Characterization of a Nonclassical Class I MHC Gene in a Reptile, the Gala´ pagos Marine Iguana (Amblyrhynchus cristatus ) Scott Glaberman1*, Louis Du Pasquier2, Adalgisa Caccone1 1 Department of Ecology and Evolutionary Biology and the Yale Institute for Biospheric Studies, Yale University, New Haven, Connecticut, United States of America, 2 Institute of Zoology and Evolutionary Biology, University of Basel, Basel, Switzerland Abstract Squamates are a diverse order of vertebrates, representing more than 7,000 species. Yet, descriptions of full-length major histocompatibility complex (MHC) genes in this group are nearly absent from the literature, while the number of MHC studies continues to rise in other vertebrate taxa. The lack of basic information about MHC organization in squamates inhibits investigation into the relationship between MHC polymorphism and disease, and leaves a large taxonomic gap in our understanding of amniote MHC evolution. Here, we use both cDNA and genomic sequence data to characterize a class I MHC gene (Amcr-UA) from the Gala´ pagos marine iguana, a member of the squamate subfamily Iguaninae. Amcr-UA appears to be functional since it is expressed in the blood and contains many of the conserved peptide-binding residues that are found in classical class I genes of other vertebrates. In addition, comparison of Amcr-UA to homologous sequences from other iguanine species shows that the antigen-binding portion of this gene is under purifying selection, rather than balancing selection, and therefore may have a conserved function. A striking feature of Amcr-UA is that both the cDNA and genomic sequences lack the transmembrane and cytoplasmic domains that are necessary to anchor the class I receptor molecule into the cell membrane, suggesting that the product of this gene is secreted and consequently not involved in classical class I antigen-presentation. The truncated and conserved character of Amcr-UA lead us to define it as a nonclassical gene that is related to the few available squamate class I sequences. However, phylogenetic analysis placed Amcr-UA in a basal position relative to other published classical MHC genes from squamates, suggesting that this gene diverged near the beginning of squamate diversification. Citation: Glaberman S, Du Pasquier L, Caccone A (2008) Characterization of a Nonclassical Class I MHC Gene in a Reptile, the Gala´pagos Marine Iguana (Amblyrhynchus cristatus). PLoS ONE 3(8): e2859. doi:10.1371/journal.pone.0002859 Editor: Robert DeSalle, American Museum of Natural History, United States of America Received January 18, 2008; Accepted June 24, 2008; Published August 6, 2008 Copyright: 2008 Glaberman et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding for this project was provided by the Yale Institute for Biospheric Studies (YIBS). Work was also supported by pilot study funds from the YIBS Center for Field Ecology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: scott.glaberman@gmail.com Introduction Class I major histocompatibility complex (MHC) molecules are well known for their pivotal role in the recognition of altered self cells (e.g. virus-infected cells) by T cytotoxic (TC) cells. In this process, short peptide fragments derived from pathogens within the host cell are bound to class I receptor structures and transported to the cell surface. Here, the paired receptor/antigen complex is recognized by CD8+ TC cells, initiating a sequence of events that ultimately leads to the lysis of the infected host cell [1,2]. Since class I antigen presentation is essential for the cell- mediated clearance of intracellular pathogens, it is not surprising that these molecules are expressed on most somatic cells and in the majority of host tissue types [3]. An additional characteristic of the genes encoding class I receptors is a high level of polymorphism, which enables the host to recognize and bind a wide array of foreign peptides [4]. However, as the number of descriptions of vertebrate MHC loci has increased, it has become clear that many class I genes do not possess the features described above. The classification of vertebrate class I loci has been divided into two general categories: classical (or class Ia) and nonclassical (or class Ib). Classical genes are those found within the MHC region, possessing high polymorphism, strong and wide expression, and are involved in the presentation of endogenous antigens to TC cells. Classical loci have been well characterized in humans (HLA- A, -B, and -C) and mice (H-2K, D, and L) but are also well described in some other mammals and in fish, and to a lesser to degree in amphibians, birds, and non-avian reptiles. Nonclassical genes can be located inside or outside of the MHC and typically possess little or no polymorphism and weak expression that is often limited to specific tissue types. Class Ib molecules are known to perform a diverse array of functions which include the recognition of antigenic lipids (human and mouse CD1) [5], the binding and transportation of classical class I molecules within the host cell (human HLA-E and mouse Qa-1b) [6], and the targeting of evolutionarily conserved protein epitopes of pathogens (mouse H2-M3) [7]. There are even nonclassical loci whose functions are not related to immunity. For example, the human HFE molecule is known to play an important role in iron metabolism [6]. Phylogenetic studies depict an extremely complex evolutionary history of class I genes, where both classical and nonclassical loci exhibit little orthology across mammals as well as among vertebrates in general [8]. Since particular species, or closely PLoS ONE | www.plosone.org 1 August 2008 | Volume 3 | Issue 8 | e2859