A soluble form of the HLA-G antigen is encoded by a messenger ribonucleic acid containing intron 4.
The HLA-G primary transcript is alternatively spliced to yield mRNAs encoding three alternative membrane bound proteins. In addition to these forms, a soluble HLA-G protein has been described which is not encoded directly by any of the three alternative mRNAs. To explain the process which might lead to the expression of a soluble HLA-G Ag, we investigated the potential roles proteolytic processing and additional alternative splicing of HLA-G RNA might play. By generating transfected cells with HLA-G cDNA expression driven by a retroviral promoter, it was possible to rule out proteolytic processing of the membrane-bound HLA-G as a mechanism of generating soluble HLA-G, resulting in our focus on alternative splicing as an explanation. Analysis of PCR-amplified cDNA revealed a relatively abundant transcript present in all samples examined which consisted of the full length HLA-G mRNA sequence interrupted by intron 4 sequence. The open reading frame in this mRNA continues into intron 4 terminating 21 amino acids after the alpha 3 domain, thus excluding the transmembrane encoding region and yielding a protein with a highly charged carboxyl terminus. Transfection of the intron 4 containing cDNA, inserted into a retroviral expression vector, into LCL .221 followed by comparison of the class I protein to native soluble G by two dimensional isoelectric focusing/SDS-PAGE analysis, demonstrated this message encoded the soluble HLA-G protein. In addition, a similar intron containing message derived from the HLA-G2 mRNA was found, suggesting the existence of a soluble form of this alternative HLA-G protein. These findings are discussed in relation to other soluble class I molecules and with regard to potential functions of the soluble HLA-G Ag.
Fujii
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T
Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98104.
Ishitani
A
A
Geraghty
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DE
D017398:Alternative Splicing / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D005434:Flow Cytometry; D006680:HLA Antigens / Q000096:biosynthesis* / Q000235:genetics*; D059951:HLA-G Antigens; D015395:Histocompatibility Antigens Class I / Q000096:biosynthesis* / Q000235:genetics*; D006801:Humans; D007438:Introns / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D014162:Transfection
D006680:HLA Antigens; D059951:HLA-G Antigens; D015395:Histocompatibility Antigens Class I; D012333:RNA, Messenger