Activated protein C N-linked glycans modulate cytoprotective signaling function on endothelial cells

Abstract

Activated protein C (APC) has potent anticoagulant and anti-inflammatory properties that limit clot formation, inhibit apoptosis, and protect vascular endothelial cell barrier integrity. In this study, the role of N-linked glycans in modulating APC endothelial cytoprotective signaling via endothelial cell protein C receptor/protease-activated receptor 1 (PAR1) was investigated. Enzymatic digestion of APC N-linked glycans (PNG-APC) decreased the APC concentration required to achieve half-maximal inhibition of thrombin-induced endothelial cell barrier permeability by 6-fold. Furthermore, PNG-APC exhibited increased protection against staurosporine-induced endothelial cell apoptosis when compared with untreated APC. To investigate the specific N-linked glycans responsible, recombinant APC variants were generated in which each N-linked glycan attachment site was eliminated. Of these, APC-N329Q was up to 5-fold more efficient in protecting endothelial barrier function when compared with wild type APC. Based on these findings, an APC variant (APC-L38D/N329Q) was generated with minimal anticoagulant activity, but 5-fold enhanced endothelial barrier protective function and 30-fold improved anti-apoptotic function when compared with wild type APC. These data highlight the previously unidentified role of APC N-linked glycosylation in modulating endothelial cell protein C receptor-dependent cytoprotective signaling via PAR1. Furthermore, our data suggest that plasma β-protein C, characterized by aberrant N-linked glycosylation at Asn-329, may be particularly important for maintenance of APC cytoprotective functions in vivo.

FIGURE 1.

Characterization of PNGase F-treated APC. A, APC and PNGase F-treated APC (PNG-APC, 1 μg) were characterized by 4–15% SDS-PAGE analysis and Western blotting, using a sheep anti-protein C/APC polyclonal antibody that detects the protein C/APC heavy chain (HC, shown). B, the anticoagulant activity of APC (○) and PNG-APC (■ 2.5–20 nm) was assessed in protein C-deficient plasma by a thrombin generation assay. Thrombin generation was initiated with 5 pm soluble tissue factor and 100 mm CaCl₂ and assessed as described under ”Experimental Procedures.“ The percentage of ETP was determined for thrombin generation in the presence and absence of APC, with 100% ETP defined as ETP in the absence of either APC species.

FIGURE 2.

PNGase F-treated APC possesses enhanced endothelial barrier-protective function and up-regulation of anti-apoptotic gene expression. A, the role of APC N-linked glycosylation in mediating APC-dependent endothelial cell signaling was assessed in a thrombin-induced endothelial cell barrier permeability assay. EA.hy926 cells were grown to confluence on polycarbonate membrane Transwell permeable supports and then incubated with either APC (○) or PNG-APC (■) (1.25–10 nm). Treated cells were then incubated with 5 nm thrombin for 10 min, and barrier permeability was determined for each APC concentration at 10 min. Endothelial barrier permeability (percentage) was calculated as described under ”Experimental Procedures.“ B, to assess the role of EPCR binding in PNGase F-treated APC cytoprotective signaling, EA.hy926 cells were incubated with PNGase F-treated APC and an anti-EPCR monoclonal antibody (RCR-252; 25 μg/ml) to prevent APC-EPCR binding. Thrombin-induced permeability was then assessed as described above. C, deglycosylation improves APC anti-apoptotic gene expression in staurosporine-treated endothelial cells. The anti-apoptotic function of PNG-APC was determined by calculation of the ratio of pro- and anti-apoptotic gene expression, using Bax and Bcl-2 expression, respectively. EA.hy926 cells were pretreated with 10 nm APC for 4 h. EA.hy926 cell apoptosis was then induced by staurosporine (20 μm, 4 h). After RNA extraction, RT-PCR was performed using bax, bcl-2, and β-actin TaqMan® gene expression assays. Experiments were performed in triplicate and plotted as a percentage of the mean staurosporine-treated bax/bcl-2 ratio (100% apoptosis). **, p < 0.005.

FIGURE 3.

Glutamine substitution of the N-linked glycan attachment site at Asn-329 causes enhanced endothelial barrier-protective and anti-apoptotic APC activity. A, each recombinant protein C (wild type protein C, PC-N248Q, PC-N313Q, PC-N329Q, and PC-L38D/N329Q) was reduced using β-mercaptoethanol and assessed by 7.5% SDS-PAGE analysis. The protein C heavy chain was subsequently detected by Western blot using a sheep anti-protein C polyclonal antibody. B, the anticoagulant activity of each APC variant was determined by a thrombin generation assay in protein C-deficient plasma (●, wild type APC; ▼, APC-N248Q; ♦, APC-N313Q; ♢, APC-N329Q; all 1.25–20 nm, except APC-N313Q, 1.25–10 nm). C, the endothelial barrier-protective properties of wild type APC (○) and APC-N329Q (□; 1.25–10 nm) were determined as described previously. D, endothelial cell pro/anti-apoptotic gene expression in the presence of wild type APC and APC-N329Q (both 5 nm) was measured by RT-PCR quantification of the relative expression of Bax/Bcl-2 mRNA transcripts, as described under ”Experimental Procedures.“ *, p < 0.05.

FIGURE 4.

Inhibition of endothelial cell apoptosis by recombinant APC N-linked glycan variants. A, endothelial cell apoptosis was measured by accumulation of apoptosis-specific dye (pink-purple) in staurosporine-treated EA.hy926 cells following incubation with wild type/variant APC. Untreated/staurosporine-treated EA.hy926 cells (top panels) and wild type/variant APC (1.25 nm, middle and bottom panels) are shown. Images are representative of three independent experiments. B, APC concentration-dependent reduction in endothelial cell apoptosis (●, wild type APC; ♢, APC-N248Q; △, APC-N313Q; □, APC-N329Q; 0.625–20 nm). Uptake of apoptosis-specific dye was quantified by converting digital photograph images into pixel counts using Adobe® Photoshop® software according to manufacturer's instructions. Average pixel counts calculated were based on analysis of at least three images per well.

FIGURE 5.

Enzymatic deglycosylation of non-anticoagulant APC variant APC-L38D with PNGase F enhances APC-L38D-mediated endothelial cell barrier protection. Endothelial cell barrier permeability was assessed in the presence of wild type APC (○) and PNG-APC-L38D (■) (1.25–10 nm) for 3 h prior to incubation with 5 nm thrombin. Endothelial barrier permeability was assessed by leakage of Evans Blue-BSA through the endothelial cell barrier, as described above.

FIGURE 6.

APC-L38D/N329Q possesses no anticoagulant activity in plasma but demonstrates enhanced cytoprotective PAR-1 signaling. A, thrombin generation in protein C-deficient plasma was assessed in the presence of wild type APC and APC-L38D/N329Q. Thrombin generation (nm × min) was initiated with platelet-poor plasma reagent and CaCl₂ as before, and the percentage of ETP (thrombin generation in the absence of APC) was determined. (○, no APC; □, 5 nm wild type APC; ●, 10 nm wild type APC; ♦, 20 nm APC-L38D/N329Q). B, EPCR-PAR1-dependent endothelial cell barrier protection by APC-L38D/N329Q is more potent than wild type APC. Barrier permeability assays using EA.hy926 cells were performed in the presence of wild type APC (○) or APC-L38D/N329Q (■; 1.25–10 nm) prior to thrombin treatment. Permeability is expressed as a percentage of total thrombin-induced endothelial cell barrier permeability. C, endothelial cell apoptosis was measured by accumulation of apoptosis-specific dye (pink-purple) in staurosporine-treated EA.hy926 cells following incubation with wild type APC or APC-L38D/N329Q (1.25 nm). D, APC concentration-dependent reduction in endothelial cell apoptosis (○, wild type APC; ■, APC-L38D/N329Q; 0.3125–20 nm).

FIGURE 7.

The N-linked glycan at Asn-329 is conserved and proximal to a PAR1-binding exosite on APC. A, amino acid alignment of known protein C (PROC) amino acid sequences indicates conservation of the unusual NXC N-linked glycan attachment site in known mammalian protein C amino acid sequences. B, N-linked glycosylation occurs at three sites (Asn-248, Asn-313, and Asn-329; turquoise) on the protein C/APC serine protease domain (blue). The Asn-329 glycan attachment site that regulates APC cytoprotective signaling is situated next to two amino acid residues (Glu-330/Glu-333; red) that are essential for PAR1 cleavage by APC (34). The APC catalytic triad (His-211, Asp-257, and Ser-360; yellow) is indicated. The model was generated based upon the Gla domainless APC crystal structure (1AUT (32)) using the PyMOL molecular visualization software.