Two Cys residues essential for von Willebrand factor multimer assembly in the Golgi

Purvis, Angie R.; Gross, Julia Christina; Dang, Luke T.; Huang, Raven H.; Kapadia, Milan; Townsend, R. Reid; Sadler, J. Evan

doi:10.1073/pnas.0705175104

Cited by 70 publications

(84 citation statements)

References 37 publications

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“…1D). Expression of preProVWF-EGFP in HUVECs gives rise to ProVWF-EGFP that exists as a dimer in the ER (Purvis et al, 2007). Its mobility was the slowest (Table 1 and Fig.…”

Section: Wpb Core Proteins Are Mobile In the Er Of Huvecsmentioning

confidence: 98%

Protein mobilities and P-selectin storage in Weibel–Palade bodies

Kiskin¹,

Hellen²,

Babich

et al. 2010

Journal of Cell Science

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Using fluorescence recovery after photobleaching (FRAP) we measured the mobilities of EGFP-tagged soluble secretory proteins in the endoplasmic reticulum (ER) and in individual Weibel–Palade bodies (WPBs) at early (immature) and late (mature) stages in their biogenesis. Membrane proteins (P-selectin, CD63, Rab27a) were also studied in individual WPBs. In the ER, soluble secretory proteins were mobile; however, following insertion into immature WPBs larger molecules (VWF, Proregion, tPA) and P-selectin became immobilised, whereas small proteins (ssEGFP, eotaxin-3) became less mobile. WPB maturation led to further decreases in mobility of small proteins and CD63. Acute alkalinisation of mature WPBs selectively increased the mobilities of small soluble proteins without affecting larger molecules and the membrane proteins. Disruption of the Proregion–VWF paracrystalline core by prolonged incubation with NH4Cl rendered P-selectin mobile while VWF remained immobile. FRAP of P-selectin mutants revealed that immobilisation most probably involves steric entrapment of the P-selectin extracellular domain by the Proregion–VWF paracrystal. Significantly, immobilisation contributed to the enrichment of P-selectin in WPBs; a mutation of P-selectin preventing immobilisation led to a failure of enrichment. Together these data shed new light on the transitions that occur for soluble and membrane proteins following their entry and storage into post-Golgi-regulated secretory organelles.

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“…1D). Expression of preProVWF-EGFP in HUVECs gives rise to ProVWF-EGFP that exists as a dimer in the ER (Purvis et al, 2007). Its mobility was the slowest (Table 1 and Fig.…”

Section: Wpb Core Proteins Are Mobile In the Er Of Huvecsmentioning

confidence: 98%

Protein mobilities and P-selectin storage in Weibel–Palade bodies

Kiskin¹,

Hellen²,

Babich

et al. 2010

Journal of Cell Science

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“…1) (13,17). Baby hamster kidney (BHK) cells that express this construct secrete a mixture of VWF propeptide (domains D1D2), DЈD3 monomers, and DЈD3 dimers (17,18). We purified each of these proteins [supporting information (SI) Fig.…”

Section: Vwf Propeptide and D D3mentioning

confidence: 99%

Assembly of Weibel–Palade body-like tubules from N-terminal domains of von Willebrand factor

Huang

Wang²,

Roth³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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144

285

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Lengthwise sections show longitudinal striations, and cross sections reveal closely spaced Ϸ20-nm diameter tubules separated by a less-dense matrix (1). Weibel-Palade bodies are composed almost entirely of von Willebrand factor (VWF) (2, 3), which is a multimeric plasma glycoprotein that can exceed 20 million Da in mass and 4 m in length. Megakaryocytes synthesize large VWF multimers and package them into platelet ␣-granules that are roughly spherical rather than cigar-shaped. Nevertheless, the VWF multimers in ␣-granules are organized into clusters of tubules with dimensions similar to those of VWF tubules in Weibel-Palade bodies (4). VWF is the largest known protein in the blood, and the very largest VWF multimers bind connective tissue and mediate platelet adhesion at sites of vascular injury. Weibel-Palade bodies play a critical role in hemostasis by delivering VWF multimers into the circulation. Defects in VWF multimer structure cause several forms of von Willebrand disease, the most common inherited bleeding disorder worldwide (5).VWF is synthesized as an Ϸ350-kDa precursor with a signal peptide and five kinds of structural domains arranged in the order D1-D2-DЈ-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK ( Fig. 1) (5). In the endoplasmic reticulum (ER), proVWF dimerizes through disulfide bonds between C-terminal CK domains (6-8).The resultant ''tail-to-tail'' proVWF dimers are transported to the Golgi, where the propeptide (domains D1D2) is cleaved by furin and additional ''head-to-head'' disulfide bonds form between D3 domains, yielding multimers that condense into tubules and form Weibel-Palade bodies (6, 9).In fact, the expression of VWF determines the existence and also the cigar-like shape of Weibel-Palade bodies. Without VWF, endothelial cells lack Weibel-Palade bodies (10, 11), and the expression of VWF in other cell types that have a regulated secretory pathway results in the generation of organelles that are indistinguishable from Weibel-Palade bodies (12, 13). This self-organizing behavior depends on a conserved set of Nterminal domains that have the dual function of promoting multimer assembly and directing tubular storage.Multimer assembly (14-16) and tubular packing (12, 17) both depend on the N-terminal D1D2DЈD3 domains and require the acidic pH of the late secretory pathway (13). Furthermore, the tubular morphology of Weibel-Palade bodies is essential for . VWF multimers are held together by intersubunit disulfide bonds between CK domains, which form in the ER, and by intersubunit disulfide bonds between D3 domains, which form in the Golgi. The structures of polypeptides encoded by the plasmids used in these studies are indicated.

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“…6 In the trans-Golgi and post-Golgi compartments, the VWF propeptide catalyzes the formation of VWF multimers via disulfide bonds near the VWF N terminus (C1099-C1099 and C1142-C1142) and is cleaved from proVWF by furin. [7][8][9] The resulting mature VWF multimers condense into long, helical structures that characterize the shape of WeibelPalade bodies and are released into the circulation as long, linear polymers with multiple concatemerized subunits. Each VWF multimer is composed of 2 to .60 subunits 10 (with each monomer containing a single FVIII-binding site).…”

Section: Introductionmentioning

confidence: 99%

“…12,13 The FVIII binding region within VWF has been localized to a proteolytic VWF fragment that spans residues S764-R1035, which compose the N terminus of the D9D3 domains and excludes the cysteines that coordinate multimerization. 8,14 Removal of the N-terminal S764-L1039 segment by granzyme M cleavage disrupts FVIII binding to multimeric VWF. 15 Multiple biochemical methods and the location of VWD type 2N mutations implicate the VWF D9 domain (S764-A865) as the site for FVIII docking.…”

mentioning

confidence: 99%

A von Willebrand factor fragment containing the D′D3 domains is sufficient to stabilize coagulation factor VIII in mice

Yee

Gildersleeve

et al. 2014

Blood

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• The D9D3 domains of VWF are sufficient to stabilize FVIII in vivo.• The prolongation of VWF D9D3 survival in vivo by Fc fusion elevates FVIII levels in the setting of VWF but not FVIII deficiency.Plasma factor VIII (FVIII) and von Willebrand factor (VWF) circulate together as a complex. We identify VWF fragments sufficient for FVIII stabilization in vivo and show that hepatic expression of the VWF D9D3 domains (S764-P1247), either as a monomer or a dimer, is sufficient to raise FVIII levels in Vwf 2/2 mice from a baseline of ∼5% to 10%, to ∼50% to 100%. These results demonstrate that a fragment containing only ∼20% of the VWF sequence is sufficient to support FVIII stability in vivo. Expression of the VWF D9D3 fragment fused at its C terminus to the Fc segment of immunoglobulin G1 results in markedly enhanced survival in the circulation (t 1/2 > 7 days), concomitant with elevated plasma FVIII levels (>25% at 7 days) in Vwf 2/2 mice. Although the VWF D9D3-Fc chimera also exhibits markedly prolonged survival when transfused into FVIII-deficient mice, the cotransfused FVIII is rapidly cleared. Kinetic binding studies show that VWF propeptide processing of VWF D9D3 fragments is required for optimal FVIII affinity. The reduced affinity of VWF D9D3 and VWF D9D3-Fc for FVIII suggests that the shortened FVIII survival in FVIII-deficient mice transfused with FVIII and VWF D9D3/D9D3-Fc is due to ineffective competition of these fragments with endogenous VWF for FVIII binding. (Blood. 2014; 124(3):445-452)

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Two Cys residues essential for von Willebrand factor multimer assembly in the Golgi

Cited by 70 publications

References 37 publications

Protein mobilities and P-selectin storage in Weibel–Palade bodies

Protein mobilities and P-selectin storage in Weibel–Palade bodies

Assembly of Weibel–Palade body-like tubules from N-terminal domains of von Willebrand factor

A von Willebrand factor fragment containing the D′D3 domains is sufficient to stabilize coagulation factor VIII in mice

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