ISSN:
1432-1211
Keywords:
Key wordsβ2
;
microglobulin
;
Biosensor
;
Kinetics
;
Peptide
;
HLA-A2
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
,
Medicine
Notes:
Abstract We used an optical biosensor to determine the relative binding affinity of peptides to purified HLA class I molecules. In this assay we monitor β2–microglobulin (β2m) exchange within the HLA-A2 molecule, whereby native β2m in the complex is replaced by β2m 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 β2m 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 β2m exchange in a sequence-specific manner. Five of six peptides increased the association rate, decreased the dissociation rate, and significantly increased the affinity (KA=1.55–1.88×109 M–1) of HLA-A2 for immobilized β2m compared with the control (KA =1.14±0.04×109 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 β2m 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.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/s002510050409
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