harrisii) immune system has recently been under scrutiny because of the emergence of a contagious cancer, which has decimated devil numbers. Here we provide a comprehensive description of the Tasmanian devil immunoglobulin variable regions. We show that heavy chain variable (VH) and light chain variable (VL) repertoires are similar to those described in other marsupial taxa: VL diversity is high, but VH diversity is restricted and belongs only to clan III. As in other mammals, one VH and one Vλ germline family and multiple incomplete Vκ germline sequences were identified in the genome. High Vκ variation was observed in transcripts and we predict that it may have arisen by gene conversion and/or somatic mutations, as it does not appear to have originated from germline variation. Phylogenetic analyses revealed that devil VL gene segments are highly complex and ancient, with some lineages predating the separation of marsupials and eutherians. These results indicate that although the evolutionary history of immune genes lead to the expansions and contractions of immune gene families between different mammalian lineages, some of the ancestral immune gene variants are still maintained in extant species. A high degree of similarity was found between devil and other marsupial VH segments, demonstrating that they originated from a common clade of closely related sequences. The VL families had a higher variation than VH both between and within species. We suggest that, similar to other studied marsupial species, the complex VL segment repertoire compensates for the limited VH diversity in Tasmanian devils.

Introduction

The adaptive/combinatorial immune system arose about 400–450 million years ago, and from its onset had the potential to recognize and distinguish all possible antigens, whether of ‘self’, ‘altered self’ or ‘non-self’ origin (Marchalonis et al., 2006). The basis for this ability lies in the structural organization and assembly of immunoglobulins: while gene segments specifying the constant regions and frameworks are genetically inherited, diversity in the recognition units are generated by somatic mutations and by somewhat random rearrangements. Immunoglobulins are composed of two identical light chains and two identical heavy chains. The heavy chain determines the subclass of each antibody, while based on small polypeptide sequence differences the light chains can be classified as either kappa (κ) or lambda (λ). The chains are held together by inter- and intra-chain disulfide bonds. Based on the variability in the amino acid sequences both the light and heavy chains can be divided into two segments, a variable and a constant region. Antibodies bind antigens primary through their variable region. Unique immunoglobulin variable regions are produced by somatic recombination of tandemly repeated subgenes, called variable (V), diversity (D) and joining (J) segments to generate an exon encoding the V domain. The heavy chain variable region is formed by random assembly of one V, one D and one J gene segment, while the light chain variable region is generated by combining one V and one J segment. The VDJ subgenes evolve via gene duplication, divergence and deletion generating multiple copies of these gene segments and providing a broad array of genes available for recombination. Mature Ig V segment diversity is created by three distinct mechanisms: (1) germline diversity, (2) gene conversion, and (3) somatic mutation (Sun et al., 2013). The combination of these processes produces large numbers of antibodies with different antigen binding specificity. The diversity of VH sequences originates from the presence of divergent germline VH families, where family clustering is determined by VH genes sharing ≥75% nucleotide identity. Based on their degrees of identity VH genes from various vertebrate species have been classified into five groups (A–E) (Sitnikova and Su, 1998), of which mammals possess three (A–C) (or clans I–III) (Kirkham et al., 1992).

The number of IgHV genes in these three groups varies among different mammals (Das et al., 2008, Sitnikova and Su, 1998): primates, rodents and bats have multiple VH families, but other mammals (including both eutherians, monotremes and marsupials) show limited VH diversity (Baker et al., 2010, Mainville et al., 1996, van Dijk et al., 1993) with only a single VH family or a set of recently derived VH families present in the germline (Baker et al., 2005, Butler, 1997, Johansson et al., 2002). The number of functional and pseudo-VH genes also varies between species: humans and mice have over 40 functional VH genes (Das et al., 2008), while rabbits only use one VH segment to generate 70–90% of the expressed antibody repertoire (Knight, 1992).

Compared to the VH genes, the mammalian VL genes most likely originated more than 470 million years ago (Rast et al., 1994) and their evolution has followed a more complex path: the genes coding the two light chains (κ and λ) are located on different chromosomes, and the λ-chain genes appear to be polyphyletic to the κ-chain genes (Rast et al., 1994, Sitnikova and Su, 1998). Mammalian VL genes are grouped into six clans (five Vλ sequences and 1 Vκ) – which are further divided into 10 subgroups (Sitnikova and Nei, 1998, Sitnikova and Su, 1998, Sun et al., 2013). Tetrapod species utilize κ and λ chains to different extents, but the overall complexity of the germline Vλ and Vκ repertoire tends to favor the preferential use of one isotype over another (Almagro et al., 1998, Sun et al., 2013). Mammals and reptiles utilize both types of light chains: κ and λ, birds possess only λ chains; and both κ and λ orthologues have been identified in amphibians and fish (reviewed in Sun et al., 2013, Wu et al., 2010).

Species that have a restricted VH repertoire tend to have limited VL repertoires as well (Sun et al., 2013, Wu et al., 2010). Divergent taxa, such as rabbits, cattle, platypus and chicken both have limited germline VH and VL diversity. In contrast, other organisms, such as humans, sheep and mice, show both high germline VH and VL diversity. The two marsupial species investigated so far, the gray short-tailed opossum (Monodelphis domestica) and the common brushtail possum (Trichosurus vulpecula) provide an exception to this rule. They show limited VH diversity but high VL diversity (Baker et al., 2005, Miller, 2010, Miller et al., 1998). In this study we characterize immunoglobulin VH and VL diversity in the Tasmanian devil, a species under threat of extinction due to a contagious cancer (Hawkins et al., 2006).

Section snippets

Analysis of the Tasmanian devil whole genome and transcriptome sequences

VH segments were identified in the whole genome sequence of the Tasmanian devil available in the Ensembl (Murchison et al., 2012) database (DEVIL7.0, GCA_000189315.1) by using the BLAT, BLASTN and BLASTX algorithms. The reference genome of Tasmanian devils was generated from fibroblast cell lines originated from a 5-year old female devil. Sequencing libraries were sequenced from both ends, yielding 2.87 × 109 pairs of 100 bp sequence reads. Additional “mate pair” libraries were generated and 50 bp

Germline VH and VL repertoire

VH and VL sequences identified by RACE-PCR were used to search the Tasmanian devil genome. Seventeen scaffolds ranging from 2.3 to 49.7 kb containing VH segments were identified. Of these, six contained stop codons and are presumably pseudogenes, and 6 sequences were partial, either the 3′ or the 5′ end of the sequences were missing. Six sequences had a leader peptide, open reading frame (ORF) and an identifiable recombination signal sequence (RSS) and are potentially functional VH segments

Discussion

The Tasmanian devil exhibits low immunoglobulin VH complexity and high VL diversity, as in other characterized marsupials. High VH sequence similarities, and close relatedness of Vλ and Vκ sequences among the three marsupials was also observed. Similar to the findings of Baker et al. (2005) the devil VH sequences belonged to the mammalian VH cluster III. Previous studies have shown that the evolution of VH segments (I, II and III) follows the birth and death process (duplication and deletion),

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgements

We thank Sarah Peck and colleagues from the Save the Tasmanian Devil Program, DPIPWE, and Menna Jones and Rodrigo Hamede from the School of Zoology, University of Tasmanian for sharing Tasmanian devil blood samples. We also acknowledge Yuanyuan Cheng from the University of Sydney and Tony Papenfuss from the Walter and Elisa Hall Institute for sharing unpublished sequence data. This research was funded by the Australian Research Council (ARC) and the University of Sydney. K.B. is supported by an

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