Discussion
The roles of the VWFpp, D1, and D2 domains, in both multimer assembly
and storage of VWF in WPBs, are well established . Each of the two D1
and D2 domains contains a CGLC amino acid motif which is considered to
be a consensus sequence of the PDI enzyme family, having a catalytic
role in N-terminus interchain disulfide bonds between the D3 domains to
form large multimers in the Golgi compartments . Furthermore, the
propeptide serves as an intramolecular chaperone, aligning the dimeric
pro-VWF subunits in the appropriate special alignment to facilitate
interdimer N-terminal disulfide bond formation during multimerization
and to ensure VWF helical assembly into tubules in nascent WPBs .
Another probable function attributed to the VWFpp is the sorting of the
mature VWF into storage granules via an intrinsic signal (linear
sequence or conformation) in the trans-Golgi .
Nonetheless, here in this study, we demonstrated that VWFpp missense
variants hamper the exit of VWF from the ER, resulting in VWF
sequestration in the ER compartment. The present study showed that four
out of the six VWFpp variants (p.Gly55Glu, p.Val86Glu, p.Trp191Arg, and
p.Cys608Trp) severely impaired secretion, along with intracellular
retention of these variants. Further, we illustrated that VWF proteins
with the mutations residing in propeptide are accumulated in the ER, and
do not appear to generate WPBs-like granules. We demonstrated an
increased co-localization of these VWF mutants with the luminal ER
marker, PDI, when is compared with wt.
Multiple QC mechanisms operate within the ER to ensure fidelity of
protein expression in cells. These mechanisms, in general, rely on a
repertoire of molecular chaperones and protein folding facilitators that
promote correct protein folding and detect defective proteins for
degradation . Additionally, ER-Golgi export QC is another checkpoint
that ensures the secretion of correctly folded proteins through cargo
receptors. By recruiting cargos for specific capture into COP I and COP
II vesicles, cargo receptors can monitor the folding status of their
clients before capturing into vesicles . Recently, Lopes-da-Silva et al
showed that the ARF family of proteins contributes to the ER-Golgi
trafficking of the VWF . In the active state, when bound to GTP, ARFs
initiate coat and cargo recruitment onto intracellular membranes to
conduct anterograde transportation . The switch between inactive and
active state occurs through GTP/GDP exchange/binding that is mediated by
guanine nucleotide exchange factors (GEFs). Lopes-da-Silva et al
demonstrated that GBF1 (one of the guanine nucleotide exchange factors)
reduction affects the rate of VWF movement between ER and Golgi,
indirectly implicating the ARF family of proteins as the major
recruitment proteins for anterograde transport of VWF . Another recent
study demonstrated that the rate of the anterograde transport between ER
and Golgi and consequently the morphology of the WPBs is dependent on
the Sec22b-containing SNARE complexes. Sec22b is a component of
ER-derived COPII-coated vesicles that direct proteins from ER to the
Golgi, and interacts with Sec23/Sec24 of the COPII coating complex,
which is essential for cargo recruitment in the ER. Both Sec22b
silencing and Sec24 deficiency were reported to result in ER retention
of the secretory proteins . Since our microscopic analysis suggested the
accumulation of VWF mutants in ER, we tested the hypothesis that the
misfolded VWF hampers its recruitment into COP I and COP II vesicles
(mediated by ARF or Sec24) by docking one of the active ARF protein (GTP
bound ARF1) and Sec24 structures onto our N-terminal VWF model. The fact
that most of the variants occur at the ARF1/or Sec24-VWFpp putative
interfaces on these docks strongly support the idea that the ER
accumulation might be a result of these mutations interfering with the
ARF/or Sec24-VWF recruitment process for anterograde transport.
Nevertheless, we propose this mechanism as a putative affected QC
pathway, and we can not deny involvement of the other QC checkpoints.
Therefore, our structural analysis results will need to be verified by
more experimental evidence in the future.
Accumulation of VWF mutants in ER was associated with reduced
intracellular packaging and defect in secretion of VWF. Nonetheless, the
homozygous expression of these propeptide variants still showed a minor
quantity of secreted VWF, only ~ 10% of wt, as well as
a trail of the oligomers (i.e. tetramers). This suggests that a small
number of dimers still succeed to rescue from the ER and reach their
destination in trans-Golgi. Further, all of the four causative variants
showed a 65%-75% fall in the secretion of VWF when heterozygously
expressed, indicating their dominant impact on secretion, along with an
increased colocalization with the ER marker (though with a lesser extent
compared with homozygously expressed variants). Besides, the
co-expression of these VWFpp variants with wt revealed a deficiency in
VWF multimer assembly, lacking large/and intermediate multimers. We
consequently assume that the limited number of the mutant homodimers/or
heterodimers rescued from the ER will successfully cross-over to the
Golgi and will interfere with the accurate orientation of the propeptide
within the dimeric bouquet and succeeding multimer elongation, based on
our observations made on the VWF N-terminal model´s orientation.
Furthermore, in the current study, the biogenesis of WPBs in
co-expressing cells of wt/mutants was explored by computerized
morphometric analysis of the whole pseudo-WPBs population using
state-of-the-art arivis Vision4D. Initially, microscopic inspections
revealed a reduction in the fraction of the cells generating pseudo-WPBs
in cells co-expressing wt/mutants. The computed average number of
pseudo-WPBs per cell was ~7 for cells expressing wt,
this number was less for the cells co-expressing p.Gly55Glu, p.Val86Glu,
and p.Cys608Trp (~ 3.4-4.0 per cell). Altogether, both
declines in the portion of cells forming pseudo-WPBs and their average
number per cell for the later co-expressed mutants indicate their
dominant-negative impact on the formation of the storage vesicles. The
reduction in the number of WPBs was previously also reported for the
mutations detected in type 1 and type 2A VWD, which was associated with
intracellular VWF retention and decreased secretion . However, these
previous reports were based on only visual inspections but not the
automated quantifications as we have done in the current study.
The WPBs have a distinct rod-shaped morphology and their length varies
between 0.5 and 5 µm in endothelial cells. Karampini et al have
quantified the length of WPBs in cultured endothelial cells (HUVECs) and
they reported the average length of ~ 2.2 µm .
Furthermore, in our lab, quantification WPBs length in the blood
outgrowth endothelial cells (BOECs) showed the various WPBs lengths with
an average length of 2.281 µm ± 0.01 (data not shown). Likewise, here,
the quantitative analysis of the whole pseudo-WPBs population in HEK293
cells showed various sizes of pseudo-WPBs (Figure 3E) with an average
length of 2.180 ± 0.09 µm in cells expressing wt. The co-expressed
p.Trp191Arg caused a reduction in both length and width of the WPBs-like
granules. It showed also slightly augmented sphericity, which indicates
the storage granules have a more rounded shape than elongated. Although
co-expression of the p.Gly55Glu, p.Val86Glu, and p.Cys608Trp seems not
to affect the size of pseudo-WPBs, the reduction in the computed mean
intensity values implies unspecified changes in their configuration,
besides their influence on the number of the generated pseudo-WPBs.
Variants p.Asn211Asp and p.Gly334Glu did not demonstrate any defect in
rVWF expression and function in vitro. Based on our observations, we
suggest these two variants should be considered as benign variants
rather than pathologic mutations.
In sum, here in this study, we demonstrated that VWFpp mutations hinder
anterograde ER-Golgi VWF export, resulting in ER accumulation.
Consequently, retention of the VWF inside the ER resulted in no storage
vesicle formation in the homozygously transfected cells, and a decrease
in the number of generated pseudo-WPBs in heterozygously transfected
cells. Furthermore, the heterozygous VWFpp mutants cause changes in the
size and configuration of the WPBs-like granules, with variability in
the extent/type of these changes. This highlights the consequence of the
VWF variants on biogenesis and configuration of WPBs, which in turn may
affect the recruitment of the other WPBs’ cargos.