Epitope mapping for monoclonal antibody reveals the activation mechanism for αVβ3 integrin.

Epitopes for a panel of anti-αVβ3 monoclonal antibodies (mAbs) were investigated to explore the activation mechanism of αVβ3 integrin. Experiments utilizing αV/αIIb domain-swapping chimeras revealed that among the nine mAbs tested, five recognized the ligand-binding β-propeller domain and four recognized the thigh domain, which is the upper leg of the αV chain. Interestingly, the four mAbs included function-blocking as well as non-functional mAbs, although they bound at a distance from the ligand-binding site. The epitopes for these four mAbs were further determined using human-to-mouse αV chimeras. Among the four, P3G8 recognized an amino acid residue, Ser-528, located on the side of the thigh domain, while AMF-7, M9, and P2W7 all recognized a common epitope, Ser-462, that was located close to the α-genu, where integrin makes a sharp bend in the crystal structure. Fibrinogen binding studies for cells expressing wild-type αVβ3 confirmed that AMF-7, M9, and P2W7 were inhibitory, while P3G8 was non-functional. However, these mAbs were all unable to block binding when αVβ3 was constrained in its extended conformation. These results suggest that AMF-7, M9, and P2W7 block ligand binding allosterically by stabilizing the angle of the bend in the bent conformation. Thus, a switchblade-like movement of the integrin leg is indispensable for the affinity regulation of αVβ3 integrin

Effect of integrin extension on the binding of the anti-αVβ3 mAbs.

<p>Wild-type αV or αV carrying the Q589NAT mutation was transiently expressed together with wild-type β3 in CHO cells. The binding of function-blocking mAbs to these cells was examined by FACS. The MFI obtained from the whole cell population with each mAb was normalized by the MFI obtained with an anti-αV mAb 17E6 that represents the αV expression. There was no significant difference in the binding of leg-binding mAbs in cells expressing Q589NAT (solid column) as compared with cells expressing wild-type αV (hatched column).</p

MAb binding to cells expressing domain-swapping chimeras.

<p>The numbers represent the percentage of the cell population stained with each mAb.</p

MAb binding to cells expressing human-to-mouse αV mutants.

<p>The numbers represent the percentage of the cell population stained with each mAb. Bindings significantly lower than those for 17E6 or SZ21 are marked in red.</p

Effect of αV-to-αIIb mutation on the binding of the leg-binding mAbs.

<p>The amino acid residues 457–467 of the αV chain were individually mutated to the corresponding αIIb residues. The mutant αV was transiently expressed together with wild-type β3 in CHO cells, and the binding of mAbs to these cells was examined by FACS. The ratio of the MFI obtained from the whole cell population with each mAb to that obtained with 7E3 is shown as normalized binding. Asterisks indicate binding that was less than 50% of the wild type.</p

Effect of anti-αVβ3 mAbs on fibrinogen binding.

<p>A. FITC-fibrinogen binding to cells expressing αVβ3 in the presence of 1 mM Ca²⁺ and 1 mM Mg²⁺ (open column) or in the presence of 2 mM Mg²⁺ and 5 µM EGTA (hatched column) is shown. The ratio of the MFI (FL1) to the MFI (FL2) in the gated cell population was used to normalize the binding with the expression of αVβ3 on the cell surface. B. FITC-fibrinogen binding to cells expressing wild-type αVβ3 in the presence of 2 mM Mg²⁺ and 5 µM EGTA was examined. Binding in the presence of 100 µg/mL of the indicated mAb is shown in the hatched column. An equivalent volume of PBS, instead of the mAb solution, was included to obtain the control binding. The asterisks indicate statistically different binding abilities, compared with the control (*<i>P</i><0.01, **<i>P</i><0.05).</p

Schematic representation of αV/αIIb chimeras.

<p>The abbreviations P, T, C1, C2, and TC in the figure stand for β-propeller, thigh, calf-1, calf-2, and transmembrane-cytoplasmic domain, respectively. The numbers indicate the domain boundaries used to create the chimeras.</p

MAb binding to cells expressing tail-swapping chimeras.

<p>MAb binding to cells expressing wild-type human β3 (β3), wild-type human αVβ3 (αVβ3), wild-type human αIIbβ3 (αIIbβ3), tail-swapping mutants (V/B, B/V), and to parent CHO cells (CHO) was examined. The numbers represent the percentage of the cell population stained with each mAb.</p