Characterization of Two Avian MHC-Like Genes Reveals an Ancient Origin of the CD1 Family
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Characterization of two avian MHC-like genes reveals an ancient origin of the CD1 family Marcia M. Miller†, Carren Wang‡, Emilio Parisini‡§, Ricardo D. Coletta¶, Ronald M. Goto†, Stella Y. Lee‡, Duarte C. Barral‡, Maria Townes‡, Carme Roura-Mir‡, Heide L. Ford¶, Michael B. Brenner‡, and Christopher C. Dascher‡ʈ ‡Division of Rheumatology, Immunology, and Allergy, Brigham and Women’s Hospital, Harvard Medical School, Smith 516C, 1 Jimmy Fund Way, Boston, MA 02115; ¶Department of Obstetrics and Gynecology, Box B198, University Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, CO 80262; †Division of Molecular Biology, Beckman Research Institute of the City of Hope, 1450 East Duarte Road, Duarte, CA 91010; and §Department of Cancer Immunology and AIDS, Dana–Farber Cancer Institute, Smith 1036, 1 Jimmy Fund Way, Boston, MA 02115 Edited by Max D. Cooper, University of Alabama, Birmingham, AL, and approved April 20, 2005 (received for review January 5, 2005) Many of the genes that comprise the vertebrate adaptive immune Although clearly related to the MHCI family of proteins, CD1 has system are conserved across wide evolutionary time scales. Most evolved to bind and present lipid antigens to T cells in a manner notably, homologs of the mammalian MHC gene family have been analogous to the paradigm established for peptides and MHC (4). found in virtually all jawed vertebrates, including sharks, bony In addition, many of the CD1-restricted T cells recognize microbial fishes, reptiles, and birds. The CD1 family of antigen-presenting lipids and glycolipids and have a proinflammatory phenotype with molecules are related to the MHC class I family but have evolved antimicrobial effector functions (5). These data suggest an impor- to bind and present lipid antigens to T cells. Here, we describe two tant role for CD1 in the immune response to infection and other highly divergent nonclassical MHC class I genes found in the diseases (6). chicken (Gallus gallus) that have sequence homology to the mam- Based on structural and sequence similarities, it is thought that malian CD1 family of proteins. One of the chicken CD1 genes CD1 and MHCI diverged from a common ancestral gene during the expresses a full-length transcript, whereas the other has multiple early evolution of vertebrates (7). One of our goals has been to splice variants. Both Southern blot and single nucleotide polymor- investigate the origin of the CD1 genes by identifying homologs in phism analysis indicates that chicken CD1 is relatively nonpoly- more primitive animal species. These studies may allow a better morphic. Moreover, cross-hybridizing bands are present in other appreciation of the role that CD1 has played in the evolution of the bird species, suggesting broad conservation in the avian class. immune system. However, early ancestral or transitional forms of Northern analysis of chicken tissue shows a high level of CD1 CD1 in extant nonmammalian vertebrate species have thus far expression in the bursa and spleen. In addition, molecular model- remained elusive. Birds are generally accepted to be the living ing predicts that the potential antigen-binding pocket is probably descendants of Theropod dinosaurs and, therefore, represent an hydrophobic, a universal characteristic of CD1 molecules. Genomic ancient lineage of reptiles (8). Here, we describe two highly analysis indicates that the CD1 genes are located on chicken divergent nonclassical MHCI genes in the chicken (Gallus gallus) chromosome 16 and maps to within 200 kb of the chicken MHC B that share a common set of features typically found in the CD1 gene locus, suggesting that CD1 genes diverged from classical MHC family. These genes reveal an ancient origin for CD1 and thus genes while still linked to the major histocompatibility complex provide a unique opportunity for comparative analysis. locus. The existence of CD1 genes in an avian species suggests that the origin of CD1 extends deep into the evolutionary history of Materials and Methods terrestrial vertebrates. Animal Tissues. Tissue samples for Northern analysis and RT-PCR were obtained from Charles River SPAFAS (Wilmington, MA). antigen presentation ͉ comparative immunology͞evolution ͉ cell surface Camperos chicken genomic DNA samples (starting from the left in molecules fig. 3A in ref. 9) with previously described genotypes were used: 14 from the female parent line (f2, f3, f5, f6, f7, f8, f10, f11, f12, f13, he essential elements that define the adaptive immune system f14, f15, f16, and f18) and three from the male parent line (m1, m2, Temerged early in the course of vertebrate evolution. This and m3). Human genomic DNA was obtained from the MCF7 cell finding is evidenced by the fact that virtually all jawed vertebrates line. Other avian genomic DNA samples used for Fig. 3C were share a conserved set of genes that have clear evolutionary ho- extracted as described in ref. 10 and are listed in Table 2, which is mologs with the extensively characterized mammalian immune published on the PNAS web site. system (1). One of the most conserved parts of the adaptive cellular immune system are the genes that make up the major histocom- Genes and Sequence Analysis. The non-human and non-mouse patibility complex (MHC) locus. The MHCI genes encode highly GenBank EST library database was searched with murine CD1d polymorphic cell-surface glycoproteins that are expressed on all protein sequence (GenBank accession no. P11609) encompassing ␣ ␣ somatic cells, whereas MHCII has a more restricted expression the 1- 2 domains. High scoring G. gallus candidates were re- pattern, being expressed primarily on professional antigen- screened against the same databases to extract other close matches presenting cells. It is well established that the MHCI and MHCII and are listed in Table 3, which is published as supporting infor- proteins have the capacity to bind and present pathogen-derived peptides to specific CD8ϩ and CD4ϩ T cells, respectively. The This paper was submitted directly (Track II) to the PNAS office. MHC gene family is therefore the core of the cell-mediated immune system’s antigen-presenting function and is critical for host resis- Abbreviation: MHC, major histocompatibility complex. tance to microbial infection. Data deposition: The sequence reported in this paper has been deposited in the GenBank database [AY375530 (chCD1-2 complete cDNA sequence), AY874074–AY874080 (chCD1-1 The CD1 genes represent a third family of cell-surface proteins, splice variants 1–7), AY939845 and AY939846 (additional G. gallus genomic DNA sequences in addition to MHCI and MHCII, which have the capacity to for chCD1-1 intron 2 and chCD1-2 intron 4), and DQ007238 and DQ007237 (probes to map present antigens to T cells. The human CD1 gene family is chicken MHCII and C4 genes)]. composed of five nonpolymorphic genes (CD1A,-B,-C,-D, and -E) See Commentary on page 8399. that are located in a small cluster on human chromosome 1 and are ʈTo whom correspondence should be addressed. E-mail: [email protected]. therefore unlinked to the MHC locus on chromosome 6 (2, 3). © 2005 by The National Academy of Sciences of the USA 8674–8679 ͉ PNAS ͉ June 14, 2005 ͉ vol. 102 ͉ no. 24 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0500105102 Downloaded by guest on September 29, 2021 mation on the PNAS web site. A candidate cDNA sequence to PCR amplify bursa-derived cDNA. Amplification of chCD1-1 (BG713169) that appeared in multiple EST libraries (designated revealed multiple products ranging in size from 700 to 1,300 bp with K24) was chosen for more extensive analysis and coded for a a prominent band of 900 bp (Fig. 1B, lane 2). We extracted the DNA SEE COMMENTARY putative protein we designated chCD1–2 (see Results and Discus- fragments, derived clones, and sequenced the inserts. Fig. 1C shows sion for nomenclature). Multiple sequence alignment and neighbor- a schematic summary of the aligned chCD1-1 sequence inserts from joining trees were made with CLUSTAL-X and MEGA3 (11). The first a random selection of seven clones. These sequence inserts reveal draft of the red jungle fowl (G. gallus) genome sequence was used multiple deletions in the chCD1-1 cDNA sequences. As shown in for genomic and single nucleotide polymorphism analysis (12, 13). Fig. 1B (lane 2, arrow), the most intense band is Ϸ900 bp, which is Sequencing of two gaps in the chCD1 locus of G. gallus red jungle consistent with the size of the predominant 872-bp splice variant fowl genomic DNA (Table 2) was accomplished with primers 7 and (Fig. 1C, clones chCD1-1.2, -1.3, -1.4, and -1.5). Variations in the 8forchCD1-1 intron 2 and primers 9 and 10 for chCD1-2 intron 4, size of CD1 mRNA transcripts have been described (15–17). Some which are listed in Table 4, which is published as supporting of these variants also lack the transmembrane domain and result in information on the PNAS web site. soluble forms of CD1 (15, 17, 18). The biological significance of these variants is unclear, but they may be a mechanism for post- RT-PCR of chCD1-1 and chCD1-2 mRNA. Total RNA prepared from transcriptional regulation. the bursa and spleen of Charles River SPAFAS chickens used for In contrast to the chCD1-1 gene described above, a single band Northern analysis (see below) was used to generate cDNA with the was obtained for the chCD1-2 PCR reaction, which sequencing Superscript RT-PCR kit by the oligo(dT) priming method accord- confirmed as a full-length 1044-bp ORF identical to the original ing to the manufacturer’s instructions (Invitrogen). All primer chCD1-2 K24 EST sequence (AY375530). A similar result to Fig. sequences are listed in Table 4. Bursa cDNA was used as template 1B was obtained for both chCD1 genes by using spleen cDNA (data for PCR of full-length chCD1-1 and chCD1-2.