Structure and Function of a Fungal Adhesin that Binds Heparin and Mimics Thrombospondin-1 by Blocking T Cell Activation and Effector Function
Figure 6
Influence of competitors on BAD-1 binding to heparin.
(A) Effect of a WxxW motif heparin-binding peptide on binding of BAD-1 (eFluor605) to immobilized heparin. “None” denotes BAD-1 binding to heparin agarose with no competitor. The WxxW peptide, or a mutant control peptide, was incubated with heparin resin at 1 mg/ml before addition of fluorescent BAD-1. Binding was quantitated by fluorescence units detected in a Filtermax F5 plate reader. Results are the mean ± SEM two experiments. (B) Effect of TR4 reduction on binding to heparin. Samples were incubated with resin directly or first boiled for 3 min in buffer alone or buffer with 5 mM DTT. TR4 has four copies of the BAD-1 tandem repeat. ΔCterm has all 41 repeats, but no C-terminal EGF-like domain. Binding was quantified by A280 measurement. Reduced TR4 bound significantly better than untreated TR4 or TR4 boiled without DTT (*, p<0.05). Results are the mean ± SEM of two experiments. (C) Effect of reduced TR4 on binding of BAD-1 (eFluor605) to immobilized heparin. BAD-1 binding to heparin agarose was quantified with or without pretreatment of resin with reduced TR4 as in panel C. BAD-1 binding was quantified by fluorescence units as above. Mannan resin is a background control. Heparin resin pre-treated with reduced TR4 at 0.2 and 0.1 mg/ml bound BAD-1 significantly better than untreated heparin resin (*, p<0.05). Soluble heparin significantly blocked binding of BAD-1 to both of these pre-treated resins (**, p<0.05).