Disulfide bond-mediated dimerization of HLA-G on the cell surface

Abstract

HLA-G is a nonclassical class I MHC molecule with an unknown function and with unusual characteristics that distinguish it from other class I MHC molecules. Here, we demonstrate that HLA-G forms disulfide-linked dimers that are present on the cell surface. Immunoprecipitation of HLA-G from surface biotinylated transfectants using the anti-␤2-microglobulin mAb BBM.1 revealed the presence of an Ϸ78-kDa form of HLA-G heavy chain that was reduced by using DTT to a 39-kDa form. Mutation of Cys-42 to a serine completely abrogated dimerization of HLA-G, suggesting that the disulfide linkage formed exclusively through this residue. A possible interaction between the HLA-G monomer or dimer and the KIR2DL4 receptor was also investigated, but no interaction between these molecules could be detected through several approaches. The cell-surface expression of dimerized HLA-G molecules may have implications for HLA-G͞receptor interactions and for the search for specific receptors that bind HLA-G.

Complexes of HLA-G Protein on the Cell Surface Are Important for Leukocyte Ig-Like Receptor-1 Function

“…Supporting our results, a recent report suggested the presence of disulfide-linked dimers of HLA-G on the cell surface that are formed via the Cys 42 residue (39). The role of residue 147 or the functional relevance of these dimers was not investigated (39).…”

“…The role of residue 147 or the functional relevance of these dimers was not investigated (39). Importantly, in this study we show that both dimers and trimers of HLA-G are expressed on the cell surface, that both Cys 42 and Cys 147 residues are involved in the formation of these high molecular HLA-G complexes on the cell surface, and that these complexes play a crucial role in recognition by the LIR-1 receptor.…”

“…The structure of the HLA-G protein is as yet unsolved; however, the critical role of residue 42 is supported by the superimposition of the HLA-G molecule onto the HLA-A2 crystal structure. Such superimposition reveals that the Cys 42 residue is located on a loop projecting out from the MHC class I molecule (39). The Cys 147 residue, on the other hand, is predicted to be located on a position pointing into the groove.…”

HLA-G-mediated NK cell senescence promotes vascular remodeling: implications for reproduction

“…39 Recombinant KIR2DL4-immunoglobulin fusion proteins bound transfectants expressing high levels of HLA-G on their surface, 34,40 and this binding was blocked with antibodies to HLA-G (G233) or KIR2DL4 (#33). 38 However, direct binding of soluble forms of HLA-G and KIR2DL4 has not been detected by surface plasmon resonance, 41 possibly due to low intrinsic affinity, as seen with the activating KIR and their MHC ligands. 42 Tetrameric HLA-G bound ILT4 on monocytic cells, but did not bind either ILT2 or KIR2DL4, 43 owing to the very low to negligible surface expression of these two receptors on NK cells.…”

“…On the cell surface, HLA-G can be expressed as either monomers or as a disulphide-linked homodimer. 41,44 While dimeric HLA-G is the preferred ligand of ILT2, whether KIR2DL4 favors monomers or dimers is unknown. From the crystal structure of dimeric HLA-G and modeling of KIR2DL4-HLA-G interactions, it is unlikely that KIR2DL4 can bind HLA-G in its dimeric state.…”