(B-74) The unfolding behaviors of von Willebrand Factor (VWF) A2 domain mutants with reduced N-linked glycosylation

Introduction:: Von Willebrand factor (VWF) is a multimeric plasma glycoprotein involved in hemostasis, facilitating platelet adhesion to damaged vascular walls under shear stress. The A2 domain of VWF, which contains an enzymatic cleavage site for the plasma metalloprotease ADAMTS13, plays a crucial role in VWF’s functional regulation. When exposed to shear flow, the A2 domain unfolds, revealing the cleavage site and allowing ultra-large VWF multimers to be digested into smaller, less thrombogenic multimers. Abnormal mechanical properties of the A2 domain may affect its unfolding, potentially leading to thrombosis and bleeding disorders. Recent research has highlighted the importance of N-linked glycans in enhancing the mechanical stability of the A2 domain. To better understand the impact of N-linked glycosylation on VWF biomechanics, single-molecule force measurements were performed on wild-type VWF A2 domain and two A2 domain mutants, N1515Q and N1574Q, with reduced N-linked glycosylation.


Materials and Methods:: Optical tweezer experiments were conducted to examine the unfolding behaviors of recombinant wildtype (WT) A2 domain and its mutants N1515Q and N1574Q. The A2 proteins were expressed in HEK293 cells and purified via affinity chromatography, followed by size-exclusion chromatography. The purity of protein samples and the reduction of N-linked glycosylation were confirmed by SDS-PAGE analysis. The unfolding extension of wild-type A2 domain and A2 domain mutants was measured by a custom-built optical tweezer apparatus at five constant loading rates of force. Streptavidin (SA) modified polystyrene beads (2.0 μm in diameter) were incubated with either SpyCatcher-DNA handle or Spytag-A2 domain at room temperature in Tris-buffered saline (10 mM Tris, 150 mM NaCl, 0.01% BSA, pH 7.5) for 15 minutes. One SA bead was fixed by a micropipette, while the other was controlled by a laser trap. The beads were allowed to approach each other and to facilitate the formation of an isopeptide covalent bond between Spytag and SpyCatcher, securing both ends of the A2 domain for the pulling study. The stretching force and extension of the molecular tether were recorded at various pulling speeds.


Results, Conclusions, and Discussions:: A minimum of 50 force-extension curves were obtained for each sample at each pulling speed. Histograms of the A2 unfolding extensions were generated and fit to Gaussian distributions to determine the most probable extensions at different force ranges, then fit to the worm-like chain (WLC) model. The WLC fit result showed that the contour length of unfolded N1515Q was 70.5 ± 3.7 nm (mean ± SEM), similar to the contour length of the WT A2 unfolding (70.2 ± 2.7 nm, mean ± SEM). Interestingly, the unfolding extension histogram of N1574Q displayed two peaks in its Gaussian distributions, differing from the histograms of the WT A2 and N1515Q (Figure.1), which is consistent with our previous result of cleavage assay, where WT and N1515Q proteins were not cleaved by the VWF protease ADAMTS13, but N1574Q protein was cleaved into 10-15 kDa fragments. This suggests a heterogeneous conformation of N1574Q and highlights the significance of N-linked glycosylation at N1574 in maintaining the A2 structural stability.



Using optical tweezers, the end-to-end extensions associated with the forced unfolding of VWF A2 domain mutants have been measured, indicating that N-linked glycosylation at N1574 influences A2 domain unfolding and ADAMTS13 regulation. This single-molecule biomechanical data will contribute to a better understanding of the mechanical mechanisms involved in the unfolding of VWF A2 domains and their related functions.


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