MIC genes in non‐human primates

Abstract

MIC molecules belong to the immunoglobulin superfamily, are encoded within the MHC region and are recognized by γ/δ T‐cell receptors. In humans, at least two functional genes (MIC‐A* and MIC‐B*) and two pseudogenes (MIC‐C* and MIC‐D*) exist. Functional MIC gene copies are characterized by a high degree of polymorphism, while pseudogenes bear several debilitating mutations either in the putative extracellular region or in the transmembrane region of the molecule. In this study we sequenced these segments of MIC genes in seven non‐human primates in order to determine whether debilitating mutations were present. All the MIC primate genes studied were highly homologous to their human counterparts, and cystein residues involved in the maintenance of the immunoglobulin‐like structure were highly conserved. Furthermore, none of the MIC genes studied contained stop codons in the extracellular or transmembrane segments of the molecule, which suggests that at least one functionnal gene copy exists in non‐human primates.A distinct family of MHC immunoglobulin‐like genes was recently identified within the MHC class I region in humans (Bahram et al., 1994; Leelayuwat et al., 1994). Members of this MIC (MHC class I chain related) gene family belong to the immunoglobulin superfamily. Similar to classical class I MHC genes, they are characterized by three distinct extracellular domains (α1–3), a transmembrane (TM) segment and a cytoplasmic segment, each encoded by a separate exon (Bahram et al., 1994; Bahram et al., 1996). Other similarities between MIC genes and classical MHC genes include a high degree of polymorphism (Fodil et al., 1996; Pellet et al., 1997) and recognition by T‐cell receptors (Groh et al., 1998). These findings suggest that the putative MIC‐A* chain has evolved for a function that is related to, but quite distinct from, that of typical MHC class I chains.Southern blot analysis showed that members of the MIC family exist in most mammalian species (Bahram et al., 1994). Nevertheless it is still unclear whether functional copies of these genes exist in these species, since MIC pseudogenes (MIC‐C* and MIC‐D*) also exist in humans. Usually, these MIC pseudogenes bear several stop codons in the putative extracellular or TM segments of the gene. In this report, we sequenced the α1, α 2, α 3 and, when possible, TM segments of one individual’s MIC genes for three Hominidae, three Cercopithecidae and one Hylobatidae species in order determine whether debilitating mutations were present in these exons.Genomic DNA was graciously provided by Professor Barré Sinoussi (Institut Pasteur, Paris, France). The MIC variants were identified by direct sequencing of polymerase chain reaction (PCR) amplified gene segments including the α1 (exon 2), α2 (exon 3), α3 (exon 4) and TM exons, as previously described for human MIC‐A* genes (Fodil et al. 1996). The sequences were obtained by using the Dye Primer Sequencing kit (ABI, USA) which enabled us to distinguish the heterozygoous positions in 99% of cases. When heterozygosity was detected, PCR products were subcloned in a plasmid vector (TA Cloning kit; Invitrogen, the Netherlands). Several clones were sequenced separately until the definitive sequence of both alleles was obtained. Optimal alignment between primate MIC genes and the human MIC‐A* gene is shown in Fig. 1.As expected, the MIC sequences obtained from Hominidae species showed a high degree of homology to their human counterparts, varying between 86 and 97% (Fig. 1). For all Hominidae species, we also obtained the sequence of the TM segment (data not shown), where stop codons occur in human MIC‐A* genes. We did not find any stop codon in any exons sequenced. Furthermore, none of the four cysteins involved in the maintenance of the immunoglobulin‐like structure of the molecule was replaced by other amino acid residues.Among the DNAs analysed, only the Pongo pygmaeus (orang utan) and the Hylobate Par (gibbon) proved to be heterozygous, which suggests that, as in humans, the MIC gene could be polymorphic in these species.For most of the non‐Hominidae species analysed (gibbon, Cercopithecus patas (patas) and Papio sp. (baboon)), optimal alignments suggest that the coding sequences are shorter than in humans. However, as in the Hominidae, the immunoglobulin‐like structure seems preserved since cysteins at positions 96, 164, 202 and 259 were not replaced by other residues even in regions of the molecule that had an important interspecies polymorphism (position 164).These data show that at least one functional MIC gene copy without debilitating mutations in regions affected by this type of modification in human MIC pseudogenes exists. Furthermore, as in humans, for some species these genes are polymorphic and there seems to be a strong evolutionary pressure for the maintenance of an immunoglobulin‐like structure for these molecules. Taken together, these data provide evidence that MIC genes are probably functional and play an important role in host defence in non‐human primates. Further studies investigating the expression of these molecules in these species will be necessary to provide definitive support for this hypothesis.