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O-linked glycans protect VWF
inhibition of MGL-mediated clearance is attributable to an increase in VWF half-life. Importantly the magnitude of this effect of MGL on plasma VWF levels is also greater than that previously reported in Asgr1-/- mice (VWF:Ag lev- els increased ~1.5 fold).24 Collectively, these data demon- strate that mMGL2 constitutes another novel macrophage clearance receptor for VWF in mice. More importantly, the findings further suggest that MGL has a greater effect than ASGPR in regulating physiological VWF clearance.
The A domains of von Willebrand factor play a critical role in regulating MGL binding
Previous studies have demonstrated that macrophages play a key role in VWF clearance, and have further shown that macrophage receptor-recognition site(s) are present within the VWF-A1A2A3 domains.13,16 To assess the specif- ic role of VWF domains in modulating MGL binding a series of variants were expressed (Figure 2A) (Online Supplementary Table S1). Since MGL is expressed on macrophages, we investigated whether the A domains of VWF influence MGL binding. Dose-dependent binding of pdVWF to recombinant human MGL was observed (Figure 2B). In keeping with the fact that MGL is a C-type lectin, this binding was ablated in the presence of EDTA (Figure 2B). Conversely, VWF-MGL binding was significantly enhanced in the presence of ristocetin (1 mg/mL) (Figure 2B). No significant effect of VWF multimer distribution on MGL-binding was observed (Online Supplementary Figure S1). Binding studies confirmed dose-dependent binding of VWF-A1A2A3 to MGL, which was again ristocetin- and calcium-dependent (Figure 2C). Finally, the relative impor- tance of the individual domains within A1A2A3 in deter- mining MGL binding was assessed. Although significant binding of the VWF-A1 domain to MGL was seen, no binding for either VWF-A2 or VWF-A3 was observed (Figure 2D). Together, these data support the hypothesis that the A1 domain of VWF plays a critical role in deter- mining VWF binding to the MGL surface receptor on macrophages.
O-linked glycans on von Willebrand factor modulate the MGL interaction
Each VWF monomer contains 13 N-linked and ten O- linked glycan structures (Figure 3A).34,35 Whereas N-glycans are distributed across the VWF monomer, eight of the ten O-glycans are clustered in two groups around the VWF-A1 domain.35,36 To study the importance of specific N- and O- glycans in regulating MGL binding, pdVWF was treated with PNGase F and/or O-glycosidase respectively. Following each digestion, residual VWF glycan expression was assessed using lectin-binding ELISA (Online Supplementary Figure S2A, B). Although treatment with PNGase F successfully removed N-linked glycans from full- length pdVWF, it did not have a significant effect on MGL binding (Figure 3B). Conversely, digestion with O-glycosi- dase was associated with a marked reduction in pdVWF binding to MGL (P<0.001) (Figure 3B). The VWF-A1A2A3 truncation contains two N-linked glycans and eight O- linked glycans (Figure 3A). In keeping with full-length VWF, PNGase treatment of A1A2A3-VWF (Online Supplementary Figure S2B, C) had no impact on the MGL interaction, whereas removal of O-glycans markedly atten- uated binding (P<0.01) (Figure 3C). Digestion of isolated A1 domain with O-glycosidase (Online Supplementary Figure S2E) was also associated with a significant reduction
in MGL binding (P<0.001) (Figure 3D). Finally, to investi- gate the relative importance of the two OLG clusters at either side of the A1 domain, isolated A1-OLG cluster 1 and A1-OLG cluster 2 were expressed (Figure 2A). Although MGL-binding was observed for both of these VWF A1 domain truncations, significantly enhanced bind- ing was observed for A1-OLG cluster 2 (Figure 3E). Cumulatively, these findings demonstrate that VWF OLG, particularly those clustered either side of the A1 domain, play a major role in regulating MGL interaction.
α2-3 sialylation on O-glycans protects von Willebrand factor against MGL-mediated clearance
Recent mass spectrometry studies have characterized the O-glycan structures expressed on human pdVWF and highlighted significant heterogeneity (Figure 4A).35-37 Critically, however, a consistent feature of these O-glycan chains is that they generally terminate with sialic acid, which may be present in either α2-3 or α2-6 linkage.35,36 In contrast, most N-linked sialic acids are α2-6 linked.34 To further investigate the role of VWF O-glycans in determin- ing MGL-mediated clearance, pdVWF was digested with a series of exoglycosidases to generate specific VWF glyco- forms (Figure 4A). Treatment with α2-3 neuraminidase to remove α2-3 linked sialylation from O-glycans (Online Supplementary Figure S2F) significantly enhanced pdVWF binding to MGL (P=0.017) (Figure 4B). Similarly, digestion with α2-3,6,8,9 neuraminidase (which removes α2-3 linked sialylation from O-glycans and α2-6 linked sialyla- tion from both N- and O-glycans) (Online Supplementary Figure S2G) was also associated with significantly increased MGL binding (P=0.006). Despite the fact that an estimated 80% of total sialylation on VWF is α2-6 linked, α2-3,6,8,9 Neu-VWF binding to MGL was not different to that observed following α2-3 neuraminidase digestion alone (Figure 4B). Significantly enhanced binding was observed for PNG-VWF following additional removal of α2-3 linked sialylation and exposure of the O-linked T antigen structure (Figure 4C). Finally, PNG-VWF was sequentially treated with α2-3 neuraminidase and β1-3 galactosidase to remove both terminal sialic acid and sub- terminal galactose (Gal) residues from VWF O-glycan chains (Online Supplementary Figure S2H). This combined digestion ablated the enhanced binding observed following α2-3 neuraminidase digestion alone (Figure 4C). These data demonstrate that α2-3 linked sialylation on VWF O- glycans specifically protects VWF against MGL-mediated clearance. Loss of this capping sialic acid results in Gal residue exposure on VWF O-glycans, which then triggers clearance through the MGL receptor. In order to consider whether other VWF domains/glycans may contribute to MGL-interaction, we compared binding for N-terminal D’A3-VWF and C-terminal A3-CK-VWF fragments. In keeping with a key role for the A1 domain, significant binding of D’A3-VWF to MGL was observed (Figure 4D). Interestingly, however, some A3-CK-VWF binding was also seen, suggesting that O-glycans (T1679 and/or T2298) downstream of the A1 domain may also play a role.
Role of MGL and ASGPR in modulating pathological enhanced clearance of desialylated von Willebrand factor
Previous studies have demonstrated altered VWF sialy- lation in patients with VWD as well as in a number of other conditions.41 To investigate the role of MGL in mediating the enhanced clearance of pathologically desia-
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