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.

Hydrogen bonds (H-bonds) are crucial for the stability of the peptide-major histocompatibility complex (MHC) complex. In particular, the H-bonds formed between the peptide ligand and the MHC class II binding site appear to have a great influence on the half-life of the complex. Here we show that functional groups with the capacity to disrupt hydrogen bonds (e.g. -OH) can efficiently catalyze ligand exchange reactions on HLA-DR molecules. In conjunction with simple carrier molecules (such as propyl or benzyl residues), they trigger the release of low affinity ligands, which permits the rapid binding of peptides with higher affinity. Similar to HLA-DM, these compounds are able to influence the MHC class II ligand repertoire. In contrast to HLA-DM, however, these simple small molecules are still active at neutral pH. Under physiological conditions, they increase the number of "peptide-receptive" MHC class II molecules and facilitate exogenous peptide loading of dendritic cells. The drastic acceleration of the ligand exchange on these antigen presenting cells suggests that, in general, availability of H-bond donors in the extracellular milieu controls the rate of MHC class II ligand exchange reactions on the cell surface. These molecules may therefore be extremely useful for the loading of antigens onto dendritic cells for therapeutic purposes.Peptide ligands bind to the peptide-binding groove of MHC 1 class II molecules by an array of intermolecular hydrogen bonds (H-bonds). These hydrogen bonds are mostly formed between the backbone of the peptide and conserved residues of the MHC class II molecule. Some of these H-bonds are particularly crucial for the stability of the ligand complex (1). It has been shown for a murine MHC class II molecule that the elimination of H-bonds between the ligand and residues His-81 or Asp-82 of the I-A d ␤-chain results in a rapid loss of the bound peptide (2). Detailed kinetic studies with these mutated MHC molecules revealed peptide dissociation rates that were increased up to 200-fold (3). This increase was in the same range observed after addition of HLA-DM to the peptide complex of the nonmutated I-A d molecule. It was therefore proposed that HLA-DM-mediated ligand release (4, 5) is also accomplished by the disruption of H-bonds (6), a hypothesis also introduced when the crystal structure of HLA-DM was published (7).Because H-bonds appear to be fundamental in maintaining the stability of the MHC class II peptide complexes, we started to investigate small molecules capable of disrupting H-bonds with the goal of achieving an HLA-DM-like catalytic effect on the kinetics of peptide binding. H-bonds require a hydrogen donor and an acceptor group, which provides a free electron pair. Some of the functional groups that can fulfill this function are hydroxyl or amino groups. They are present in a variety of natural and synthetic molecules, such as lipids, metabolites, amino acids, and pharmaceutical drugs. One example is ethanol, where the well known physiological effects appear to resul...

Class II MHC molecules undergo conformational changes on shifts of the pH. As a consequence, low-affinity peptides tightly bound at pH 7.0 can be released at pH 5.0. The imidazole group of histidine is the only amino acid side chain affected within this range. At pH 5.0 the group is positively charged, polar, and hydrophilic, whereas at pH 7.4 it is neutral, apolar, and hydrophobic. In this study, we used soluble forms of HLA-DR and substituted conserved histidine residues with tyrosine, an isosteric analogue to the uncharged form of histidine. The goal of this substitution was to identify crucial His residues by an increase in pH stability of the ligand complex. HLA-DM-mediated release experiments revealed that substitution of His-33 in the ␣ 1 domain of the HLA-DR molecule almost doubled the half-life of HLA-DR1͞class II-associated invariant-chain peptide complexes. The divergence in the off-rate of WT and H33Y mutated complex was strictly pH-dependent and correlated with the theoretical titration curve of the imidazole group. For both HLA-DR1 and HLA-DR4 molecules the mutation resulted in a shift of class II-associated invariant-chain peptide release curves by up to 0.5 pH units. His-33␣1 is present in all HLA-DR and H-2E molecules. It connects the ␣1 and ␣2 domains in its noncharged form by hydrophobic interactions with residue Val-136␣2. It is located in close proximity to the putative interface with HLA-DM and may function as a pH-sensitive ''button,'' which is closed at pH 7.0 but opens below pH 6.0 to allow conformational transitions necessary for ligand exchange.