Figure 3.

Peptide 15-1 binds to HA oligosaccharides and blocks their interaction with recombinant RHAMM protein. A: The graph shows quantification of the interaction between P15-1 and HA oligosaccharides by ITC. P15-1 bound to HA oligosaccharides (avg. 10 kDa) with a binding constant of kDa = 10⁻⁷ mol/L. A 0.9 mmol/L solution of HA was injected into MES (pH 6.0) containing 0.6 mmol/L peptide 15-1, and the energy released on interaction with P15-1 was determined by ITC. Binding curves of two independent experiments (red or black) are depicted. B: HA oligosaccharide:P15-1 interactions were confirmed with ELISA. ELISA plates were coated with HA oligosaccharides (1 mg/mL), followed by incubation with either biotinylated P15-1 or Scr Pep (50 μg/mL). PBS alone was used as control. Bound biotinylated peptides were detected by streptavidin-horseradish peroxidase. Peptide 15-1 binds to HA oligosaccharides. Values represent the mean ± SD of three ELISA wells. C: ELISA plates were coated with 1 μg/mL recombinant RHAMM followed by incubation with either 10 μg/mL 30mer HA or HA oligosaccharides (10 kDa) together with P15-1 or Scr Pep (1 μg/mL). Bound HA was detected with biotinylated aggrecan followed by streptavidin-horseradish peroxidase. P15-1 reduces RHAMM/HA binding. Values represent the mean ± SD of n = 3. *P < 0.001.