SPLICING OF MAMU AG MRNA SUGGESTS EXISTENCE OF SOLUBLE MHC CLASS I MOLECULE

University Of Wisconsin Madison, Madison WI

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Abstract

OBJECTIVE To determine whether mRNAs are expressed in the rhesus placenta which could encode a soluble MHC class I molecule. RESULTS An unusual feature of both the human placental MHC class I molecule HLA-G and the rhesus molecule Mamu-AG is expression of multiple protein isoforms and splice variants. Splice variants also exist for HLA-G which encode a soluble protein but the presence of a mRNA encoding a soluble rhesus MHC class I molecule has not previously been detected. We cloned a fragment of the Mamu-AG gene from rhesus monkey genomic DNA to define the sequences of intron 4 and 5 and noted that the sequence of intron 4 was highly homologous to that of HLA-G (88% identical), which is similar to exon 4 homology with HLA-A sequences (~91%), but higher than that for intron 5, suggesting that evolutionary drift of intron 4 has been restrained and implying selection for functionally important sequences. We then conducted RT-PCR analyses with placental mRNA using an upstream primer specific for Mamu-AG exon 4, and a downstream primer which would amplify through the 3U-untranslated region and allow diagnostic identification of Mamu-AG transcripts via the premature stop codon homologous to HLA-G. RT-PCR products were amplified from placental RNA, cloned and sequenced. A stop codon (TAG) is present in the exact codon location of the TAA stop codon in the soluble HLA-G reading frame of intron 4. The predicted amino acid sequences of the carboxy-terminal end of the 4th exon and the intron 4 peptides indicate identity with the human sequence at 16/21 residues, and conservative substitutions (e.g., G to S) at the other 5 residues. Further studies cloned and defined the sequences of the other introns of Mamu-AG. FUTURE DIRECTIONS These results suggest that the rhesus monkey will be a model for addressing the physiology of soluble Mamu-AG. We will evaluate 1) the expression of soluble mRNA in the fetal membranes, 2) whether antibodies against soluble HLA-G recognize the rhesus molecule, and 3) rhesus amniotic fluid and fetal serum for the presence of soluble MHC class I molecules. KEY WORDS maternal-fetal immune tolerance, placenta, amniotic fluid, splice variant FUNDING NIH HD 26458, HD34216