Transcripts of the MHC class I HLA-G gene and their...

Details Transcripts of the MHC class I HLA-G gene and their... Transcripts of the MHC class I HLA-G gene and their...

1997-10-27

2001-09-18

Whisenant, Ethan (Department: 1655)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S024300, C435S006120

Reexamination Certificate

active

06291659

ABSTRACT:

The present invention relates to transcripts of the major histocompatibility complex (MHC) class I HLA-G gene TwhIch are present in foetal trophoblasts and/or in adult circulating mononuclear cells, and to their applications.
The antigens of the major histocompatibility complex (MHC) divide into several classes, i.e. class I antigens (HLA-A, HLA-B and HLA-C) which exhibit 3 globular domains (&agr;1, &agr;2 and &agr;3), of which the &agr;3 domain is associated with &bgr;2 microglobulin, class II antigens (HLA-DP, HLA-DQ and HLA-DR) and class III antigens (complement).
In addition to the aforementioned antigens, the class I antigens include other antigens, termed non-classical class I antigens, in particular the antigens HLA-E, HLA-F and HLA-G; this latter, in particular, is expressed by the extravillous trophoblasts of the normal human placenta.
The sequence of the HLA-G gene (HLA-6.0 gene) has been described by GERAGHTY et al., (Proc. Natl. Acad. Sci. USA, 1987, 84, 9145-9149): it comprises 4,396 base pairs and exhibits an intron/exon organization which is homologous to that of the HLA-A, HLA-B and HLA-C genes. More precisely, this gene comprises 8 exons and an untranslated, 3′UT, end, with the following respective correspondence: exon 1: signal sequence, exon 2: &agr;1 domain, exon 3: &agr;2 domain, exon 4: &agr;3 domain, exon 5: transmembrane region, exon 6: cytoplasmic domain I, exon 7: cytoplasmic domain II, exon 8: cytoplasmic domain III and 3′ untranslated region (GERAGHTY et al., mentioned above, ELLIS et al., J. Immunol., 1990, 144, 731-735). However, the HLA-G gene differs from the other class I genes in that the in-frame translation termination codon is located at the second codon of exon 6; as a result, the cytoplasmic region of the protein encoded bv this aene HLA-6.0 is considerably shorter than that of the cytoplasmic regions of the HLA-A, HLA-B and HLA-C proteins.
Contrary to the other class I antigens, this HLA-G antigen (G 6.0 and BeWO.G7 clones) is apparently not polymorphic and is not expressed in cell types other than trophoblasts (ELLIS et al., J. Immunol., 1990, mentioned above).
Other HLA-G clones have been isolated (TAMAKI et al., Microbiol. Immunol., 1993, 37, 8, 633-640); in particular, the HLA-G clone designated 7.0E was isolated from a Japanese placenta and its amino acid sequence was found to be identical to that of the abovementioned G6.0 and BeWO.G7 clones. Furthermore, the authors of this article demonstrate that a certain heterogeneity can exist within the HLA-G genes.
These HLA-G antigens are mainly expressed by the cytotrophoblast cells of the placenta; however, HLA-G mRNA has been encountered in the tissues of the eye and in foetal liver (ISHITANI et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 3947-3951; with the numeration corresponding to that of the HLA 6.0 sequence as described in SHUKLA et al., Nucleic Acid Research, 1990, 18, 8, 2189).
The HLA-G antigens expressed by the cytotrophoblasts are regarded as playing a role in the protection of the placenta (absence of rejection). Furthermore, in so far as the HLA-G antigen is monomorphic, it can also be implicated in the growth or the function of the placental cells (KOVATS et al., Science, 1990, 248, 220-223).
Other research studies relating to this nonclassical class I antigen (ISHITANI et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 3947-3951) have demonstrated that the primary transcript of the HLA-G gene can be spliced in various ways and produces at least 3 distinct mature 3 mRNAs; the primary HLA-G transcript supplies a complete copy (G1) of 1,200 bp, a fragment of 900 bp (G2) and a fragment of 600 bp (G3).
The G1 transcript does not include exon 7 and corresponds to the sequence described by ELLIS et al. (mentioned above), that is, it encodes a protein which comprises a leader sequence, three external domains, a transmembrane region and a cytoplasmic sequence. The G2 mRNA does not include exon 3, that is, it encodes a protein in which the &agr;1 and &agr;3 domains are directly connected; the G3 mRNA contains neither exon 3 nor exon 4, that is it encodes a protein in which the &agr;1 domain and the transmembrane sequence are directly connected.
The splicing which prevails for obtaining the HLA-G2 antigen leads to an adenine (A) (originating from the domain encoding &agr;1) being joined to an AC sequence (from the domain encoding &agr;3), resulting in the creation of an AAC (asparagine) codon in place of the GAC (aspartic acid) codon which is encountered at the beginning of the sequence encoding the &agr;3 domain in HLA-G1.
The splicing which is generated in order to obtain HLA-G3 does not result in a novel codon being formed in the splicing zone.
The authors of this article have also analysed the different proteins which are expressed: the 3 mRNAs are translated into protein in the cell line .221-G.
The authors of this article conclude that HLA-G plays a fundamental role in protecting the placenta from a maternal immune response (induction of immune tolerance). However, it is made clear that the role of the G3 protein, which does not contain the &agr;3 domain, has not been established.
The complexity of the MHC and the role of the HLA-G antigen in tolerance mechanisms have led the inventors to search at least for an HLA-G transcript which could, at one and the same time:
readily be demonstrated in peripheral blood,
be suitable for expressing, under appropriate conditions, a protein, preferably soluble, which is suitable as a tolerance agent,
enable immature stem cells to be selected which are suitable for being employed in bone marrow transplants,
and also enable foetal cells in which this gene is being expressed to be demonstrated in the maternal blood.
Consequently, the underlying object of the present invention is to provide sequences derived from an mRNA of the HLA-G gene which is suitable for solving all the problems set out above.
Such sequences can be applied, in particular:
in the separation, from a sample of maternal blood, of foetal cells,
in a procedure for enriching with immature stem cells which are suitable for being employed in marrow transplants, and
in the specific separation of circulating mononuclear cells,
and in the preparation of a immunomodulating drug.
The present invention relates to a cDNA sequence derived from an mRNA of the human MHC HLA-G gene, which sequence is characterized in that it comprises, in succession in the 5′ to 3′ direction:
a fragment encoding the signal peptide (exon 1),
a fragment encoding the &agr;1 domain (exon 2),
a fragment encoding the &agr;2 domain (exon 3),
a fragment encoding the transmembrane TM domain (exon 5),
a fragment encoding the cytoplasmic domain (exon 6), and
the untranslated 3′ fragment (exon 8), with this sequence being designated HLA-G3-5 or according to the now in force nomenclature HLA-G4.
The distinctive feature of a sequence of this nature is that it does not include exon 4 and demonstrates all of the properties enumerated above and is, in particulier, able to be detected in adult circulating mononuclear cells.
Mononuclear cells mean all mononuclear cells of the peripheral blood, except natural killer cells (NK cells and other large granular lymphocytes (LGL)).
According to an advantageous embodiment of the invention, the said sequence comprises, in succession in the 5′ to 3′ direction:
the fragment encoding the &agr;2 domain (exon 3),
the fragment encoding the transmembrane TM domain (exon 5),
the fragment encoding the cytoplasmic domain (exon 6), and
the untranslated 3′ fragment (exon 8).
In conformity with the invention, such a sequence which encodes a protein in which the &agr;2 domain and the HLA-G transmembrane sequence are directly connected exhibits the following SEQ ID NO:1: CC AAT GTG GCT GAA CAA AGG AGA GCC TAC CTG GAG GGC AGC TGC GTG GAG TGG CTC CAC AGA TAC CTG GAG AAC GGG AAG GAG ATG CTG CAG CGC GCG G
3
/
5
AG CAG TCT TCC CTG CCC ACC ATC CCC ATC ATG GGT ATC GTT GCT GGC CTG GTT GTC CTT GCA GCT GTA GTC ACT GGA GCT GCG GTC GCT GCT GTG CTGT TGG AGX
1
AAG AAG A

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