The 33mer peptide anginex is a potent inhibitor of angiogenesis and tumor growth.
Its biological activity is dependent on the beta-galactoside-binding protein galectin-1
(gal-1), which has been reported to be the main receptor for anginex. The gal-1-anginex
interaction has been observed using surface plasmon resonance and mass spectrometric
methods, but the stoichiometry and affinity in the solution remain elusive. Our aim
was to characterize the gal-1-anginex interaction via isothermal titration calorimetry.
In order to ensure protein purity and integrity, native gel electrophoresis, Western
blot analysis, mass spectrometric measurements, and ultracentrifugation were carried
out for the recombinant wild-type human gal-1 and V5D gal-1 expressed in E. coli.
Two stages were identified in the titration curves: (i) formation of a 4:1 galectin-1-anginex
complex with low nM affinity, and (ii) a complex with 1:1 stoichiometry exhibiting
K (D) > 200 nM. The 4:1 complex was robust at different concentrations, and neither
the oxidation state nor the V5D mutation (a monomeric gal-1 mutant) of gal-1 affected
this stoichiometry. The presence of the high-affinity 4:1 interaction may have implications
for the biological applications of anginex.
