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,
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Departments of
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Stomatology,
Anatomy,
Pharmaceutical Chemistry, and
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Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco, CA 94143;
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The Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98104; and
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Adeza Biomedical, Sunnyvale, CA 94089
The human placenta expresses HLA-G, a nonclassical (class Ib) MHC molecule that could play a central role in maternal tolerance of the semiallogeneic fetus. In this work, we report the production of a new mAb, 4H84, that specifically reacts with HLA-G in two formats: immunocytochemistry and immunoblotting. Immunolocalization experiments with 4H84 confirmed our previous finding that cytotrophoblasts within the uterine wall are the only cells in tissue sections of placenta that express the HLA-G protein. Additional experiments showed that both amniocytes and cytotrophoblasts in the amnion-chorion express this protein. Since multiple HLA-G transcripts have been described, we used immunoblotting to study the HLA-G isoforms produced by cytotrophoblasts in vitro and by the amnion-chorion in vivo. Cytotrophoblasts, their conditioned medium, and amniotic fluid samples contained heterodisperse immunoreactive bands (Mr 35,000–50,000). N-deglycosylation by peptide-N-glycosidase F digestion resolved these isoforms into two distinct bands. Cell samples contained primarily an Mr 37,000–42,000 protein, most likely encoded by the full-length mRNA. Conditioned medium and amniotic fluid contained a slightly smaller protein, most likely the secreted form lacking the transmembrane and cytoplasmic regions. Removal of polylactosamine chains by endo-ß D-galactosidase digestion significantly reduced the electrophoretic mobility of the immunoreactive bands, suggesting that HLA-G, unlike class Ib molecules studied to date, carries N-acetyllactosamine units. These data show that Mr heterogeneity of HLA-G is due to its novel glycosylation, rather than to the translation of alternatively spliced mRNAs. We postulate that the unusual carbohydrate structures this molecule carries could interact with maternal immune cells and/or stabilize the molecule.
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