BACKGROUND: von Willebrand factor (VWF) contains free thiols that mass spectroscopy has located to nine cysteines: two in the D3 domain (Cys889 and Cys898) and seven in the C domains (Cys2448, Cys2451, Cys2453, Cys2490, Cys2491, Cys2528, and Cys2533) (J Biol Chem, 7, 2007, 35604; Blood, 118, 5312). It has been suggested that these free thiols function to regulate the self-association of VWF through thiol-disulfide exchange (J Biol Chem, 7, 2007, 35604; Blood, 118, 5312). However, recent structural modeling has predicted that these cysteines are, in fact, disulfide-bonded (Blood, 118, 5312; Blood, 120, 449). OBJECTIVES: To use mutation and expression analyses to investigate how these conflicting reports might be compatible with the synthesis and expression of VWF. METHODS AND RESULTS: Both full-length VWF and VWF fragments with cysteine to alanine mutations of the nine cysteines and two predicted binding partners (Cys2431 and Cys2468) failed to secrete. Mutation of a cysteine pair, C2431A/C2453A, similarly resulted in a failure to secrete, indicating that this is not secondary to creation of an unpaired thiol. Deletion mutants containing seven of these cysteines, conforming to hypothesized domain boundaries, also failed to secrete: ∆C1C6 (2255-2720), ∆C3C4 (2429-2577), ∆C3 (2429-2496), and ∆C4 (2497-2577). Analysis of cell lysates and immunofluorescence confirmed that the mutants were retained within the endoplasmic reticulum (ER). Coexpression with wild-type VWF rescued secretion of some mutants to a limited extent. CONCLUSIONS: These data suggest: first, that pairing of cysteines implicated in free thiol exchange is essential for correct folding of the VWF molecule, and unpairing must occur following exit from the ER or secretion from the cell; and second, that intact C domains are essential for efficient VWF secretion and must interact in the ER.
BACKGROUND: von Willebrand factor (VWF) contains free thiols that mass spectroscopy has located to nine cysteines: two in the D3 domain (Cys889 and Cys898) and seven in the C domains (Cys2448, Cys2451, Cys2453, Cys2490, Cys2491, Cys2528, and Cys2533) (J Biol Chem, 7, 2007, 35604; Blood, 118, 5312). It has been suggested that these free thiols function to regulate the self-association of VWF through thiol-disulfide exchange (J Biol Chem, 7, 2007, 35604; Blood, 118, 5312). However, recent structural modeling has predicted that these cysteines are, in fact, disulfide-bonded (Blood, 118, 5312; Blood, 120, 449). OBJECTIVES: To use mutation and expression analyses to investigate how these conflicting reports might be compatible with the synthesis and expression of VWF. METHODS AND RESULTS: Both full-length VWF and VWF fragments with cysteine to alanine mutations of the nine cysteines and two predicted binding partners (Cys2431 and Cys2468) failed to secrete. Mutation of a cysteine pair, C2431A/C2453A, similarly resulted in a failure to secrete, indicating that this is not secondary to creation of an unpaired thiol. Deletion mutants containing seven of these cysteines, conforming to hypothesized domain boundaries, also failed to secrete: ∆C1C6 (2255-2720), ∆C3C4 (2429-2577), ∆C3 (2429-2496), and ∆C4 (2497-2577). Analysis of cell lysates and immunofluorescence confirmed that the mutants were retained within the endoplasmic reticulum (ER). Coexpression with wild-type VWF rescued secretion of some mutants to a limited extent. CONCLUSIONS: These data suggest: first, that pairing of cysteines implicated in free thiol exchange is essential for correct folding of the VWF molecule, and unpairing must occur following exit from the ER or secretion from the cell; and second, that intact C domains are essential for efficient VWF secretion and must interact in the ER.
Authors: Shanice S Webster; Marion Mathelié-Guinlet; Andreia F Verissimo; Daniel Schultz; Albertus Viljoen; Calvin K Lee; William C Schmidt; Gerard C L Wong; Yves F Dufrêne; George A O'Toole Journal: mBio Date: 2022-02-01 Impact factor: 7.867