Detergent enhances binding of a secreted HLA-A2 molecule to solid phase peptides.
We have constructed a secreted analogue (sA2) of the human class I molecule HLA-A2. sA2 was affinity purified both in the presence and absence of detergent and the effects of detergent on the magnitude and specificity of A2 binding to solid phase peptides tested. sA2 purified in the presence of detergent and detergent-solubilized A2 are shown to function comparably in the binding of the synthetic peptide M.Y + 57-68, a known T-cell epitope derived from the influenza A matrix protein. The molecules binding to M.Y + 57-68 typically represent 8% to 10% of the added protein. In contrast, less than 1% of sA2 protein purified in the absence of detergent binds M.Y + 57-68. This reduced binding is not due to a change in the affinity of sA2 for M.Y + 57-68. Addition of detergent at various stages of the purification and iodination procedures indicates that the longer the sA2 molecules are exposed to detergent the better they bind. However, the concentration of detergent during the actual binding assay does not appear to be critical. We also find that while the sA2-detergent and the sA2-no detergent molecules differ in the extent to which they bind various peptides, they do not differ in their patterns of binding. We conclude that detergent probably does not influence the specificity of class I/peptide binding but does increase the number of sA2 molecules that can participate in the binding of peptide either by generating and stabilizing "empty" sA2 molecules or by stabilizing a structure that is more amenable to binding peptide.
Tussey
L G
LG
Department of Microbiology and Immunology, University of North Carolina, Chapel Hill 27599-7290.
Frelinger
J A
JA
D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D015488:HIV Antigens / Q000737:chemistry; D015789:HLA-A2 Antigen / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D009980:Influenza A virus / Q000737:chemistry*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D012635:Secretory Component / Q000737:chemistry* / Q000235:genetics; D014162:Transfection; D014763:Viral Matrix Proteins / Q000737:chemistry*
D003902:Detergents; D015488:HIV Antigens; D015789:HLA-A2 Antigen; C031540:M-protein, influenza virus; D010455:Peptides; D012635:Secretory Component; D014763:Viral Matrix Proteins