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The Journal of Immunology, 1999, 163: 3826-3833.
Copyright © 1999 by The American Association of Immunologists

Two Ancient Allelic Lineages at the Single Classical Class I Locus in the Xenopus MHC1

Martin F. Flajnik2,*,{dagger}, Yuko Ohta*,{dagger}, Andrew S. Greenberg*, Luisa Salter-Cid*, Ana Carrizosa*, Louis Du Pasquier{ddagger} and Masanori Kasahara§

* Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, FL 33136; {dagger} Department of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201; {ddagger} Basel Institute for Immunology, Basel, Switzerland; and § Department of Biosystems Science, Graduate University for Advanced Studies, Hayama, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Unlike all other vertebrates examined to date, there is only one detectable class I locus in the Xenopus MHC. On the bases of a nearly ubiquitous and high tissue expression, extensive polymorphism, and MHC linkage, this gene is of the classical or class Ia type. Sequencing analysis of class Ia cDNAs encoded by eight defined MHC haplotypes reveals two very old allelic lineages that perhaps emerged when humans and mice diverged from a common ancestor up to 100 million years ago. The unprecedented age of these lineages suggests that different class Ia genes from ancestors of the laboratory model Xenopus laevis are now expressed as alleles in this species. The lineages are best defined by their cytoplasmic and {alpha}2 peptide-binding domains, and there are highly diverse alleles (defined by the {alpha}1 peptide-binding domain) in each lineage. Surprisingly, the {alpha}3 domains are homogenized in both lineages, suggesting that interallelic gene conversion/recombination maintains the high sequence similarity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The amphibian Xenopus is an attractive model for comparative studies of the MHC. Over the past few years, the types of genes present in the MHC have been shown to be similar to that of mammals (1, 2, 3, 4, 5, 6, 7). In addition, there is a large number of non-MHC-linked class Ib genes in Xenopus that, similar to mammals, may complement the function of the classical molecules (8).

The Xenopus MHC, however, has two characteristics that make the model unique. First, MHC genes are silenced in polyploid individuals to a diploid number, even in higher order polyploids that potentially harbor eight copies of the MHC (9, 10). This limiting of MHC gene number may place unique pressures on the frog immune system, reflected in the number and diversity of all types of MHC genes. Second, immunocompetent tadpoles express class Ia proteins and the immune proteasome element lmp7 only at low levels until metamorphosis when the genes become expressed ubiquitously (11, 12), while class II molecules are expressed in high amounts by B cells and macrophages (13, 14). After metamorphosis, class II is expressed by all lymphocytes and APC. This differential expression of MHC during development may be important for limiting autoaggressive responses when new adult-type proteins are expressed at metamorphosis (12, 15).

Concerning the general evolution of the class I gene family, there are several other unusual features of Xenopus class Ia genes and proteins: 1) unique among all vertebrates studied to date, the MHC proper encodes only one class I gene, of the class Ia type, as detected by Southern blotting under low stringency conditions (1); 2) the single serologically detected class I molecule has unusual biochemical properties, including an association with non-ß2-microglobulin proteins on erythrocytes (16), allelic size heterogeneity, and differential affinity of association with ß2-microglobulin among allelic products (17); and 3) the class I gene appears to be sandwiched between the putative class II and class III regions, probably in close linkage to the TAP2 and lmp7 genes (6, 7). To gain more insight into the Xenopus model, and to better understand the evolutionary history of the MHC, we embarked on further analyses of the frog class I genes. Specifically, we asked whether the sequences of some class Ia alleles might reveal the basis for their unusual biochemical and genetic characteristics.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Xenopus laevis with the MHC haplotypes f, g (18), j (19), or r (17), and X. laevis-Xenopus gilli (LG) interspecies hybrid frogs with the haplotypes a/c (LG15) and b/d (LG3) (20) were used in all of these studies. X. laevis and X. gilli are both pseudotetraploid frogs (2n = 36) that are capable of cross-fertilization and that are believed to have diverged from a common ancestor between 1 and 10 million years ago (20). Outbred Rana pipiens purchased from Carolina Biological Supply (Burlington, NC) were used to prepare the cDNA library for isolation of class I cDNA clones.

Immunoprecipitation

The cell surface labeling of erythrocyte proteins, preparation of lysates, and immunoprecipitation procedures have been described previously in detail (17). The gel in Fig. 1Go displays radioiodinated cell surface class I proteins immunoprecipitated with Xenopus alloantisera. The proteins were first electrophoresed under nonreducing conditions, and then excised from the gel and run on another gel under reducing conditions (see legend).



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  		FIGURE 1. Xenopus class I proteins encoded by different haplotypes migrate differently on SDS gels. The gel on the left displays the cell surface-iodinated class I proteins under nonreducing conditions, with the bottom arrow denoting class I heavy chains (the top arrow identifies a molecule that associates with class Ia on the surface of RBC (16 )). The right panel displays the class I proteins excised from the nonreducing gel and run under reducing conditions. Under reducing conditions, the f and r allelic products migrate more slowly than the g and j proteins. (The lower m.w. species in each panel of REDUCED display the class I-associated molecule on erythrocytes.)

 
Cloning of alleles

The original class Ia clone, encoded from the f haplotype, was published (1). cDNA libraries were prepared from LG3 (b/d) liver and spleen RNA and LG15 (a/c) intestine and spleen RNA, as was done previously for f/f (21). The libraries were screened under high stringency conditions (1) with the presumed exon encoding the {alpha}3 domain. Only one unique clone was isolated from the LG3 library (b/d-2 in Fig. 3Go). a/c-1, a/c-2, and a/c-2' were isolated from the LG-15 library (Figs. 3Go and 4Go). The other class I cDNAs were cloned by PCR using spleen first strand cDNA as template (2). r/r was amplified with primers in the 5' and 3' untranslated regions ending on the start and stop codons, respectively. j/j and g/g were cloned by 3' RACE (2, 22); the 5' untranslated primer ending on the start codon and an adapter primer (2, 22) at the 3' end were used in the amplifications. The 5'UT primer was not successful with b/d-1 (LG3) cDNA; thus, an internal primer at the beginning of the {alpha}1 domain (1) and the 3' primer ending on the stop codon had to be used to obtain a PCR product. Thus, the a/c-2, b/d-2, g/g, and j/j class I cDNA clones are ~1.8 kb, a/c-2' is 2.7 kb (see text), a/c-1 is 3 kb, and the b/d-1 and r/r clones encompass only the coding regions (the mRNA encoding the r/r clone is 3 kb, see Fig. 2Go; b/d-1 was not selected from the cDNA library because a relatively low number of primary recombinants was obtained from this library and (also) because of the large size of the mRNA). The PCR products were cloned into the EcoRV site of pBluescript, and the coding regions were sequenced on both strands with universal (T3 and T7) and gene-specific primers (23). Sequences were manually aligned; dS and dN were calculated by the method of Nei and Gojobori (24).



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  		FIGURE 3. Protein alignment of class I allelic products from the eight defined Xenopus haplotypes and from R. pipiens. Sequences were aligned by eye and the presumed ß strand (S), and {alpha} helical (H) structure is derived from crystal structures of human and mouse class I molecules (37 ).*, Polymorphic residue in mammals; ~, evolutionarily conserved residue. Bold residues in the cytoplasmic domains can be phosphorylated in mammals (38 39 ).

 


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  		FIGURE 4. Nucleotide sequences of the 3' UT regions of f/f, g/g, and LG15 (a/c) alleles. a/c-1 is in the f/f (class I-1) allelic set, while a/c-2 (1.8 kb) and a/c-2' (2.7 kb) are alternatively spliced versions of the g/g (class I-2) allelic set. Note that the two forms of a/c-2 are identical in sequence until the poly(A) tail of the short form; nucleotides that differ from the g/g sequence are bolded.

 


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  		FIGURE 2. A, Northern blotting of class I alleles reveals two major classes of mRNAs in the intestine (3 and 1.8 kb) that correlate with the migration of proteins on the SDS gels (f and r at 3 kb, and g and j at 1.8 kb). The actin probe (36 ) was included as a positive control. The g* lane at the far right displays a longer exposure of the other g lane. B, Northern blotting of LG 15 (a/c) RNA and erythrocytic RNA from g/g reveals an alternatively spliced form of one of the class I alleles (top arrow in the class I-2 bracket). This higher m.w. species in the a/c lanes corresponds to the a/c-2' sequence in Fig. 4Go.

 
Rana pipiens library

A cDNA library was prepared from the poly(A)+ RNA from pooled spleens of eight outbred R. pipiens adults (21). Screening was performed under low stringency conditions with a probe encoding all three external domains of the X. laevis f/f class I cDNA (1). The coding sequences of two clones were sequenced in their entirety.

Northern blotting

Fifteen micrograms of total splenic RNA from animals homozygous for the f, g, j, and r haplotypes and from LG-15 (a/c) were electrophoresed and blotted, as described (12). Hybridizations were performed under high stringency conditions with the exon encoding the {alpha}3 domains as probe, as it was assumed (correctly, in retrospect) to be the most conserved region of class I mRNA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Recapitulation

Previously, we have shown that class I proteins encoded by the a, b, c, d, f, g, j, and r haplotypes migrate to different positions by SDS-PAGE (radioiodinated r, f, g, and j class I molecules in Fig. 1Go; also see Refs. 16, 17). f, r, and one allele from the LG15 and LG3 hybrids (a and b) migrate to a relatively higher position under reducing conditions than g, j, and the other allele from the LG hydrids (c and d). In previous experiments with defined crosses of Xenopus, all of these alleles have been shown to segregate as alleles at a single locus (1, 7, 16, 17). Such differences in the migration of allelic sets on SDS gels are attributable to some other biochemical property(ies) besides glycosylation since the proteins, after endoglycosidase-F digestion, still display the same m.w. differences (17). After the cloning of the class Ia gene from the f haplotype (1), we embarked on sequence analysis of all eight alleles to examine: 1) the biochemical basis for these two sizes of class I proteins; 2) the previously described unusual serological properties; and 3) their level of diversity.

Northern blot analysis

The original class Ia cDNA clone encoded by the f haploytpe is ~3 kb (1), about the size of f class Ia mRNA, as detected by Northern blotting (Fig. 2GoA). Surprisingly, while r class I mRNA is also of this high m.w., g and j display smaller-sized mRNAs (~1.8 kb). Note that the very weak band in the f and r lanes that is approximately the same size as the g and j bands is not a class I transcript, but the leading edge of 18S ribosomal RNA; no low m.w. transcript for class I has been cloned from either the f or r haplotypes, and a repeat of the northern with f/f RNA reveals no band at ~1.8 kb (Fig. 2GoB). Surprisingly, for the LG15 (a/c) interspecies hybrid, three bands are always detected by Northern blotting, one coincident with the f (3-kb) band and one with the g (1.8-kb) band, and a third band at ~2.7 kb (upper band marked with an arrow in the class I-2 bracket in Fig. 2GoB). This upper class I-2 band is also found in RNA isolated from g/g erythrocytes, but not from intestine (compare Fig. 2Go, A and B). Sequence of the 3'UT of the LG-15 2.7-kb band shows it to be an alternatively spliced form of the lower m.w. transcript (a/c-2' in Fig. 4Go); this species is found in high amounts in erythrocyte RNA for all class I-2 haplotypes (e.g., j and g) and in all tissues for one LG15 haplotype (Fig. 2GoB and data not shown). In total, the Northern blotting data suggest that there are distinct classes of mRNA for the Xenopus alleles (3 kb for f and r, and 1.8 (and 2.7) kb for g and j), and further that the X. gilli and X. laevis alleles also conform to these two classes; this is perhaps not surprising considering the relatively recent divergence time for X. laevis and X. gilli. The groupings detected by Northern blotting correspond precisely to the f, g, j, and r (and LG) protein data described above (without protein sequences or segregation analyses of the LG alleles, however, we have not shown definitively that all of the lower m.w. mRNAs encode the lower m.w. proteins detected under reducing conditions).

Sequencing of alleles

The coding regions of the eight alleles were sequenced, and the deduced amino acid sequences were aligned. There is great diversity in the PBD3 and, conversely, near invariance in the Ig-like {alpha}3 domain (Fig. 3Go). Surprisingly, the cytoplasmic, and to a lesser extent the transmembrane regions are divergent, and the alleles segregate into the same two groups resolved by the Northern blotting: f/f, r/r, a/c-1, and b/d-1 fall into one group and g/g, j/j, a/c-2, and b/d-2 into the other. This segregation into two classes is also apparent in the majority of the {alpha}2 PBD, but is not as noticeable in the {alpha}1 PBD; indeed, the {alpha}1 domain is much more diverse than the {alpha}2 domain, and even within allelic sets there is a high degree of diversity (Figs. 3Go and 5Go).



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  		FIGURE 5. Phylogenetic trees of the three extracellular domains employing the neighbor-joining method of Saito and Nei (40 ). Polymorphic residues were removed or not for analysis of the {alpha}1 and {alpha}2 domains. Besides the Xenopus alleles and R. pipiens sequences, the following class I genes were employed: human HLA-A2 (41 ) and HLA-B7 (42 ); mouse Kb (43 ) and Db (44 ); Salmon (sasa, 45 ); bird BF19 (26 ); lizard LC (46 ); and Xenopus nonclassical xnc1 (7 ).

 
Phylogenetic trees were constructed with the amino acid sequences of individual domains (Fig. 5Go). For the polymorphic {alpha}1 and {alpha}2 PBD trees were made with and without amino acids that bind peptides and are polymorphic in other species; because these residues are very diverse even within a species, it was logical to remove them from the analysis when comparing sequences over extremely long distances. In this way we hoped to reveal true phylogenetic distances in the absence of positive selection (25, 26). The distances between the Xenopus alleles in the {alpha}1 domain are similar to the distance between human and mouse genes. In contrast, the distances between the mouse and human sequences in the {alpha}3 domains are nearly the same as that in the other two domains, while the Xenopus {alpha}3 sequences cluster very tightly.

The frequency of mutations in the PBD, at least for the {alpha}1 domains, is higher than any set of alleles characterized to date (Table IGo and Fig. 6Go; see Ref. 25). However, while the number of nonsynonymous (dN) changes is very high, their frequency is the same as ({alpha}1) or not significantly greater than ({alpha}2) the synonymous substitutions (dS). Surprisingly, given the great distance between alleles in the {alpha}1 domain, and especially the cytoplasmic domains, the {alpha}3 domains are extremely well conserved, suffering neither synonymous nor nonsynonymous changes.


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  		Table I. Synonymous and nonsynonymous substitutions in the Xenopus class Ia alleles1

 


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  		FIGURE 6. Pairwise comparisons of the number of nucleotide differences among the eight Xenopus alleles in the three extracellular domains (see Ref. 47 ). Windows of five substitutions are indicated in each bar.

 
There are no major features of these sequences to result in the g and j proteins migrating faster than f and r on SDS gels, i.e., no differences in glycosylation sites, disulfide bonds, etc. It is likely that some property of the highly divergent CYT (not obvious!) is responsible for the differential gel migration.

R. pipiens sequences

Because of the interesting features of the Xenopus MHC, we thought it appropriate to begin to analyze MHC of other amphibians to determine whether the same basic phenomena occur in evolution. In addition, we hoped to provide stability to our phylogenetic trees by including sequences from other anuran amphibians. R. pipiens MHC class I cDNAs were obtained by cross-hybridization with the Xenopus class Ia probe under low stringency conditions. Sequences of the two clones show them to be allelic and most likely class Ia clones: these sequences are more similar to the Xenopus class Ia alleles in the PBD than the Ig-like {alpha}3 domain (Figs. 3Go and 5Go), suggesting, as was previously observed in chickens and Xenopus, that the PBD are more conserved over large phylogenetic distances (1, 26). In addition, amino acid differences between the two presumed Rana alleles are primarily concentrated in the amino acids that interact with peptide, and the invariant class Ia amino acids that anchor the ends of peptides in the groove are generally conserved or substituted for by amino acids of a similar composition. The genetic distance between the Rana and the Xenopus class Ia sequences is large, especially in the {alpha}3 domain, consistent with an old divergence between the two anuran families (at least 150 million years) (27).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Ancient alleles: diverse yet conserved

It is clear from our work that different functional regions of the Xenopus class Ia alleles evolve at different rates, a result consistent with other evolutionary studies (e.g., see Ref. 26). Three findings particularly deserve attention: 1) the class I domains that are most dissimilar are the cytoplasmic regions, followed by {alpha}1, {alpha}2, and {alpha}3; 2) while there is great diversity in the PBD among alleles, the number of nonsynonymous substitutions in the codons that encode peptide-binding residues is not significantly higher than synonymous ones; and 3) despite the high rate of divergence, there are very few substitutions of any type in the {alpha}3 domain.

Although it is difficult to interpret all of these data, we can understand some facets of the work. Comparisons among the Xenopus alleles are more similar to data obtained when sequences are compared between mouse and human class I genes; in fact, the distances between the {alpha}1 domains (and the CYT regions) of the Xenopus alleles, as mentioned, are similar to those between mouse and human class I isotypes (Figs. 3Go and 5Go). Considering that mice and humans diverged at least 60–65 million years ago with some estimates of up to 100 million years (28), the Xenopus alleles may have emerged at around the same time as the common ancestor of most MHC-linked mammalian class I genes.

If the alleles and the allelic lineages are so old, then why are the {alpha}3 domains of the Xenopus alleles so well conserved, to the point of homogenization? One might argue that since all of the class Ia allelic forms must associate with ß2-microglobulin and perhaps other frog-specific proteins, that the {alpha}3 domain must remain essentially invariant. However, the distances between the mouse and human isotypes are not that much smaller in {alpha}3 as compared with {alpha}1 and {alpha}2 domains, and class I proteins from these species presumably also have similar requirements in their associations with other proteins. Furthermore, this interpretation cannot account for the extremely low number of synonymous changes detected, which are expected to be similar to the frequency found in residues in the {alpha}1 and {alpha}2 domains. Thus, it is difficult to even speculate at this time as to why the Xenopus {alpha}3 domains are so remarkably similar. It appears that the sequences are homogenized by interallelic conversion/recombination, as has been shown for intronic regions of mammalian class I genes (29). Such homogenization can also be seen between the allelic lineages in some areas of the 3'UT regions (Fig. 4Go). Because Ig-like {alpha}3 domains are often used to detect phylogenetic distances among class I genes, one obviously must be wary of the significance of such trees.

The general divergence trend of CYT > {alpha}1 > {alpha}2 > {alpha}3 is also true of the Xenopus class Ib (XNC) genes, suggesting that the pressures placed on class Ia alleles also act on different class I genes (7). The differences in the sequence diversity in the {alpha}1 and {alpha}2 PBD suggest that the former domain is selected primarily for its diversity and the latter for another function, perhaps for interactions with other components of the class I-processing machinery. Furthermore, it should be pointed out that although the sequences of CYT regions of the class Ia alleles are very different between the two allelic classes, within each class the transmembrane/CYT, like the {alpha} 3 domains, are very conserved. The one exception to this rule is that the g and j genes have accumulated a few changes not found in b/d-2 and a/c-2; the most likely explanation for this finding is that the two alleles from the LG hybrids are derived from X. gilli and have been separated from their counterparts in X. laevis, g and j, for a longer time than any of the alleles from the other (f, r) group, which are probably all derived from X. laevis. Perhaps the cytoplasmic tails of the two lineages are important for a particular function, e.g., interaction with cytosolic proteins.

In mammalian class I molecules, there are significantly more nonsynonymous changes over synonymous ones in the codons specifiying residues that interact with peptides, supporting the idea of overdominant selection on MHC proteins (25, 30). In the Xenopus {alpha}1 domains, the frequency of mutation is 2–3 times that of the human and mouse, and it is likely that the synonymous sites have become saturated. Thus, while dN is not significantly greater than dS, we think this is most likely due to the age of the alleles, not to a lack of selection. More recently derived Xenopus alleles must be examined to test this notion.

Genes vs alleles

The cytoplasmic domains and 3'UT are quite different between the two sets of alleles, suggesting that these sets were originally different genes rather than alleles, although they can segregate as alleles at a single locus. The same lineages (f, r on the one hand, and g, j on the other) have been revealed for lmp7 (31) and TAP2 genes (7). The simplest interpretation is that there was a long separation of these lineages, followed by an introgression sometime in the history of Xenopus as a genus (32). Because the genus Xenopus has been estimated to be more than 100 x 106 yr old, such a finding might not be that surprising and introgression indeed has been documented in this taxon (33); speciation via allopolyploidization, postulated to be common in Xenopus, would favor introgression. However, our preliminary work has shown that the two lmp7 allelic lineages are maintained in approximately equal numbers in a large number of species examined (M. Nonaka, M. Flajnik, and L. Du Pasquier, unpublished), suggesting rather that the lineages may be under balancing selection. We hypothesize that the two allelic sets may function in presentation of very different sets of peptides. The closer genetic linkage of lmp7/TAP2/class Ia in Xenopus (and other vertebrates such as birds (34) and zebrafish (35)) as compared with mammals may have allowed coselection of certain allelic lineages that are perpetuated and maintained in the population (6). Future studies will be aimed at examining the presence of the allelic lineages among diverse Xenopus species, and further to analyze whether there is a functional pressure to maintain the class I processing and presenting genes in a tight linkage group.


   Acknowledgments
 
We thank Jim Kaufman for his suggestions in a review of an early version of the manuscript, especially for the idea of removing polymorphic amino acids in the phylogenetic tree analysis. We also thank Marilyn Diaz for discussions of the data and Pat Washington for secretarial assistance.


   Footnotes
 
1 M.F.F. is supported by National Institutes of Health Grant AI 27877. L.D.P. is supported by the Basel Institute for Immunology, which was founded and is totally supported by Hoffman La-Roche. M.K. is supported by CREST of Japan Science and Technology Corporation and by grants-in-aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan. Back

2 Address correspondence and reprint requests to Dr. Martin F. Flajnik, Department of Microbiology and Immunology, University of Maryland, 655 West Baltimore Street, Room 3-056, Baltimore, MD 21201-5159. E-mail address: Back

3 Abbreviations used in this paper: PBD, peptide-binding domain; CYT, cytoplasmic region; UT, untranslated. Back

Received for publication July 10, 1998. Accepted for publication July 19, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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