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Mutations in the D1 domain of von Willebrand factor impair their propeptide-dependent multimerization, intracellular trafficking and secretion.

Yin J, Ma Z, Su J, Wang JW, Zhao X, Ling J, Bai X, Ouyang W, Wang Z, Yu Z, Ruan C - J Hematol Oncol (2015)

Bottom Line: All four mutations impaired multimerization of von Willebrand factor, due to reduced oxidoreductase activity of isomeric propeptide.These mutations resulted in the endothelial reticulum retention and impaired basal and stimulated secretions of von Willebrand factor.Our results support that the mutations in the D1 domain lead to defective multimerization, intracellular trafficking, and secretion of von Willebrand factor and result in bleeding of patients.

View Article: PubMed Central - PubMed

Affiliation: Collaborative Innovation Center of Hematology, MOH Key Lab of Thrombosis and Hemostasis, Jiangsu Institute of Hematology, the First Affiliated Hospital, Soochow University, Suzhou, 215006, China. yjjessie@163.com.

ABSTRACT
We identified three novel mutations (p.Gly39Arg, p.Lys157Glu, p.Cys379Gly) and one previously known mutation (p.Asp141Asn) in the von Willebrand factor propeptide from three von Willebrand disease patients. All four mutations impaired multimerization of von Willebrand factor, due to reduced oxidoreductase activity of isomeric propeptide. These mutations resulted in the endothelial reticulum retention and impaired basal and stimulated secretions of von Willebrand factor. Our results support that the mutations in the D1 domain lead to defective multimerization, intracellular trafficking, and secretion of von Willebrand factor and result in bleeding of patients.

No MeSH data available.


Related in: MedlinePlus

Defective multimerization of mutant VWF and reduced dimerization of mutant truncated VWF. a HEK293 cells were transiently transfected by equal WT or mutant full-length VWF plasmid. Seventy hours after transfection, the supernatant of cells were collected for multimer analysis. In order to detect whether the mutations have abnormal multimer pattern, we measured equal VWF antigen of the mutations, wild type, and NP. Lane 1 is the negative control of the mimic empty vector. Lanes 2–6 show the supernatant obtained from VWF mutant transfections and indicate decreases in high to medium-sized multimers to varying degrees. Exclusively, dimers formed with the Gly39Arg mutant (lane 2). The ability to form multimers was partly recovered by co-transfection of Gly39Arg and Asp141Asn (lane 4). b In the lysates of transfected cells, a similar decrease in the formation of VWF multimers was seen in the variants. However, WT-VWF exhibited a multimer pattern as observed for normal plasma. c HEK293 cells were transiently transfected by equal WT or mutant truncated VWF. In the media of transfected cells, dimerizations of truncated recombinant VWF were detected by Western blotting under the non-reducing condition. Lane 1 shows the empty pSecTag2/Hygro B vector. Dimers were absent in the Gly39Arg mutant (lane 2) while lanes 3–6 tapered in the dimer to different degrees. d In the supernatant of the transfected cells, the truncated VWF mutations and WT D1D2D′D3 fragment were examined under the reducing condition, and only D′D3 monomers were found
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Fig1: Defective multimerization of mutant VWF and reduced dimerization of mutant truncated VWF. a HEK293 cells were transiently transfected by equal WT or mutant full-length VWF plasmid. Seventy hours after transfection, the supernatant of cells were collected for multimer analysis. In order to detect whether the mutations have abnormal multimer pattern, we measured equal VWF antigen of the mutations, wild type, and NP. Lane 1 is the negative control of the mimic empty vector. Lanes 2–6 show the supernatant obtained from VWF mutant transfections and indicate decreases in high to medium-sized multimers to varying degrees. Exclusively, dimers formed with the Gly39Arg mutant (lane 2). The ability to form multimers was partly recovered by co-transfection of Gly39Arg and Asp141Asn (lane 4). b In the lysates of transfected cells, a similar decrease in the formation of VWF multimers was seen in the variants. However, WT-VWF exhibited a multimer pattern as observed for normal plasma. c HEK293 cells were transiently transfected by equal WT or mutant truncated VWF. In the media of transfected cells, dimerizations of truncated recombinant VWF were detected by Western blotting under the non-reducing condition. Lane 1 shows the empty pSecTag2/Hygro B vector. Dimers were absent in the Gly39Arg mutant (lane 2) while lanes 3–6 tapered in the dimer to different degrees. d In the supernatant of the transfected cells, the truncated VWF mutations and WT D1D2D′D3 fragment were examined under the reducing condition, and only D′D3 monomers were found

Mentions: To determine whether and how these mutations impair VWF expression and function, we transiently transfected the human embryonic kidney 293 cells (HEK293) with wild type (WT) or VWF mutant constructs and analyzed VWF multimer (Fig. 1a,b). In the supernatant, the WT-VWF showed a full range of multimers similar to NP. Asp141Asn, Lys157Glu, and Cys379Gly mutants each exhibited different degrees of the loss of large- and medium-sized multimers. We also co-transfected Asp141Asn and Gly39Arg mutants and found that a partial VWF protein multimerization was readily detectable. When mutant and WT-VWF were co-transfected into HEK293 cells, the abnormal VWF multimers were all restored (Additional file 3: Figure S2). We examined cell lysates for VWF multimer and found that multimerization from all VWF mutations were inhibited in different levels as showed in the media. To determine the underlying mechanisms of defective VWF multimerization, we employed a previously established dimerization model using D1D2D′D3 fragment of VWF [7, 8]. In this model, the decreased dimerization of D1D2D′D3 indicates the reduced oxidoreductase of mutant VWFpp. Under the non-reducing condition (Fig. 1c), truncated WT (T-WT) VWF formed both D′D3 monomers and dimers in the supernatant. The dimerizations of truncated four variants (T-Gly39Arg, T-Asp141Asn, T-Lys157Glu, and T-Cys379Gly) were reduced, compared to that observed with T-WT. Co-transfection of the T-Gly39Arg and T-Asp141Asn resulted in a modest restoration of dimer formation compared to the single transfection of truncated Gly39Arg. This result indicates that the impaired multimerization is caused by decreased oxidoreductase activity of the isomeric propeptide. Under the reducing condition (Fig. 1d), D′D3 dimers were reduced to D′D3 monomers in both mutant and wild type D1D2D′D3 fragment.Fig. 1


Mutations in the D1 domain of von Willebrand factor impair their propeptide-dependent multimerization, intracellular trafficking and secretion.

Yin J, Ma Z, Su J, Wang JW, Zhao X, Ling J, Bai X, Ouyang W, Wang Z, Yu Z, Ruan C - J Hematol Oncol (2015)

Defective multimerization of mutant VWF and reduced dimerization of mutant truncated VWF. a HEK293 cells were transiently transfected by equal WT or mutant full-length VWF plasmid. Seventy hours after transfection, the supernatant of cells were collected for multimer analysis. In order to detect whether the mutations have abnormal multimer pattern, we measured equal VWF antigen of the mutations, wild type, and NP. Lane 1 is the negative control of the mimic empty vector. Lanes 2–6 show the supernatant obtained from VWF mutant transfections and indicate decreases in high to medium-sized multimers to varying degrees. Exclusively, dimers formed with the Gly39Arg mutant (lane 2). The ability to form multimers was partly recovered by co-transfection of Gly39Arg and Asp141Asn (lane 4). b In the lysates of transfected cells, a similar decrease in the formation of VWF multimers was seen in the variants. However, WT-VWF exhibited a multimer pattern as observed for normal plasma. c HEK293 cells were transiently transfected by equal WT or mutant truncated VWF. In the media of transfected cells, dimerizations of truncated recombinant VWF were detected by Western blotting under the non-reducing condition. Lane 1 shows the empty pSecTag2/Hygro B vector. Dimers were absent in the Gly39Arg mutant (lane 2) while lanes 3–6 tapered in the dimer to different degrees. d In the supernatant of the transfected cells, the truncated VWF mutations and WT D1D2D′D3 fragment were examined under the reducing condition, and only D′D3 monomers were found
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4487848&req=5

Fig1: Defective multimerization of mutant VWF and reduced dimerization of mutant truncated VWF. a HEK293 cells were transiently transfected by equal WT or mutant full-length VWF plasmid. Seventy hours after transfection, the supernatant of cells were collected for multimer analysis. In order to detect whether the mutations have abnormal multimer pattern, we measured equal VWF antigen of the mutations, wild type, and NP. Lane 1 is the negative control of the mimic empty vector. Lanes 2–6 show the supernatant obtained from VWF mutant transfections and indicate decreases in high to medium-sized multimers to varying degrees. Exclusively, dimers formed with the Gly39Arg mutant (lane 2). The ability to form multimers was partly recovered by co-transfection of Gly39Arg and Asp141Asn (lane 4). b In the lysates of transfected cells, a similar decrease in the formation of VWF multimers was seen in the variants. However, WT-VWF exhibited a multimer pattern as observed for normal plasma. c HEK293 cells were transiently transfected by equal WT or mutant truncated VWF. In the media of transfected cells, dimerizations of truncated recombinant VWF were detected by Western blotting under the non-reducing condition. Lane 1 shows the empty pSecTag2/Hygro B vector. Dimers were absent in the Gly39Arg mutant (lane 2) while lanes 3–6 tapered in the dimer to different degrees. d In the supernatant of the transfected cells, the truncated VWF mutations and WT D1D2D′D3 fragment were examined under the reducing condition, and only D′D3 monomers were found
Mentions: To determine whether and how these mutations impair VWF expression and function, we transiently transfected the human embryonic kidney 293 cells (HEK293) with wild type (WT) or VWF mutant constructs and analyzed VWF multimer (Fig. 1a,b). In the supernatant, the WT-VWF showed a full range of multimers similar to NP. Asp141Asn, Lys157Glu, and Cys379Gly mutants each exhibited different degrees of the loss of large- and medium-sized multimers. We also co-transfected Asp141Asn and Gly39Arg mutants and found that a partial VWF protein multimerization was readily detectable. When mutant and WT-VWF were co-transfected into HEK293 cells, the abnormal VWF multimers were all restored (Additional file 3: Figure S2). We examined cell lysates for VWF multimer and found that multimerization from all VWF mutations were inhibited in different levels as showed in the media. To determine the underlying mechanisms of defective VWF multimerization, we employed a previously established dimerization model using D1D2D′D3 fragment of VWF [7, 8]. In this model, the decreased dimerization of D1D2D′D3 indicates the reduced oxidoreductase of mutant VWFpp. Under the non-reducing condition (Fig. 1c), truncated WT (T-WT) VWF formed both D′D3 monomers and dimers in the supernatant. The dimerizations of truncated four variants (T-Gly39Arg, T-Asp141Asn, T-Lys157Glu, and T-Cys379Gly) were reduced, compared to that observed with T-WT. Co-transfection of the T-Gly39Arg and T-Asp141Asn resulted in a modest restoration of dimer formation compared to the single transfection of truncated Gly39Arg. This result indicates that the impaired multimerization is caused by decreased oxidoreductase activity of the isomeric propeptide. Under the reducing condition (Fig. 1d), D′D3 dimers were reduced to D′D3 monomers in both mutant and wild type D1D2D′D3 fragment.Fig. 1

Bottom Line: All four mutations impaired multimerization of von Willebrand factor, due to reduced oxidoreductase activity of isomeric propeptide.These mutations resulted in the endothelial reticulum retention and impaired basal and stimulated secretions of von Willebrand factor.Our results support that the mutations in the D1 domain lead to defective multimerization, intracellular trafficking, and secretion of von Willebrand factor and result in bleeding of patients.

View Article: PubMed Central - PubMed

Affiliation: Collaborative Innovation Center of Hematology, MOH Key Lab of Thrombosis and Hemostasis, Jiangsu Institute of Hematology, the First Affiliated Hospital, Soochow University, Suzhou, 215006, China. yjjessie@163.com.

ABSTRACT
We identified three novel mutations (p.Gly39Arg, p.Lys157Glu, p.Cys379Gly) and one previously known mutation (p.Asp141Asn) in the von Willebrand factor propeptide from three von Willebrand disease patients. All four mutations impaired multimerization of von Willebrand factor, due to reduced oxidoreductase activity of isomeric propeptide. These mutations resulted in the endothelial reticulum retention and impaired basal and stimulated secretions of von Willebrand factor. Our results support that the mutations in the D1 domain lead to defective multimerization, intracellular trafficking, and secretion of von Willebrand factor and result in bleeding of patients.

No MeSH data available.


Related in: MedlinePlus