Identification of HLA-G7 as a new splice variant of the HLA-G mRNA and expression of soluble HLA-G5, -G6, and -G7 transcripts in human transfected cells
Introduction
Various features distinguish nonclassical MHC class Ib HLA-G from classical class Ia genes. These features include lower polymorphism, and different gene regulation mechanisms that lead to restricted tissue distribution of protein expression. The description of HLA-G1 protein isoform as a ligand for distinct killing inhibitory receptors expressed by immunocompetent cells has enlightened the specific role played by these nonclassical HLA class I molecules in modulation of immune responses. The immunotolerant role of the HLA-G antigen may be further specified in trophoblast and thymus tissues in consequence of its restricted pattern of expression (reviewed in 1, 2).
Analysis of HLA class I sequences has led different groups to propose specific means of analyzing transcription of nonclassical HLA class I genes by RT-PCR, RNase protection, and Northern Blot analysis using locus specific probes. These approaches led to the identification of mechanisms of mRNA alternative splicing that seem to be characteristic for the nonclassical HLA-G gene. The HLA-G primary transcript has been shown to generate six alternative mRNAs that have the capacity to encode four membrane isoforms (HLA-G1, -G2, -G3, and -G4) and two soluble isoforms (HLA-G5 and -G6) 3, 4, 5, 6. HLA-G1 encodes a full length protein that contains α1, α2, and α3 extracellular domains linked to the transmembrane domain encoded by exon 5 and to a shortened cytoplasmic tail due to a premature stop codon in exon 6 [7]. HLA-G2, -G3, and -G4 transcripts that, respectively, exclude exon 3, exon 3 and 4, or exon 4 generate truncated isoforms that retain only the α1 for -G3, the α1 and α3 domains for -G2, and the α1 and α2 domains for -G4, joined to the transmembrane region.
Unusual mechanisms for generating alternate soluble HLA-G proteins include the use of an intron 4 retaining mRNA. The soluble full length HLA-G5 molecule thus retains identical leader, α1, α2, α3 domains, but includes intron 4 sequences, yielding to a soluble HLA-G5 specific open reading frame that encodes 21 amino-acids linked to the α3 domain and excludes the transmembrane domain. A similar messenger retaining intron 4, HLA-G2, was also described suggesting the presence of a truncated HLA-G6 isoform [3] that lacks the α2 domain of the protein.
HLA-G transcripts that correspond to both soluble and membrane bound isoforms have been detected in various cell types but are most abundant in placental tissues, and placental-derived cell line JEG-3. Development of various antibodies that specifically detect HLA-G protein expression 8, 9, 10, 11, 12, 13 has allowed detection of HLA-G1 and -G5 isoforms in subpopulation of extravillous cytotrophoblastic cells but also in thymic epithelial cells 14, 15, in endothelial cells of chorionic blood vessels [16], amniochorion and amniotic fluid 17, 18, 19, tumor tissues 20, 21, 22, 23, in oocytes [24] and preimplantation embryo [25], activated macrophage cell lines, and in peripheral blood monocytes 26, 27, 28, 29.
Although intracellular translated proteins encoded by truncated alternative HLA-G2, -G3, and -G4 cDNAs have been detected mostly intracellularlly in transfected cells 30, 31, their physiologic expression and function remains to be characterized.
The specific role of intron retaining soluble molecules also remains largely unexplored, but presence of soluble molecules in body fluids, tumor biopsies [20], in the serum of transplanted patients [32], and recent implication of HLA-G5 as an inducer of activated CD8+ T-cell apoptosis [33] may provide new insights on the role of HLA-G isoforms in mediating immune tolerance during pregnancy, transplantation of tumor progression.
In this article, we examined the capacity of HLA-G cDNA corresponding to both full length and truncated soluble alternative transcripts to be translated into mammalian cells. This study enabled us to identify a novel, alternatively spliced variant of the HLA-G mRNA, which also uses intron retention mechanisms and has the capacity to encode a soluble HLA-G3 molecule.
Section snippets
Cell lines and tissues
The human HLA-G-positive choriocarcinoma JEG-3 and Teratocarcinoma cell line Tera-2 were obtained from the American Type Culture Collection and cultured in DMEM (Sigma, St. Louis, MO, USA) with 2 mM L-glutamine, 10 μg/ml gentamicin, and fungizone (Sigma) supplemented with 10% heat-inactivated fetal calf serum. M8 is an HLA class I positive (HLA-A1, -A2, -B12, and -B40/male) but HLA-G-negative melanoma cell line [34] and was cultured in RPMI 1640 with Glutamax-I (Life Technologies, Gaithersburg,
Identification of HLA-G7 as a novel alternative splicing variant of the HLA-G mRNA
In order to clone HLA-G transcripts corresponding to HLA-G soluble proteins, cDNAs were obtained by RT-PCR amplification of RNA extracted from the JEG-3 choriocarcinoma cell line using primers derived from the 5′ promoter and intron 4 HLA-G sequences. Three predominant bands of approximate size 960, 680, and 430 bp were observed. The 960 and 680 bp cDNA correspond to expected size of cDNA products encoding HLA-G5 and -G6 soluble isoforms and were further gel purified to be introduced into
Discussion
In this report, we identify HLA-G7 as a new splicing variant of the HLA-G mRNA that retains intron 2 sequences, yielding a 117 amino-acid HLA-G7 protein isoform that only contains the α1 domain of the HLA-G protein linked to two amino-acids at the carboxyl terminal specifically encoded by intron 2. HLA-G7 transcripts are detected in human tissues and their distribution matches that of other HLA-G transcripts with high levels of HLA-G7 transcripts in trophoblast, term placenta, and amniotic
Acknowledgements
We thank Luc Gourand and Michel Rami for providing us with sera and fetal tissues, and Stephane Prost for cord blood. We are very gratefull to M. McMaster and S. Fisher for the generous gift of 4H84 mAb. We also want to thank M.F. Avril and F.A. Le Gal for their precious collaboration in obtaining melanoma biopsies. We also thank Elisabeth Nelson for excellent technical assistance and the “service photographique de l’institut d’Hématologie.” This work was supported by Association pour la
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P.P. and F.A.C. contributed equally to this work.