Abstract
Abstract 1078
Von Willebrand Factor (VWF) is a large multimeric plasma glycoprotein that mediates platelet adhesion under high shear stress and is the carrier molecule for FVIII. VWF contains a large number of cysteine residues that were previously thought to all participate in either intra- or inter-molecular disulphide bonds. However, recently it has been shown that VWF contains a proportion of unpaired cysteine residues, or free thiols and these may be involved in lateral self association of VWF. Initially Choi et al concluded that two free thiols are present in the D3 domain C889 and C898) and seven in the C domains (C2448, C2451, C2453, C2490, C2491, C2528 and C2533). Moreover, Ganderton et al recently showed that expression of the isolated VWF C2 domain resulted in the formation of disulphide linked oligomers and suggested that lateral self-association of VWF involved the C2431-C2453 bond located in the VWF C2 domain. When they mutated C2453 to Alanine the extent of oligomerisaton was enhanced. However it is not clear how this relates to the full length VWF molecule. In the present study we investigated the effect of mutating the predicted unpaired cysteine residues on the expression of full length VWF. Initially we used the binding of MPB to VWF to compare the relative amount of free thiol on plasma derived and recombinant VWF (rVWF). Interestingly, rVWF presented an increased free thiol content compared to purified plasma derived VWF indicating that free thiols, at least in rVWF are formed without exposure to the circulation. Next we created nine individual point mutations, based the observations of Choi et al, changing the predicted unpaired cysteine residues to alanine in full length VWF and analysed their expression in HEK293T cells. Interestingly, all of the point mutations failed to secrete from HEK293T cells, with the protein being retained within the cell lysate. A double point mutant, C2431A-C2453A, similarly failed to secrete. Analysis of the pro-VWF:mature-VWF ratio and Endo-H digestion of intracellular VWF demonstrated that all the mutants were retained within the endoplasmic reticulum (ER). Co-expression experiments with wild type VWF partially restored expression of some mutants, however co-expression with a deletion A1/A3 construct, demonstrated that the molecules containing the cysteine point mutations were retained predominately in the ER. Together these data suggest that in full length VWF, correct disulphide bonding within the ER is required for protein secretion. Since the point mutations did not express we created a series of deletion mutants to remove portions of the C-terminus of VWF. While VWF with either its A1, A2, A3 or D4 domain was expressed at comparable levels to wild type, all of the created C-terminal deletion variants: ΔC1C6(2255–2720), ΔC1C2(2255–2428), ΔC3(2431–2494), ΔC3C4-(2400–2515) and ΔC3C6-(2400–2662) also failed to secrete at significant levels. This data suggest that in the full length VWF molecule an intact sequence of C-domains is required for proper expression. To establish if the cysteine mutants could be expressed in smaller VWF constructs we introduced the same mutations into VWF molecules spanning the A2-CK, A2C6 and C3-CK domains. Interestingly, the mutants failed to express in VWF-A2CK and A2C6, again being retained in the ER, but were secreted in VWF-C3CK although to a significantly less extent than wild type C3CK. Furthermore, the A2C6 construct only expressed as monomers with very few dimers. Together these data demonstrate that correct disulphide bonding and an intact series of C domains are required for passage through the ER into the Golgi and efficient VWF secretion. The location and mechanism by which certain disulphide bonds break, forming free thiols remains to be established.
Disclosures:
No relevant conflicts of interest to declare.
Formation of one or more intrachain disulphide bonds is required for the intracellular processing and transport of CD36
