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 the se forms, a soluble HLA-G protein has been described which is not enco ded directly by any of the three alternative mRNAs. To explain the pro cess which might lead to the expression of a soluble HLA-G Ag, we inve stigated the potential roles proteolytic processing and additional alt ernative splicing of HLA-C RNA might play. By generating transfected c ells with HLA-C cDNA expression driven by a retroviral promoter, it wa s possible to rule out proteolytic processing of the membrane-bound HL A-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 fu II length HLA-G mRNA sequence inter rupted by intron 4 sequence. The open reading frame in this mRNA conti nues into intron 4 terminating 21 amino acids after the alpha 3 domain , thus excluding the transmembrane encoding region and yielding a prot ein with a highly charged carboxyl terminus. Transfection of the intro n 4 containing cDNA, inserted into a retroviral expression vector, int o LCL.221 followed by comparison of the class I protein to native solu ble G by two dimensional isoelectric focusing/SDS-PAGE analysis, demon strated this message encoded the soluble HLA-G protein. In addition, a similar intron containing message derived from the HLA-G2 mRNA was fo und, suggesting the existence of a soluble form of this alternative HL A-G protein. These findings are discussed in relation to other soluble class I molecules and with regard to potential functions of the solub le HLA-G Ag.