A covalent oxidoreductase intermediate in propeptide-dependent von Willebrand factor multimerization

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Von Willebrand factor (VWF) multimerization is essential for its hemostatic function.
• The precise mechanism of VWF multimer assembly in the Golgi apparatus remains incompletely understood.
• The VWF propeptide plays a critical role in VWF maturation and secretion.

Purpose of the Study:

• To investigate the role of the VWF propeptide in VWF multimer assembly.
• To identify intermediate disulfide-linked species during VWF multimerization.
• To elucidate the oxidoreductase activity of the VWF propeptide.

Main Methods:

• Expression of truncated VWF subunits (D1D2D'D3) in baby hamster kidney cells.
• Two-dimensional gel electrophoresis to identify disulfide-linked intermediates.
• Proteolytic cleavage with thrombin or furin to analyze protein fragments.

Main Results:

• An intrachain disulfide-linked intermediate between the VWF propeptide and D3 domain was detected in the endoplasmic reticulum.
• This intermediate rearranged in the Golgi apparatus to form secreted propeptide and D'D3 dimers.
• Similar disulfide-linked species were observed in cells expressing full-length VWF or separate propeptide and D'D3 proteins.

Conclusions:

• The VWF propeptide functions as an oxidoreductase, promoting disulfide bond formation necessary for VWF multimerization.
• A transient propeptide-D3 disulfide bond intermediate is formed and rearranged during VWF assembly.
• These findings support a model where the propeptide actively facilitates VWF multimerization in the Golgi apparatus.