1926 Richard Apps et al. Eur. J. Immunol. 2007. 37: 1924–1937 Figure 1. HLA-G is present on the surface of normal trophoblast cells as a disulphide-linked homodimer of the conventional b2m- associated class I complex. 721.221 + HLA-G1 transfectants (A, B), JEG-3 cell line (C–E) and trophoblast (F, G) were surface biotinylated, immunoprecipitated with the indicated antibodies and resolved by reducing (A, C, F) or non-reducing (B, D, E, G) PAGE before Western blotting with streptavidin-HRP. All were exposed for less than 1 min, with the exception of (E) which was exposed for 10 min. Complexes immunoprecipitated with G233 from 721.221 HLA-G+ transfectants (H) and trophoblast (J) were also analysed by 2D-PAGE. These gels are representative of at least three different experiments for each cell type, using a total of 20 pooled samples for trophoblast cells. Images in this figure include lanes from several gels. monomeric form of HLA-G predominates on JEG-3 cells, a finding consistent with previous reports [17, 19]. The dimeric HLA-G complex is expressed at the surface of normal trophoblast cells The outstanding question is whether the dimeric form of HLA-G is present on the surface of normal trophoblast cells. Cells isolated from normal first-trimester placen- tae, in which only trophoblast cells have been shown to express HLA-G [25], were surface biotinylated and immunoprecipitated as before. Immunoprecipitation with HLA-G-specific antibodies detected 80-kDa dimeric HLA-G as well as the conventional 39-kDa form under non-reducing conditions (Fig. 1G). In contrast, the 80-kDa species was not seen in reducing PAGE (Fig. 1F). By densitometry, 40% of the HLA-G molecules appear to be in the dimeric complex, although the mAb 87G displays a greater bias to the monomer as before. W6/32 and BBM.1 antibodies immunoprecipitated both the monomeric and dimeric forms of HLA-G, and the 45-kDa HLA-C molecule was also expressed by normal tropho- blast cells [23, 24]. As with JEG-3, HLA-C-specific antibodies confirm the identity of the bands in this doublet (Fig. 1F, G). To confirm that the 80-kDa complex consists of homodimers of HLA-G molecules and not of hetero- dimers of different HLA-I molecules, or is a cross- f 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim reaction of the Western reagents with an unrelated 80-kDa protein, two-dimensional (2D)-PAGE was per- formed. Surface-labelled HLA-G was specifically im- munoprecipitated from transfectants (Fig. 1H) and trophoblast (Fig. 1J) with the mAb G233. Non-reducing PAGE in the horizontal dimension resolved the 80-kDa complex, 39-kDa conventional HLA-G and 13.7-kDa b2m. After reduction on the gel followed by reducing PAGE in the vertical dimension, the previously 80-kDa species migrated the same distance as conventional 39-kDa HLA-G. This confirms that the 80-kDa species is a disulphide-linked homodimer of the 39-kDa HLA-G molecule. To summarise, these results show that HLA-G can form disulphide-linked homodimers that are b2m associated and that a significant proportion of the HLA-G molecules at the surface of normal trophoblast cells in vivo are present in this dimeric conformation. Similar results were obtained for HLA-G expressed by transfected 721.221 and Ltk cells. Preferential binding of 87G to the monomer in both transfectants and trophoblast cells suggests that the dimer has a similar conformation in both contexts, and therefore these transfectants represent a good model of HLA-G as displayed at the surface of the trophoblast. In contrast, HLA-G is found on the surface of the cell line, JEG-3, predominantly as the conventional monomeric class I complex. www.eji-journal.eu