T. Fujii et al., A SOLUBLE FORM OF THE HLA-G ANTIGEN IS ENCODED BY A MESSENGER-RIBONUCLEIC-ACID CONTAINING INTRON 4, The Journal of immunology, 153(12), 1994, pp. 5516-5524
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.