Analysis of intracellular storage and regulated secretion of 3 von Willebrand disease–causing variants of von Willebrand factor

Michaux, Grégoire; Hewlett, Lindsay; Messenger, Sarah L.; Goodeve, Anne; Peake, I. R.; Daly, Mary E.; Cutler, Daniel F.

doi:10.1182/blood-2003-02-0599

Cited by 76 publications

(139 citation statements)

References 32 publications

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“…Consistent with previous observations, cells transfected with wtVWF demonstrated 3 distinct staining patterns: (1) diffuse granular staining that colocalized with the ER marker calnexin (data not shown), (2) diffuse granular staining with a number of elongated or rounded punctate structures that have previously been shown to be pseudo-WPBs, and (3) little or no diffuse staining with just the presence of pseudo WPB. 26 After 5 days transfection the majority of wtVWF transfected cells demonstrated little granular staining and numerous WPBs indicating correct processing and storage of VWF (Figure 4Ai). For mutants VWF-N99Q, N857Q, and N2790Q, most of the cells demonstrated diffuse granular ER, staining confirming that these mutants impaired normal processing of the VWF molecule resulting in ER retention (Figures 4Aii-iv) Interestingly though, for all 3 mutants a small number of cells were able to form VWFcontaining inclusion bodies (Figures 4Av-vii).…”

Section: Glycosylation Mutants Vwf-n99q N857q and N2790q Are Retainmentioning

confidence: 99%

“…Expression of VWF in HEK293 cells has previously been shown to induce the formation of storage vesicles indistinguishable from WP bodies and these cells were therefore used for this investigation. 26 Five days after transfection, HEK293 cells were fixed and permeabilized, stained for VWF then analyzed by immunofluorescence confocal microscopy ( Figure 4). Consistent with previous observations, cells transfected with wtVWF demonstrated 3 distinct staining patterns: (1) diffuse granular staining that colocalized with the ER marker calnexin (data not shown), (2) diffuse granular staining with a number of elongated or rounded punctate structures that have previously been shown to be pseudo-WPBs, and (3) little or no diffuse staining with just the presence of pseudo WPB.…”

Section: Glycosylation Mutants Vwf-n99q N857q and N2790q Are Retainmentioning

confidence: 99%

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Specific N-linked glycosylation sites modulate synthesis and secretion of von Willebrand factor

McKinnon¹,

Goode²,

Birdsey

et al. 2010

Blood

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We examined the role that N-linked glycans play in the synthesis and expression of von Willebrand Factor (VWF). Blocking the addition of N-linked glycans (NLGs) or inhibiting initial glycan processing prevented secretion of VWF. To determine whether specific glycosylation sites were important, the 16 VWF N-linked glycosylation sites were mutated followed by expression in HEK293T cells. Four NLG mutants affected VWF expression: N99Q (D1 domain), N857Q (D' domain), N2400Q (B1 domain), and N2790Q (CK domain) either abolished or reduced secretion of VWF and this was confirmed by metabolic labeling. Multimer analysis of mutant N2790Q cell lysate revealed an increase in VWF monomers, which was also observed when the isolated CK domain was expressed with N2790 mutated. Immunofluorescence microscopy showed that mutants N99Q, N857Q, and N2790Q were primarily retained within the ER, producing only few pseudo Weibel-Palade bodies over longer time periods compared with wtVWF. All the variants also showed an increase in free thiol reactivity. This was greatest with N857Q and D4-C2 NLG mutants, which had approximately 6-fold and 3-to 4-fold more free thiol reactivity than wtVWF. These data provide further evidence of the critical role that individual N-linked glycans play in determining VWF synthesis and expression. (Blood. 2010;116(4):640-648) Introductionvon Willebrand factor (VWF) is a large multimeric plasma glycoprotein essential for normal hemostasis, acting firstly by supporting platelet adhesion to surfaces at sites of vascular injury and secondly as the carrier molecule for pro-coagulant Factor VIII (FVIII). 1,2 Synthesis of VWF is limited to megakaryocytes and endothelial cells. 3 The pre-pro-VWF molecule comprises a 22 amino acid signal peptide, a 741 amino acid propeptide, and a 2050 amino acid mature subunit. The pro-VWF monomer is composed of 4 types of domains (A-D) arranged as follows: NH 2 -D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK-COOH. 1 VWF multimers are formed by C-and N-terminal intermolecular disulphide bonds, 4,5 with the largest multimers exceeding 2 ϫ 10 4 kDa and having the greatest adhesive activity. 6 During synthesis, VWF undergoes extensive posttranslational modification resulting in the addition of 12 N-linked and 10 O-linked glycosylation sites per mature monomer. 7 Furthermore, the propeptide also contains 4 potential N-linked glycosylation sites although it is not known if these are used. In total, carbohydrate accounts for approximately 20% of the molecular weight of VWF. 8 N-linked glycosylation is one of the most common co/ posttranslational modifications. It is characterized by the addition of a carbohydrate moiety to the protein via a beta-glycosidic linkage between an N-acetylglucosamine residue and the ␦-amide of an asparagine residue in the sequence NXS/T (or in some cases NXC), 9 where X is any amino acid except proline. 10 This takes place after translocation into the endoplasmic reticulum (ER) when the enzyme oligosaccharyltransferase (OST) transfers a preformed 14-saccharide core...

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Section: Glycosylation Mutants Vwf-n99q N857q and N2790q Are Retainmentioning

confidence: 99%

Section: Glycosylation Mutants Vwf-n99q N857q and N2790q Are Retainmentioning

confidence: 99%

Specific N-linked glycosylation sites modulate synthesis and secretion of von Willebrand factor

McKinnon¹,

Goode²,

Birdsey

et al. 2010

Blood

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“…We find that when early response (10 min after activation) is analyzed, a loss of 40% of normal release occurs, whereas at 30 min, the difference, while still statistically significant, is much lower, at around 10%. Assays of VWF release have shown a quasi-linear increase in released VWF over 60 min [32], but this new dataset clearly implies that there is a machinery involved that can differentially affect early and late events. Whether, as in neuro/ neuroendocrine or the pancreatic b-cell systems, there are readily-released and reserve pools with all the associated complexity of exocytic machineries is unclear, but now that their presence is suggested, experiments will surely follow.…”

Section: Discussionmentioning

confidence: 99%

“…VWF secretion from HUVECs was analysed as previously described [32]. Cells were rinsed in release medium (M199 medium with EarleÕs modified salts (GIBCO BRL), 10 mM Hepes, pH 7.4, and 0.2% BSA); 1 mL of release medium containing 100 ng mL )1 phorbol 12-myristate-13-acetate (PMA) was then added to each well for the required time (10-30 min) at 37°C.…”

Section: Secretion Assaysmentioning

confidence: 99%

A role for Rab10 in von Willebrand factor release discovered by an AP‐1 interactor screen in C. elegans

Michaux

Dyer

Nightingale

et al. 2011

Journal of Thrombosis and Haemostasis

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Summary. Background: Endothelial von Willebrand factor (VWF) mediates platelet adhesion and acts as a protective chaperone to clotting factor VIII. Rapid release of highly multimerized VWF is particularly effective in promoting hemostasis. To produce this protein, an elaborate biogenesis is required, culminating at the trans-Golgi network (TGN) in storage within secretory granules called Weibel-Palade bodies (WPB). Failure to correctly form these organelles can lead to uncontrolled secretion of low-molecular-weight multimers of VWF. The TGN-associated adaptor AP-1 and its interactors clathrin, aftiphilin and c-synergin are essential to initial WPB formation at the Golgi apparatus, and thus to VWF storage and secretion. Objectives: To identify new proteins implicated in VWF storage and/or secretion. Methods: A genomewide RNA interference (RNAi) screen was performed in the Nematode C. elegans to identify new AP-1 genetic interactors. Results: The small GTPase Rab10 was found to genetically interact with a partial loss of function of AP-1 in C. elegans. We investigated Rab10 in human primary umbilical vein endothelial cells (HUVECs). We report that Rab10 is enriched at the Golgi apparatus, where WPB are formed, and that in cells where Rab10 expression has been suppressed by siRNA, VWF secretion is altered: the amount of rapidly released VWF was significantly reduced. We also found that Rab8A has a similar function. Conclusion: Rab10 and Rab8A are new cytoplasmic factors implicated in WPB biogenesis that play a role in generating granules that can rapidly respond to secretagogue.

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“…To quantify pseudo-WPBs, 100 VWF-positive cells from two independent experiments were randomly selected and analysed. We used similar morphological criteria to those reported by Michaux et al (2003). Briefly, we categorized the pseudo-WPBs with a ratio of length over diameter <2 as 'round pseudo-WPBs' and those with a ratio ≥2 as 'elongated pseudo-WPBs.…”

Section: Immunostainingmentioning

confidence: 99%

Storage and secretion of naturally occurring von Willebrand factor A domain variants

et al. 2014

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SummaryVon Willebrand disease (VWD) is a bleeding disorder characterized by reduced plasma von Willebrand factor (VWF) levels or functionally abnormal VWF. Low VWF plasma levels in VWD patients are the result of mutations in the VWF gene that lead to decreased synthesis, impaired secretion, increased clearance or a combination thereof. However, expression studies of variants located in the A domains of VWF are limited. We therefore characterized the biosynthesis of VWF mutations, located in the VWF A1-A3 domains, that were found in families diagnosed with VWD. Human Embryonic Kidney 293 (HEK293) cells were transiently transfected with plasmids encoding full-length wild-type VWF or mutant VWF. Six mutations in the A1-A3 domains were expressed. We found that all mutants, except one, showed impaired formation of elongated pseudo-Weibel-Palade bodies (WPB). In addition, two mutations also showed reduced numbers of pseudo-WPB, even in the heterozygous state, and increased endoplasmic reticulum retention, which is in accordance with the impaired regulated secretion seen in patients. Regulated secretion upon stimulation of transfected cells reproduced the in vivo situation, indicating that HEK293 cells expressing VWF variants found in patients with VWD can be used to properly assess defects in regulated secretion.

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Analysis of intracellular storage and regulated secretion of 3 von Willebrand disease–causing variants of von Willebrand factor

Cited by 76 publications

References 32 publications

Specific N-linked glycosylation sites modulate synthesis and secretion of von Willebrand factor

Specific N-linked glycosylation sites modulate synthesis and secretion of von Willebrand factor

A role for Rab10 in von Willebrand factor release discovered by an AP‐1 interactor screen in C. elegans

Storage and secretion of naturally occurring von Willebrand factor A domain variants

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