Eur. J. Immunol. 2007. 37: 1924–1937 Soluble HLA-G also forms a disulphide-linked homodimer We also investigated whether the two soluble forms of HLA-G form dimers. Full-length membrane-bound HLA- G1 molecules can be shed intact or cleaved from the surface by metalloproteinase activity [26, 27]. Also, a splice variant, HLA-G5, retains intron 4, introducing a premature stop codon before the transmembrane domain [28]. Both of these soluble forms of HLA-G were investigated by immunoprecipitation with mAb G233 and W6/32 from culture supernatant of 721.221 cells transfected with either HLA-G1 or HLA-G5 cDNA. Immunoprecipitated complexes were analysed by redu- cing or non-reducing PAGE and detected on a Western blot with the HLA-G-specific mAb MEM-G/1. Under reducing conditions, secreted HLA-G5 was detected at *37 kDa, as expected (Fig. 2A). A soluble HLA-G band was immunoprecipitated from supernatants of HLA-G1- transfected cells, but this was at much lower concentra- tion and only detected in 30-fold concentrated samples (Fig. 2B). We looked for the presence of dimers using non-reducing conditions and found that a small proportion of HLA-G5 was present as a dimer, using both G233 (Fig. 2C) and W6/32 mAb (not shown). This confirms that soluble HLA-G can form a disulphide- Immunomodulation 1927 linked homodimer of the b2m-associated class I complex similarly to the transmembrane molecule. Immunopre- cipitations were repeated using concentrated super- natant of cultured primary trophoblast and pre- implantation embryos, but no soluble HLA-G1 or HLA-G5 was detected (Fig. 2B). In conclusion, the HLA-G5 soluble splice variant of HLA-G can exist as a dimer identically to HLA-G1 at the cell surface. However, no soluble forms of HLA-G were detected in vivo. Artefactual multimerisation can occur due to in- complete denaturation under non-reducing conditions permitting hydrophobic associations. However, cysteine 42 substitution [18] and the dimer crystal structure [20] have confirmed that HLA-G homodimers are disulphide linked. Cytoplasmic tail cysteine residues have also been observed to mediate post-lysis dimerisa- tion of classical HLA-I molecules when released from the reducing intracellular environment [29]. To avoid post- lysis disulphide bond formation, in our experiments a lysis buffer containing the alkylating agent iodoaceta- mide was used that prevents de novo disulphide bond formation between HLA-I molecules [30]. In addition, the HLA-G sequence lacks cytoplasmic cysteine residues and we have also shown that the soluble HLA-G5 splice variant product forms dimers (Fig. 2). We note that the Figure 2. Soluble HLA-G can also exist as a disulphide-linked homodimer of the conventional b2m-associated class I complex. Soluble HLA-G molecules were immunoprecipitated with the indicated antibodies from supernatants of 721.221 cells transfected with HLA-G1 or HLA-G5 cDNA compared to untransfected cells. The JEG-3 cell line, isolated primary trophoblast, or pre- implantation embryo culture supernatants were also immunoprecipitated compared to unconditioned medium. Precipitated complexes were separated by reducing (A, B) or non-reducing PAGE (C) and detected by Western blotting with biotinylated mAb MEM-G/1 followed by streptavidin-HRP. HLA-G1+ transfectant, JEG-3 and trophoblast culture supernatants were concentrated 30 times before immunoprecipitation. Gels are representative of four independent experiments. The image in (C) includes lanes from separate gels. f 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu