Figure Legends
Figure 1. Impact of the VWF propeptide mutations on secretion, function and multimer structures of the VWF protein. (A) HEK293T cells were transiently transfected with rVWF-wt, p.Gly55Glu, p.Val86Glu, p.Trp191Arg, p.Asn211Asp, p.Gly334Glu and p.Cys608Trp (left side); the mutant constructs were co-transfected with VWF-wt at a 1:1 ratio (right side). Recombinant VWF antigen levels (VWF:Ag) in medium (dark gray bars) and lysate (light gray bars) were measured and expressed as a percentage relative to the VWF-wt. Each bar represents the average value and \(\pm\) SEM of three independent experiments in triplicate.(B) The VWF binding activities, VWF:GPIb and VWF:CB. The functional assessments are presented as mean and \(\pm\) SEM of ratios VWF:GPIb and VWF:CB to VWF:Ag from three independent experiments in triplicate. The n/a indicates where the amount of mutated VWF antigen in media was not adequate for reliable quantification evaluations.(C) The multimer pattern of rVWF-wt and mutants in the medium of transfected HEK293T cells (both single and co-transfected cells) analyzed by electrophoresis on 1.6% SDS-agarose gel. Lines 1 and 2 show normal pool plasma and rVWF-wt, respectively. Lines 3, 5, 7, 9, 11 and 13 represent single-expressed VWF mutants, whereas lines 4, 6, 8, 10, 12 and 14 illustrate co-expressed mutants with VWF-wt in a ratio of 1:1.
wt, wild type; VWF, von Willebrand factor;rVWF , recombinant VWF; VWF:Ag, VWF antigen;VWF:GPIb, VWF binding to platelet GPIb; VWF:CB, VWF binding to collagen type I.
Figure 2. Intracellular localization and storage of recombinant VWF (rVWF) variants in transfected HEK293 cells. (A and B) rVWF-wt and pathogenic VWF variants were expressed in HEK293, subsequently, intracellular trafficking and storage of VWF were investigated. Cells were fixed and immunostained to visualize VWF (green, left channels) and endoplasmic reticulum (ER) using the PDI marker (red, middle channels). Right channels illustrate the merge of green and red staining, showing co-localization of the two proteins VWF and PDI. The punctate staining (green) represents a storage of VWF in pseudo-WPBs, whereas the diffuse green staining indicates VWF retained in ER. Scale bar = 10µm. (A) HEK293 cells were transiently transfected with rVWF-wt or the pathogenic VWF variants p.Gly55Glu, p.Val86Glu, p.Trp191Arg, and p.Cys608Trp. (B) HEK293 cells were co-transfected with VWF mutant and wt in a 1:1 ratio to imitate heterozygous expression. (C ) Degree of co-localization of VWF mutants with ER. This bar graph represents the mean of Pearson’s coefficient for rVWF-wt and pathologic variants (single and co-expressed) analyzed in this study. Error bars represent the SEM. The Pearson’s coefficient has been calculated from at least 100 cells for each construct on the Zeiss ZEN 2 (blue edition) software.
Figure 3. Immunostaining and quantitative morphological analysis of WPBs-like vesicles in co-transfected HEK293 cells. (A)Immunofluorescence images show a 3D model of formation of the WPBs-like granules in transiently transfected HEK293 cells with recombinant VWF wt as well as transiently co-transfected wt with variants p.Gly55Glu, p.Val86Glu, p.Trp191Arg, and p.Cys608Trp in a 1:1 ratio imitating heterozygous expression. Scale bar = 10µm. Boxes represent close-up views of the processed 3D models of the WPBs-like granules surrounding with oriented bounds which are generated by arivis Vision4D 3.2 software. The size measurements (length, depth, and width) of some wt and mutant pseudo-WPBs are provided jus as examples; the sizes are presented as µm (B) The fraction of cells forming WPBs-like granules are presented as % of the cells detected with green signal (indicating expressing VWF) after inspecting at least 100 cells for each HEK293 cells transfected with wt VWF and HEK293 cells co-transfected with wt and VWF variants. Bars represent means ± SEM from a minimum of eight fields of view. (C) The relative average number of WPBs-like organelles per cell in transfected HEK293 cells, calculated by dividing the global count of the WPBs in each acquired image arena by the cells encompassing at least one WPB (D) The mean intensity signal value (green, VWF) for every single WPB-like organelle was calculated. Bars display average ± SEM from the whole population of WPBs-like granules (hundreds of WPBs). (E, F, and G) panels demonstrate the average value of the longest (length), middle (depth), and shortest sides (width) from the whole population of WPBs-like granules (hundreds of WPBs), respectively. Each bar represents the average value and \(\pm\) SEM. (H) The Sphericity factor (Ψ), describing the roundness of the 3D granules were computed. It is represented as a value between 0 and 1, where 1 is an ideal sphere. Each value implies the average rate ± SEM of the whole population of WPBs-like granules.
ns: non-significant; *: P<0.05; **: P<0.002; ***: P<0.0001 by Student t-test.
Figure 4. The generated merged model of the D1-D2-D´-D3 domains and its docking with ARF1 protein and Sec24. (A) The structural model represents the joined models of the D1-D2-D´-D3 domains. D1 and D2 domains (VWFpp) models (orange and green, respectively) were generated individually on the ITASSER threading server, and the crystal structure of the D´-D3 assembly (cyan and yellow, respectively) was downloaded from the structure database (PDB ID: 6N29; Resolution: 2.5 Å). Subsequently, the joined model of the VWFpp and the D´-D3 assembly was created. All propeptide cysteines are represented in blue colored stick format. The mutated residues have been depicted with their molecular surface, and are colored red. (B) The 3-dimensional model illustrates an orientational depiction of the joined model of the propeptide and the D´-D3 domains. The model itself is depicted by its molecular surface area. The molecular surface area of the linearly contiguous D1,D2,D’ and D3 domains are colored in a darker shade of grey while the D3 domain of an adjacent dimer and part of the complete model is colored in a lighter shade of grey. (C) The schematic illustration of the multimeric arrangement and tubular packing of the N-terminal of the VWF. In the ER, VWF monomers dimerized through disulfide bonds formation between C-terminal cysteine-knot (CK) domains, later in trans-Golgi each pro-VWF dimer zip up into a dimeric bouquet (transparent light blue boxes display the arrangement of the D1 (orange) D2 (green) D´ (cyan) D3 (yellow) domains in each dimeric bouquet). Forming the dimeric bouquets are crucial for accurate VWF dimer incorporation into growing tubules inside WPBs. Homotypic interactions between the D1 and D2 domains (non-covalent dimerization of N-terminal ends within proVWF dimeric bouquet, shown with broken arrow) places one D´D3 domain within a dimer apart from one another, and brings one D´D3 nearby to one D´D3 domain from another assembled dimer, facilitating N-terminal disulfide bond between dimers . (D) panel shows the ten best docking poses resulting from docking the crystal structure of ARF1 on our propeptide model. The backbone of both participating proteins is depicted in ribbon format with ARF1 colored magenta and VWF colored grey. The residues on which causative mutations have been reported in our study have been depicted with their molecular surface are colored red. (E) panel shows a close-up view of the four mutated residues reported in our study on the model, especially for their position vis-à-vis the ARF1 interaction interfaces (residues Gly55, Val86, Trp191) and also free unpaired Cysteines (residue Cys608). All protein structures are depicted in ribbon format, with VWF colored grey and ARF-1 colored magenta. The regions on ARF-1 which undergoes a conformational change during its transition from an inactive to an active state (upon GTP binding) are colored yellow. The mutated wild type residues are depicted in their stick formats. The wild type residues of Gly55, Val86, and Trp191 are colored red. The Cysteines forming disulfide bonds are also depicted in blue colored stick format, while the unpaired cysteines are depicted in green colored stick formats. (F) panel shows the ten best docking poses resulting from docking the crystal structure of Sec24 on our propeptide model. The backbone of both participating proteins is depicted in ribbon format with Sec24 colored magenta and VWF colored grey. The residues on which causative mutations have been reported in our study have been depicted with their molecular surface are colored red. (G) panel shows a close-up view of the four mutated residues reported in our study on the model, especially for their position vis-à-vis the Sec24 interaction interfaces (residues Gly55, Val86, Trp191 and Cys608).