Biogenesis of Weibel-Palade bodies in von Willebrand's disease variants with impaired von Willebrand factor intrachain or interchain disulfide bond formation

Abstract

Background: Mutations of cysteine residues in von Willebrand factor are known to reduce the storage and secretion of this factor, thus leading to reduced antigen levels. However, one cysteine mutation, p.Cys2773Ser, has been found in patients with type 2A(IID) von Willebrand's disease who have normal plasma levels of von Willebrand factor. We hypothesize that disruption of either intra- or interchain disulfide bonds by cysteine mutations in von Willebrand factor has different effects on the biogenesis of Weibel-Palade bodies.

Design and methods: The effect of specific cysteine mutations that either disrupt intrachain (p.Cys1130Phe and p.Cys2671Tyr) or interchain (p.Cys2773Ser) disulfide bonds on storage and secretion of von Willebrand factor was studied by transient transfection of human embryonic kidney cell line 293. Upon expression of von Willebrand factor these cells formed endothelial Weibel-Palade body-like organelles called pseudo-Weibel-Palade bodies. Storage of von Willebrand factor was analyzed with both confocal immunofluorescence and electron microscopy. Regulated secretion of von Willebrand factor was induced by phorbol 12-myristate 13-acetate.

Results: p.Cys1130Phe and p.Cys2671Tyr reduced the storage of von Willebrand factor into pseudo-Weibel-Palade bodies with notable retention of von Willebrand factor in the endoplasmic reticulum, whereas p.Cys2773Ser-von Willebrand factor was stored normally. As expected, wild-type von Willebrand factor formed proteinaceous tubules that were seen under electron microscopy as longitudinal striations in pseudo-Weibel-Palade bodies. p.Cys2773Ser caused severe defects in von Willebrand factor multimerization but the factor formed normal tubules. Furthermore, the basal and regulated secretion of von Willebrand factor was drastically impaired by p.Cys1130Phe and p.Cys2671Tyr, but not by p.Cys2773Ser.

Conclusions: We postulate that natural mutations of cysteines involved in the formation of interchain disulfide bonds do not affect either the storage in Weibel-Palade bodies or secretion of von Willebrand factor, whereas mutations of cysteines forming intrachain disulfide bonds lead to reduced von Willebrand factor storage and secretion because the von Willebrand factor is retained in the endoplasmic reticulum.

Figure 1.

Intracellular localization of VWF variants in transfected HEK293 cells. HEK293 cells were transiently transfected with WT-VWF or VWF mutants as indicated. Fixed cells were stained for VWF (left panel, green) and for PDI (the ER marker, middle panel, red). In the right panel (merge of green and red channels), the pseudo-WPB show up in green (VWF staining only), and the ER containing VWF shows up in yellow as a result of double staining for VWF and PDI. Scale bar = 5 μm. Between 175 and 370 cells expressing each of the VWF variants from two independent experiments were analyzed and the percentage of cells that showed retention of VWF in the ER (cells displaying yellow) is indicated on the right.

Figure 2.

Ultrastructure of transfected HEK293 cells visualized by transmission electron microscopy. HEK293 cells were transiently transfected as indicated. Pseudo-WPB displaying VWF tubules were formed by WT-VWF (A) and the three mutants (B-E). Dilated ER was observed in cells expressing p.Cys1130Phe (F) or p.Cys2671Tyr (G) but not in cells expressing p.Cys2773Ser (H) or WT-VWF (not shown). Note that the VWF tubules were clearly visualized in the cross sections of pseudo-WPB (indicated by the arrows in panels B and E). The ER (indicated by the arrowheads) shown in panels F, G and H was imaged in the same cells shown in panels B, C and D, respectively. The stars in panels C, G and H indicate mitochondria. In panel A-E, scale bar = 200 nm; in panels F-H, scale bar = 500 nm.

Figure 3.

Subunit composition of VWF under reducing conditions. HEK293 cells were transiently transfected with WT-VWF or VWF mutants as indicated. VWF secreted into the medium (A: Medium) or VWF retained in lysate (B: Lysate) was reduced with DTT and analyzed with SDS-PAGE and western blotting. In panel C, the ratio of proVWF (p) and mature VWF subunit (m) in the cell lysate was analyzed with Image J (NIH software, version 1.44P). Note that in panels A and B the parts separated by the space between p.Cys2671Tyr and p.Cys2773Ser are from the same western blot.

Figure 4.

Regulated secretion of WT-VWF or VWF variants. HEK293 cells were transiently transfected with WT-VWF, p.Cys1130Phe, p.Cys2671Tyr and p.Cys2773Ser, respectively (Panel A). In panel B, p.Cys1130Phe, p.Cys2671Tyr or p.Cys2773Ser was co-transfected with WT-VWF at a 1:1 ratio. Seventy-two hours after transfection HEK293 cells were rinsed twice with the release medium and incubated at 37°C for 60 min in the release medium without (Ctr) or with 160 nM PMA (PMA). Each bar represents VWF secreted into the release medium as a fraction of total VWF (medium plus lysate) times 100%. The mean and SEM are based on three independent experiments in duplicate. The numbers above the bars indicate the fold increase of secreted VWF comparing the stimulated (PMA) and control (Ctr) samples.

Figure 5.

Multimer analysis of secreted VWF. Multimers are shown for single transfections (A) and co-transfections with WT-VWF at a 1:1 ratio (B). The N-terminal dimers and odd-numbered multimers formed by VWF p.Cys2773Ser are indicated by the arrow and arrowheads, respectively. In panel A, the multimers of VWF p.Cys2671Tyr are from a different gel, the other separated parts are from the same gel. In panel B, the separated parts are from the same gel. The multimer patterns of secreted VWF were analyzed by SDS-agarose gel electrophoresis and western blotting under non-reducing conditions.

Figure 6.

Schematic illustration of VWF intracellular trafficking. WT-VWF (left) is synthesized and dimerized in the ER, and passes the quality control in the Golgi apparatus to multimerize and tubulize therein, and is eventually packed into WPB. Some of the VWF mutants with impaired intrachain disulfide bond formation (middle) escape the quality control and are stored in WPB, the others are retained in the ER undergoing aggregation or intracellular degradation. VWF mutants with impaired C-terminal interchain disulfide bond formation (right) fail to form C-terminal dimers in the ER. The monomers can get through the ER to the Golgi and partly form N-terminal dimers which are tubulized and stored into WPB. The remaining monomers are presumably stored into WPB; however, whether they are incorporated into VWF tubules is unknown. This hypothesis may also apply to the mutants lacking N-terminal interchain disulfide bonds. TGN: trans Golgi network; ER: endoplasmic reticulum.