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O-linked glycans protect VWF
A
BC
DE
Figure 3. O-linked glycans on von Willebrand factor modulate the interaction with MGL. (A) Each von Willebrand factor (VWF) monomer contains 13 N-linked and ten O-linked glycan structures. The diagram also shows the most common VWF N-linked carbohydrate structure (a monosialylated, biantennary, core fucosylated com- plex glycan) and O-linked carbohydrate structure (core 1 sialylated T-antigen). (B) To investigate the role of VWF carbohydrate determinants in modulating the inter- action with MGL, plasma-derived (pd)VWF (10 mg/mL) was treated with either PNGase F (PNGase VWF) to remove N-glycans or PNGase F and O glycosidase (PNGase- OGly VWF) to remove both N- and O-glycans. Binding of the pdVWF glycoforms to human MGL was then compared to binding to untreated pd-VWF as before (100% binding = OD450 obtained for 10 mg/mL pdVWF). (C) To study a potential role for glycans in the A domains of VWF in regulating MGL binding, A1A2A3-VWF (150 nM VWF) was treated with either PNGase F or O-glycosidase. Binding to human MGL was then assessed compared to to the binding to WT A1A2A3-VWF (100% binding = OD450 obtained for 150 nM A1A2A3-VWF). (D) Since A1-VWF does not contain any N-linked glycan determinants, MGL binding studies were examined for WT-A1- VWF compared to O-glycosidase-treated VWF-A1 (100% binding = OD450 obtained for 150 nM A1-VWF). (E) Eight O-linked glycans are located in two clusters of four either side of the VWF A1 domain. To investigate the importance of these O-glycans in modulating the MGL interaction, two A1-VWF variants were generated each of which contained only one O-glycan cluster (A1-OLG cluster 1 contained T1248A, T1255A, T1256A, S1263A, while A1-OLG cluster 2 contained T1468A, T1477A, S1486A, T1487A). MGL-binding studies were compared for these two cluster variants as previously described (100% binding = OD450 obtained for A1-OLG cluster 1). All data are represented as mean ± standard error of mean of three independent experiments. *P<0.05, **P<0.01, ***P<0.001. PNGase F: peptide N-glycosi- dase F; OLG: O-linked glycans; OD450: absorbance at 450 nm.
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