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Characterisation of RT1-E2, a multigenic family of highly conserved rat non-classical MHC class I molecules initially identified in cells from immunoprivileged sites.

Lau P, Amadou C, Brun H, Rouillon V, McLaren F, Le Rolle AF, Graham M, Butcher GW, Joly E - BMC Immunol. (2003)

Bottom Line: The RT1n MHC haplotype (found in BN rats) carries a single RT1-E2 locus, which lies in the RT1-C/E region of the MHC and displays the typical exon-intron organisation and promoter features seen in other rat MHC class I genes.Compared to other class I molecules, this suggests that RT1-E2 molecules may associate with well defined sets of ligands.Several characteristics point to a certain similarity to the mouse H2-Qa2 and human HLA-G molecules.

View Article: PubMed Central - HTML - PubMed

Affiliation: IFR Claude de Préval, INSERM U563, CHU Purpan, 31300 Toulouse, France. laupoui@pasteur.fr

ABSTRACT

Background: So-called "immunoprivileged sites" are tissues or organs where slow allograft rejection correlates with low levels of expression of MHC class I molecules. Whilst classical class I molecules are recognised by cytotoxic T lymphocytes (CTL), some MHC class I molecules are called "non-classical" because they exhibit low polymorphism and are not widely expressed. These last years, several studies have shown that these can play different, more specialised roles than their classical counterparts. In the course of efforts to characterise MHC class I expression in rat cells obtained from immunoprivileged sites such as the central nervous system or the placenta, a new family of non-classical MHC class I molecules, which we have named RT1-E2, has been uncovered.

Results: Members of the RT1-E2 family are all highly homologous to one another, and the number of RT1-E2 loci varies from one to four per MHC haplotype among the six rat strains studied so far, with some loci predicted to give rise to soluble molecules. The RT1n MHC haplotype (found in BN rats) carries a single RT1-E2 locus, which lies in the RT1-C/E region of the MHC and displays the typical exon-intron organisation and promoter features seen in other rat MHC class I genes. We present evidence that: i) RT1-E2 molecules can be detected at the surface of transfected mouse L cells and simian COS-7 cells, albeit at low levels; ii) their transport to the cell surface is dependent on a functional TAP transporter. In L cells, their transport is also hindered by protease inhibitors, brefeldin A and monensin.

Conclusions: These findings suggest that RT1-E2 molecules probably associate with ligands of peptidic nature. The high homology between the RT1-E2 molecules isolated from divergent rat MHC haplotypes is particularly striking at the level of their extra-cellular portions. Compared to other class I molecules, this suggests that RT1-E2 molecules may associate with well defined sets of ligands. Several characteristics point to a certain similarity to the mouse H2-Qa2 and human HLA-G molecules.

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Alignment of RT1-E2 protein sequences deduced from the cDNA sequences summarised in Table 1with those of other rat class I sequences. For E2ba (acc. AJ537439), the translated sequence ends prematurely because of a frameshift at the start of exon 5 (see text). The line E2ba* shows the notional downstream translation of the cDNA sequence if this frameshift was not present. In the sequences from which the RT1-E2g (acc. AJ243338) sequence was compiled (p57, p4.4, p4.6), underlined residues correspond to positions that differ from all the other RT1-E2 sequences, and could therefore be due to either PCR mutations or true allelic differences. Others accession numbers are as follows: E2bu: AJ537420, E2bl: AJ537417, E2an (deduced from genomic): AJ315490, E2al: AJ276126, E2cl: AJ537418, E2dl: AJ537419, E2dc: AJ537441, Eav1: AJ537440, Eu: AJ306619, Al: L26224, Aa: M31018, Au: X82106. Alignment of the corresponding DNA sequences is available as supplementary data or upon request.
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Figure 2: Alignment of RT1-E2 protein sequences deduced from the cDNA sequences summarised in Table 1with those of other rat class I sequences. For E2ba (acc. AJ537439), the translated sequence ends prematurely because of a frameshift at the start of exon 5 (see text). The line E2ba* shows the notional downstream translation of the cDNA sequence if this frameshift was not present. In the sequences from which the RT1-E2g (acc. AJ243338) sequence was compiled (p57, p4.4, p4.6), underlined residues correspond to positions that differ from all the other RT1-E2 sequences, and could therefore be due to either PCR mutations or true allelic differences. Others accession numbers are as follows: E2bu: AJ537420, E2bl: AJ537417, E2an (deduced from genomic): AJ315490, E2al: AJ276126, E2cl: AJ537418, E2dl: AJ537419, E2dc: AJ537441, Eav1: AJ537440, Eu: AJ306619, Al: L26224, Aa: M31018, Au: X82106. Alignment of the corresponding DNA sequences is available as supplementary data or upon request.

Mentions: To identify which MHC class Ib molecules were predominantly expressed, we carried out RT-PCR on mRNA isolated from early stage LEW neurospheres, using PCR primers designed to anneal to regions that are conserved in most rat MHC class Ia and Ib transcripts [27-29] (see M&M). A first screen was performed by transient transfection of COS-7 cells with 18 plasmid clones that contained DNA inserts of the expected 1.1 kb size. Thirteen clones drove expression of a molecule that behaved serologically like the RT1-Al molecule, whilst the other five led to at best very faint staining with the MRC OX-18 antibody, and no staining with other RT1-Al-reactive mAbs, including F16.4.4. Sequencing of these five clones revealed that one corresponded to the RT1-Cl antigen [30], while the other four were highly homologous (>98%) to one another and to a sequence we had previously reported and designated RT1-Eg [31]. These clones, and many others identified from other sources representing a total of six MHC haplotypes as well as from additional screening of PCR-generated cDNA clones from LEW neurospheres and from LEW placenta, are listed in Table 1. Comparative analysis of all these sequences allowed us to identify and discount the numerous PCR-generated mutations, as well as the PCR-generated hybrids, and to attempt to classify them according to their relatedness over the sequences from exons 4 to 8 (residues 181–356 of the protein alignment shown on Figure 2). From previous experience [27,29], we know that this portion of the genes for MHC class I molecules, which codes for the α3 domain, the trans-membrane and the intra-cytoplasmic regions is often the best locus indicator when comparing sequences from different MHC haplotypes. As can be seen from Figure 3, phylogenetic analysis of our collection of sequences confirmed their close relatedness, and their separation from the group formed by RT1-A molecules. For 11 out of 13 sequences within this group, there is an even more striking level of homology over the first 180 amino acids which constitute the antigen binding groove, with very rare variations observed at only five positions. Over the same region, the remaining two sequences, RT1-Eu and RT1-Eav1, fall on the same branch of the tree, but with a relatively low support value. These two sequences are, however, more closely related to other classical RT1-A sequences, or even to human HLA-A2 or -G, or mouse H2-Q7 or -Q8/Q9.


Characterisation of RT1-E2, a multigenic family of highly conserved rat non-classical MHC class I molecules initially identified in cells from immunoprivileged sites.

Lau P, Amadou C, Brun H, Rouillon V, McLaren F, Le Rolle AF, Graham M, Butcher GW, Joly E - BMC Immunol. (2003)

Alignment of RT1-E2 protein sequences deduced from the cDNA sequences summarised in Table 1with those of other rat class I sequences. For E2ba (acc. AJ537439), the translated sequence ends prematurely because of a frameshift at the start of exon 5 (see text). The line E2ba* shows the notional downstream translation of the cDNA sequence if this frameshift was not present. In the sequences from which the RT1-E2g (acc. AJ243338) sequence was compiled (p57, p4.4, p4.6), underlined residues correspond to positions that differ from all the other RT1-E2 sequences, and could therefore be due to either PCR mutations or true allelic differences. Others accession numbers are as follows: E2bu: AJ537420, E2bl: AJ537417, E2an (deduced from genomic): AJ315490, E2al: AJ276126, E2cl: AJ537418, E2dl: AJ537419, E2dc: AJ537441, Eav1: AJ537440, Eu: AJ306619, Al: L26224, Aa: M31018, Au: X82106. Alignment of the corresponding DNA sequences is available as supplementary data or upon request.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
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Figure 2: Alignment of RT1-E2 protein sequences deduced from the cDNA sequences summarised in Table 1with those of other rat class I sequences. For E2ba (acc. AJ537439), the translated sequence ends prematurely because of a frameshift at the start of exon 5 (see text). The line E2ba* shows the notional downstream translation of the cDNA sequence if this frameshift was not present. In the sequences from which the RT1-E2g (acc. AJ243338) sequence was compiled (p57, p4.4, p4.6), underlined residues correspond to positions that differ from all the other RT1-E2 sequences, and could therefore be due to either PCR mutations or true allelic differences. Others accession numbers are as follows: E2bu: AJ537420, E2bl: AJ537417, E2an (deduced from genomic): AJ315490, E2al: AJ276126, E2cl: AJ537418, E2dl: AJ537419, E2dc: AJ537441, Eav1: AJ537440, Eu: AJ306619, Al: L26224, Aa: M31018, Au: X82106. Alignment of the corresponding DNA sequences is available as supplementary data or upon request.
Mentions: To identify which MHC class Ib molecules were predominantly expressed, we carried out RT-PCR on mRNA isolated from early stage LEW neurospheres, using PCR primers designed to anneal to regions that are conserved in most rat MHC class Ia and Ib transcripts [27-29] (see M&M). A first screen was performed by transient transfection of COS-7 cells with 18 plasmid clones that contained DNA inserts of the expected 1.1 kb size. Thirteen clones drove expression of a molecule that behaved serologically like the RT1-Al molecule, whilst the other five led to at best very faint staining with the MRC OX-18 antibody, and no staining with other RT1-Al-reactive mAbs, including F16.4.4. Sequencing of these five clones revealed that one corresponded to the RT1-Cl antigen [30], while the other four were highly homologous (>98%) to one another and to a sequence we had previously reported and designated RT1-Eg [31]. These clones, and many others identified from other sources representing a total of six MHC haplotypes as well as from additional screening of PCR-generated cDNA clones from LEW neurospheres and from LEW placenta, are listed in Table 1. Comparative analysis of all these sequences allowed us to identify and discount the numerous PCR-generated mutations, as well as the PCR-generated hybrids, and to attempt to classify them according to their relatedness over the sequences from exons 4 to 8 (residues 181–356 of the protein alignment shown on Figure 2). From previous experience [27,29], we know that this portion of the genes for MHC class I molecules, which codes for the α3 domain, the trans-membrane and the intra-cytoplasmic regions is often the best locus indicator when comparing sequences from different MHC haplotypes. As can be seen from Figure 3, phylogenetic analysis of our collection of sequences confirmed their close relatedness, and their separation from the group formed by RT1-A molecules. For 11 out of 13 sequences within this group, there is an even more striking level of homology over the first 180 amino acids which constitute the antigen binding groove, with very rare variations observed at only five positions. Over the same region, the remaining two sequences, RT1-Eu and RT1-Eav1, fall on the same branch of the tree, but with a relatively low support value. These two sequences are, however, more closely related to other classical RT1-A sequences, or even to human HLA-A2 or -G, or mouse H2-Q7 or -Q8/Q9.

Bottom Line: The RT1n MHC haplotype (found in BN rats) carries a single RT1-E2 locus, which lies in the RT1-C/E region of the MHC and displays the typical exon-intron organisation and promoter features seen in other rat MHC class I genes.Compared to other class I molecules, this suggests that RT1-E2 molecules may associate with well defined sets of ligands.Several characteristics point to a certain similarity to the mouse H2-Qa2 and human HLA-G molecules.

View Article: PubMed Central - HTML - PubMed

Affiliation: IFR Claude de Préval, INSERM U563, CHU Purpan, 31300 Toulouse, France. laupoui@pasteur.fr

ABSTRACT

Background: So-called "immunoprivileged sites" are tissues or organs where slow allograft rejection correlates with low levels of expression of MHC class I molecules. Whilst classical class I molecules are recognised by cytotoxic T lymphocytes (CTL), some MHC class I molecules are called "non-classical" because they exhibit low polymorphism and are not widely expressed. These last years, several studies have shown that these can play different, more specialised roles than their classical counterparts. In the course of efforts to characterise MHC class I expression in rat cells obtained from immunoprivileged sites such as the central nervous system or the placenta, a new family of non-classical MHC class I molecules, which we have named RT1-E2, has been uncovered.

Results: Members of the RT1-E2 family are all highly homologous to one another, and the number of RT1-E2 loci varies from one to four per MHC haplotype among the six rat strains studied so far, with some loci predicted to give rise to soluble molecules. The RT1n MHC haplotype (found in BN rats) carries a single RT1-E2 locus, which lies in the RT1-C/E region of the MHC and displays the typical exon-intron organisation and promoter features seen in other rat MHC class I genes. We present evidence that: i) RT1-E2 molecules can be detected at the surface of transfected mouse L cells and simian COS-7 cells, albeit at low levels; ii) their transport to the cell surface is dependent on a functional TAP transporter. In L cells, their transport is also hindered by protease inhibitors, brefeldin A and monensin.

Conclusions: These findings suggest that RT1-E2 molecules probably associate with ligands of peptidic nature. The high homology between the RT1-E2 molecules isolated from divergent rat MHC haplotypes is particularly striking at the level of their extra-cellular portions. Compared to other class I molecules, this suggests that RT1-E2 molecules may associate with well defined sets of ligands. Several characteristics point to a certain similarity to the mouse H2-Qa2 and human HLA-G molecules.

Show MeSH