Citation
- Authors: Anderson JR. et al.
- Year: 2022
- Journal: Blood 140 1419-1430
- Applications: in vitro / DNA / FectoPRO
- Cell type: Expi293F
Description: Human embryonic kidney Fibroblast
Known as: Expi 293-F, Expi, HEK-293 Expi
Method
Sequences encoding VWF D1-D3 (residues 1-1264), D1-A1 (1-1464), and D1-A2 (1-1671) with a C-terminal hexahistidine tag and a furin cleavage site mutated to ASAS were cloned into PD2529 vectors. 1 L of Expi293 cells in Expi293 medium were transfected using FectoPRO (Polyplus, #101000014). Valproic acid and glucose were added to the culture 24 hours after transfection to final concentrations of 3 mM and 0.4%, respectively.
Six days after transfection, secreted proteins were purified from the media by His-affinity in 20 mM Tris-HCl pH 8, 300 mM NaCl, and 10 mM CaCl2.
Abstract
von Willebrand factor (VWF) is an adhesive glycoprotein that circulates in the blood as disulfide-linked concatemers and functions in primary hemostasis. The loss of long VWF concatemers is associated with the excessive bleeding of type 2A von Willebrand disease (VWD). Formation of the disulfide bonds that concatemerize VWF requires VWF to self-associate into helical tubules, yet how the helical tubules template intermolecular disulfide bonds is not known. Here, we report electron cryomicroscopy (cryo-EM) structures of VWF tubules before and after intermolecular disulfide bond formation. The structures provide evidence that VWF tubulates through a charge-neutralization mechanism and that the A1 domain enhances tubule length by crosslinking successive helical turns. In addition, the structures reveal disulfide states before and after disulfide bond-mediated concatemerization. The structures and proposed assembly mechanism provide a foundation to rationalize VWD-causing mutations.