Function of the residues Met~(76) and Gin~(79) in HLA-G α1 domain in KIR2DL4 recognition
Objective To investigate the biological function of the unique residues Met~(76)and Gln~(79)in HLA-Gα1 domain in HLA-G specific receptor KIR2DL4 recognition.Methods Eukaryotic vector CD51neg1 was applied to generate the soluble fusion protein IR2DL4-IgG Fc.The codons encoding Met~(76)and Gln~(79)were mutated to Ala~(76,79)in HLA-Gα1 domain with the methods of nest-PCR and point to point mutagenesis.Then,the wild and mutant HLA-G gene products were transfected into the HLAⅠmolecules negative cell line K562 with retrovirus vector.The KIR2DL4 and HLA-G binding activity was measured with fluorescent density of KIR2DL4-IgG Fc bind- ing to the wild and mutant HLA-G protein expressed in K562 cells.The function of the residues Met~(76)and Gln~(79)in KIR2DL4 recognition was evaluated by comparing the mean fluorescent density(MFI)of KIR2DL4-IgG Fc binding to the wild and mutant HLA-G molecules respectively.Results Western blot data showed that the fusion protein of KIR2DL4-IgG Fc protein was successfully expressed and the FACS results revealed that the wild and mutant HLA-G was expressed in K562 cells.Mutating Met~(76),Gln~(79)to Ala~(76,79)could remarkably effect the binding activity of the KIR2DL4 to HLA-G molecules.Conclusion Met~(76),Gln~(79)in the HIA-Gα1 domain could be the key resi- dues involved in the KIR2DL4 recognition.