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A soluble form of the HLA-G antigen is encoded by a messenger ribonucleic acid containing intron 4
T Fujii1, A Ishitani, D E Geraghty
1Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98104.
Abstract:
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.
Insights
Researchers discovered that alternative splicing of HLA-G RNA, specifically intron 4 inclusion, generates soluble Human Leukocyte Antigen-G (HLA-G) proteins. This splicing mechanism, not proteolytic processing, explains soluble HLA-G expression and function.
Area of Science:
- Immunology
- Molecular Biology
- Genetics
Background:
- The Human Leukocyte Antigen-G (HLA-G) plays a crucial role in immune tolerance, particularly during pregnancy.
- Three membrane-bound HLA-G protein isoforms are produced through alternative splicing of the primary transcript.
- A soluble HLA-G protein has been observed, but its encoding mechanism remained unclear.
Purpose of the Study:
- To elucidate the molecular mechanism responsible for the generation of soluble HLA-G protein.
- To investigate the potential roles of proteolytic processing and alternative splicing in soluble HLA-G production.
Main Methods:
- Transfected cells expressing HLA-G cDNA under a retroviral promoter were utilized.
- Polymerase Chain Reaction (PCR) amplification of cDNA was performed to analyze HLA-G transcripts.
- Intron 4-containing cDNA was inserted into a retroviral expression vector and transfected into LCL.221 cells.
- Two-dimensional isoelectric focusing/SDS-PAGE was employed to compare expressed proteins with native soluble HLA-G.
Main Results:
- Proteolytic processing of membrane-bound HLA-G was ruled out as a source of soluble HLA-G.
- A novel HLA-G mRNA transcript containing intron 4 sequence was identified and found to be abundant.
- This intron 4-containing transcript encodes a soluble HLA-G protein lacking a transmembrane domain.
- A similar intron-containing message derived from HLA-G2 mRNA suggests a soluble HLA-G2 protein also exists.
Conclusions:
- Alternative splicing of HLA-G RNA, specifically the inclusion of intron 4, is the primary mechanism for generating soluble HLA-G protein.
- This alternative splicing event results in a soluble protein with a distinct carboxyl terminus.
- The findings provide insight into the expression and potential functions of soluble HLA-G in immune regulation.