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AP-3 dependent trafficking of VAMP8 in endothelial cells
and stained with FITC-conjugated anti-CD63 antibody at 4oC for 30 minutes (min) in the dark. After incubation, cells were washed with PBS, spun down at 250xg, 4oC for 2 min and sus- pended in PBS containing 0.01% (v/v) NaN3. Samples were measured by flow cytometry (BD FACSCANTO II, BD Biosciences). In order to measure CD62P levels on the plasma membrane upon stimulation, cells were pre-incubated in release medium for at least 30 min. Stimulation was performed in RM supplemented with 100 μM histamine for 5 min. After stimula- tion, cells were treated with ice-cold PBS and detached by Accutase. Staining for CD62P exposure was performed at 4oC in the dark with a PE-conjugated anti-CD62P antibody for 20 min. Cells were centrifuged at 4oC at 250xg for 2 min and subse- quently resuspended in PBS containing 0.01% (v/v) NaN3.
Further details on materials and methods are available in the
Online Supplementary Appendix.
Results
AP3B1 deficient endothelial cells exhibit impaired intracellular protein trafficking from endosomes to Weibel-Palade bodies
To study the role of the AP-3 complex in primary endothelial cells, we isolated BOEC from peripheral blood mononuclear cells of an HPS-2 patient with muta- tions in the AP3B1 gene that encodes for the β1 subunit of the heterotetrameric AP-3 complex. The patient, who has previously been described by de Boer et al.,16 is a com- pound heterozygote with both mutations (exon 2: c.177delA, p.K59Nfs4; exon 17: c.1839-1842delTAGA, p.D613Efs*38) leading to a frame shift and a premature stop codon (Figure 1A). As expected, western blot analy- sis of lysates of BOEC confirmed that the expressed levels of AP-3 β1 were not detectable (Figure 1B). The truncated AP-3β1 protein products, generated in the HPS-2 BOEC, are most likely rapidly degraded as previously reported in HPS-2 fibroblasts.13 In order to test whether there is any remaining functionality of the AP-3 complex in HPS-2 BOEC, we determined the localization of proteins that are subject to AP-3 dependent trafficking. Both the tetraspanin CD63 and the leukocyte receptor CD62P con- tinuously cycle between plasma membrane and WPB.5,6 However, after endocytic retrieval, CD63 is incorporated in maturing WPB through transfer from AP-3 positive endosomes, while P-selectin diverges to the trans-Golgi network where it is incorporated in nascent WPB.7 Therefore, we stained HPS-2 and healthy control BOEC for vWF and CD63 or P-selectin. We observed that CD63 is not detectable in WPB in HPS-2 BOEC and can only be observed in round endosome-like structures (Figure 1E, left). Quantitative evaluation of our imaging data showed that in WT BOEC, nearly a third of cellular CD63 is asso- ciated with WPB (Figure 1 right top), and that on average about 60% of the WPB are CD63 positive (1E right bot- tom), numbers that are both in close accordance with pre- vious studies.7,22 We observed a sharp reduction in these parameters in HPS-2 BOEC pointing to a defect in CD63 trafficking. P-selectin trafficking to WPB is unaffected by the lack of AP-3 β1 (Online Supplementary Figure S1A). We also measured CD63 surface expression in HPS-2 and healthy control BOEC under steady state conditions using flow cytometry. We observed that CD63 surface levels were significantly increased in HPS-2 BOEC (Online
Supplementary Figure S1B). Taken together these data point to defective AP-3 dependent trafficking mecha- nisms in HPS-2 BOEC.
To further corroborate the role of AP-3 in trafficking of CD63, we also generated AP3B1 knock-out primary endothelial cells using CRISPR-Cas9 gene editing. Using lentiviral transduction of cord blood BOEC (cbBOEC), with guide (gRNA) targeting the first exon of the AP3B1 gene, we generated 3 clonal cbBOEC lines containing indels that led to frame shifts and subsequent premature stop codons in the AP3B1 gene (Figure 1C). This resulted in complete abolishment of AP-3β1 expression in all three clonal lines (Figure 1D). Similar to what we observed in HPS-2 BOEC, and to what has been reported in endothe- lial cells after siRNA-mediated AP-3 β1 silencing,7 WPB of AP3B1-/- cbBOEC did not contain CD63 (Figure 1F and Online Supplementary Figure S2).
AP3B1-deficient HPS-2 blood outgrowth endothelial cells have an unstable AP-3 complex and lack the Weibel-Palade body v-SNARE VAMP8
To better understand the phenotypic effect of loss of AP-3 β1 expression, we performed a comparative analy- sis of the whole proteome between HPS-2 and healthy control BOEC by means of label-free LC-MS/MS. We identified 5812 proteins, from which 4323 were quantifi- able following the selection criteria. To include the natu- ral variation in our analysis, we compared HPS-2 BOEC to four independent healthy control donors. Z-scored LFQ values of the proteins with the highest variation between samples (S0=0.4, FDR = 0.05) are shown in the heatmap in Figure 2A. Following this approach, we were able to confirm the AP-3 β1 depletion in HPS-2 BOEC (Figure 2B). Moreover, we observed that the expression of AP-3 μ1, an AP-3 subunit encoded by AP3M1, was also significantly reduced. However, expression levels of AP- 3δ1 and AP-3 s1, the other two subunits of the AP-3 complex, were not significantly different and only mod- estly reduced (Figure 2B). A similar observation has previ- ously been made in HPS-2 fibroblasts23 and in murine pe fibroblasts (pearl, pe, mouse model of HPS-2), where δ1 and s1 subunits remained as a heterodimer and showed cytosolic rather than membrane-associated localization.24 The tight correlation of β1 and μ1 expression levels sug- gests that loss of AP-3 β1 and consequential disintegra- tion of the AP-3 complex destabilizes the AP-3μ1 subunit. This was supported by the observation that lentivirally expressed mEGFP-AP-3 β1 was able to rescue the expres- sion of AP-3 μ1 in HPS-2 BOEC (Figure 2C).
Interestingly, among the down-regulated proteins, we discovered that the expression of the WPB-localized SNARE protein VAMP8 is severely diminished (Figure 2A and Online Supplementary Figure S3). This was specific for VAMP8 as the expression levels of other SNARE proteins that have been implicated in WPB exocytosis were not sig- nificantly altered (Online Supplementary Figure S3). Western blot analysis also revealed a complete depletion of VAMP8 expression in HPS-2 BOEC compared to healthy controls (Figure 2D). Moreover, immunofluorescent staining in healthy control and HPS-2 BOEC for VAMP8 and vWF, showed that WPB in HPS-2 BOEC lack VAMP8 immunore- activity (Figure 2E). In addition, in the AP3B1 knockout BOEC lines, VAMP8 was strongly reduced when compared to the control cbBOEC (Online Supplementary Figure S4).
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