Intracellular Cysteine Residues in the Tail of MHC Class I Proteins Are Crucial for Extracellular Recognition by Leukocyte Ig-Like Receptor 1

Abstract

The activity of NK cells is regulated by activating receptors that recognize mainly stress-induced ligands and by inhibitory receptors that recognize mostly MHC class I proteins on target cells. Comparing the cytoplasmic tail sequences of various MHC class I proteins revealed the presence of unique cysteine residues in some of the MHC class I molecules which are absent in others. To study the role of these unique cysteines, we performed site specific mutagenesis, generating MHC class I molecules lacking these cysteines, and demonstrated that their expression on the cell surface was impaired. Surprisingly, we demonstrated that these cysteines are crucial for the surface binding of the leukocyte Ig-like receptor 1 inhibitory receptor to the MHC class I proteins, but not for the binding of the KIR2DL1 inhibitory receptor. In addition, we demonstrated that the cysteine residues in the cytoplasmic tail of MHC class I proteins are crucial for their egress from the endoplasmic reticulum and for their palmitoylation, thus probably affecting their expression on the cell surface. Finally, we show that the cysteine residues are important for proper extracellular conformation. Thus, although the interaction between leukocyte Ig-like receptor 1 and MHC class I proteins is formed between two extracellular surfaces, the intracellular components of MHC class I proteins play a crucial role in this recognition.

Interaction of the LILRB1 inhibitory receptor with HLA class Ia dimers

“…HLA-B51) pointed to the intrinsic influence of the individual HLA-I structure as the primary determinant for receptor binding, these results further supported a role for noncanonical HLA-I dimers in enhancing LILRB1 recognition. Mutation of cytoplasmic Cys in HLA-B7 was previously shown to impair binding of LILRB1-Fc and inhibition of LILRB1-expressing NK cells [19]. Consistent with these observations, LILRB1-Fc poorly stained the different .221-B7 Cys mutants ( Fig.…”

“…Cytoplasmic Cys were reported to be required for HLA-I recognition by the inhibitory receptor [19], and HLA-B27 and -A2 molecules were shown to form β2m-associated dimers in exosomes through specific intracellular Cys [20,21]. Thus, the possibility that this conformational feature might enhance the interaction of classical HLA-I molecules with LILRB1, as previously shown for HLA-G [17], was considered.…”

“…HLA-B7 cytoplasmic Cys residues were shown to play a crucial role in LILRB1 recognition [19]; moreover, Cys325 and Cys339 were reported to be respectively involved in the formation of β2m-associated HLA-B27 and -A2 dimers in exosomes [20,21].…”

“…In this context, HLA-G displays a unique Cys42 residue, allowing the formation of disulphide-linked dimers, which were shown to establish a high-affinity interaction with LILRB1 [15][16][17]. HLA-B27 β2m-free heavy chains (FHC) dimerizing through Cys67, efficiently interacted with LILRB2 [18].HLA-B7 cytoplasmic Cys residues were shown to play a crucial role in LILRB1 recognition [19]; moreover, Cys325 and Cys339 were reported to be respectively involved in the formation of β2m-associated HLA-B27 and -A2 dimers in exosomes [20,21].Altogether, these observations suggest that these noncanonical HLA class Ia conformers might enhance the interaction with LILRB1. In the present study experimental evidence supporting this hypothesis is provided.…”

“…On the other hand, cytoplasmic Cys residues were shown to be crucial for LILRB1 interaction with classical HLA-I molecules (e.g. HLA-B7) and their dimerization in exosomes [19][20][21]. To address whether such structural features might contribute to the LILRB1 interaction with classical HLA-I molecules, we analyzed with a soluble LILRB1-Fc fusion protein a panel of different HLA-I alleles transfected in the 721.221 cell line.…”