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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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Sequence of the RT1-E2n gene. The sequence was derived after subcloning the upstream 2.8 kb Pst I and 4.5 kb BamH I DNA fragments obtained by restriction digest of both the 473K19 and 303P13 PAC. Corresponding restriction sites are boxed. The coding sequence is presented in black whilst non-coding regions (introns, 5' UTR and 3' UTR) are in grey. Splice donors (GT) and splice acceptors (AG) are underlined, as well as the polyadenylation site. The fifteen nucleotides at the start of exon 6 which are boxed in dashed lines are predicted to be systematically present in the cDNAs for RT1-E2n, like those from the RT1-Eu and RT1-E2al loci because the 'canonical' second splice acceptor (AG) is mutated to AA. For E2cl and E2dl, the second splice acceptor is conserved as AG, and the corresponding 15 nucleotide segment is present in only a small proportion of the clones obtained (see Table 1).
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Figure 4: Sequence of the RT1-E2n gene. The sequence was derived after subcloning the upstream 2.8 kb Pst I and 4.5 kb BamH I DNA fragments obtained by restriction digest of both the 473K19 and 303P13 PAC. Corresponding restriction sites are boxed. The coding sequence is presented in black whilst non-coding regions (introns, 5' UTR and 3' UTR) are in grey. Splice donors (GT) and splice acceptors (AG) are underlined, as well as the polyadenylation site. The fifteen nucleotides at the start of exon 6 which are boxed in dashed lines are predicted to be systematically present in the cDNAs for RT1-E2n, like those from the RT1-Eu and RT1-E2al loci because the 'canonical' second splice acceptor (AG) is mutated to AA. For E2cl and E2dl, the second splice acceptor is conserved as AG, and the corresponding 15 nucleotide segment is present in only a small proportion of the clones obtained (see Table 1).

Mentions: - Second, out of the five clones for RT1-E2bl also obtained from LEW placenta, one carries a dinucleotide deletion at the exon 4 to exon 5 boundary, due to the use of the first two nucleotides (AG) of exon 5 as a splice acceptor instead of the one that lies just in front (see Fig. 4). This results in a frameshift and in a termination after a stretch of 30 amino acids that are not particularly hydrophobic and do not match significantly any known protein in databases. The protein translated from such a transcript would therefore most probably be soluble rather than membrane bound. This observation suggests that, within the same locus, alternate use of one or the other of these two adjacent splice acceptors can lead to either a membrane-bound or a soluble molecule. It is worthy of note that the very same type of frameshift was also found in the single RT1-E2bav1 clone (Clone DA9) isolated from a cDNA library from DA lymphoblasts. Since only a single clone was obtained, we cannot be certain that the upstream, 'canonical' AG acceptor site of exon 5 is present in the RT1-E2av1 locus, and therefore whether such membrane-bound molecules can be produced from it.


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)

Sequence of the RT1-E2n gene. The sequence was derived after subcloning the upstream 2.8 kb Pst I and 4.5 kb BamH I DNA fragments obtained by restriction digest of both the 473K19 and 303P13 PAC. Corresponding restriction sites are boxed. The coding sequence is presented in black whilst non-coding regions (introns, 5' UTR and 3' UTR) are in grey. Splice donors (GT) and splice acceptors (AG) are underlined, as well as the polyadenylation site. The fifteen nucleotides at the start of exon 6 which are boxed in dashed lines are predicted to be systematically present in the cDNAs for RT1-E2n, like those from the RT1-Eu and RT1-E2al loci because the 'canonical' second splice acceptor (AG) is mutated to AA. For E2cl and E2dl, the second splice acceptor is conserved as AG, and the corresponding 15 nucleotide segment is present in only a small proportion of the clones obtained (see Table 1).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC183868&req=5

Figure 4: Sequence of the RT1-E2n gene. The sequence was derived after subcloning the upstream 2.8 kb Pst I and 4.5 kb BamH I DNA fragments obtained by restriction digest of both the 473K19 and 303P13 PAC. Corresponding restriction sites are boxed. The coding sequence is presented in black whilst non-coding regions (introns, 5' UTR and 3' UTR) are in grey. Splice donors (GT) and splice acceptors (AG) are underlined, as well as the polyadenylation site. The fifteen nucleotides at the start of exon 6 which are boxed in dashed lines are predicted to be systematically present in the cDNAs for RT1-E2n, like those from the RT1-Eu and RT1-E2al loci because the 'canonical' second splice acceptor (AG) is mutated to AA. For E2cl and E2dl, the second splice acceptor is conserved as AG, and the corresponding 15 nucleotide segment is present in only a small proportion of the clones obtained (see Table 1).
Mentions: - Second, out of the five clones for RT1-E2bl also obtained from LEW placenta, one carries a dinucleotide deletion at the exon 4 to exon 5 boundary, due to the use of the first two nucleotides (AG) of exon 5 as a splice acceptor instead of the one that lies just in front (see Fig. 4). This results in a frameshift and in a termination after a stretch of 30 amino acids that are not particularly hydrophobic and do not match significantly any known protein in databases. The protein translated from such a transcript would therefore most probably be soluble rather than membrane bound. This observation suggests that, within the same locus, alternate use of one or the other of these two adjacent splice acceptors can lead to either a membrane-bound or a soluble molecule. It is worthy of note that the very same type of frameshift was also found in the single RT1-E2bav1 clone (Clone DA9) isolated from a cDNA library from DA lymphoblasts. Since only a single clone was obtained, we cannot be certain that the upstream, 'canonical' AG acceptor site of exon 5 is present in the RT1-E2av1 locus, and therefore whether such membrane-bound molecules can be produced from it.

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