The influence of exogenous peptide on beta2-microglobulin exchange in the HLA complex: analysis in real-time.
We used an optical biosensor to determine the relative binding affinity of peptides to purified HLA class I molecules. In this assay we monitor beta2-microglobulin (beta2m) exchange within the HLA-A2 molecule, whereby native beta2m in the complex is replaced by beta2m immobilized at the surface of the biosensor. Quantitative kinetic measurements permit us to obtain association rate (kass), dissociation rate (kdiss) and affinity constants (KA) for the beta2m exchange reaction, alone, (control) and in the presence of exogenous peptide. We tested a panel of six peptides which had been designed and synthesized with an HLA-A2 binding motif, and had also been tested by the T2-cell binding assay, along with control peptides. The biosensor results demonstrate that exogenous peptide influences the dynamics of beta2m exchange in a sequence-specific manner. Five of six peptides increased the association rate, decreased the dissociation rate, and significantly increased the affinity (KA=1. 55-1.88x10(9) M-1) of HLA-A2 for immobilized beta2m compared with the control (KA =1.14+/-0.04x10(9)M-1), demonstrating stabilization of the complex. One peptide was unable to stabilize the complex, as also shown in the T2 binding assay. However, analysis of peptide sequences demonstrated that the HLA-A2 secondary motif as well as primary motif residues are required for HLA-A2 stabilization. Further experiments demonstrated that beta2m exchange alone cannot stabilize the HLA class I complex at the cell surface until a peptide of sufficient binding affinity is bound. Hence kinetics equal to or below the control values in our biosensor assay probably represent an unstable complex in vivo. Unlike other methods described for the analysis of peptide stabilization, this approach is significantly faster, provides full kinetic analysis, and is simpler, since it requires no labeling of peptides. Furthermore, this may have important implications in the assessment of peptide vaccines.
Morgan
C L
CL
Anthony Nolan Research Institute, The Royal Free Hospital, London NW3 2QG, UK.
Ruprai
A K
AK
Solache
A
A
Lowdell
M
M
Price
C P
CP
Cohen
S B
SB
Parham
P
P
Madrigal
J A
JA
Newman
D J
DJ
D015374:Biosensing Techniques; D002460:Cell Line; D015789:HLA-A2 Antigen / Q000378:metabolism*; D006801:Humans; D008214:Lymphocytes / Q000166:cytology; D009842:Oligopeptides / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D001613:beta 2-Microglobulin / Q000378:metabolism*
D015789:HLA-A2 Antigen; D009842:Oligopeptides; D001613:beta 2-Microglobulin