Recombinant human (rh)PDGF-C (Vigenebio, China) was inserted into a site upstream of the IRES in the plasmid vector pAAV-IRES-ZsGreen (Biowit Technologies Ltd., China). Site-directed mutagenesis was performed to construct Asn→Ala (NA) mutations at potential PDGF-C glycosylation sites. All constructs were verified by DNA sequencing. The expression and sizes of the recombinant proteins were verified by Western blot (see Results).

The sequences of the site-directed mutagenesis primers are as follows:

PDGF-C-N25A-s: 5′ GGG​ACT​CAG​GCG​GAA​AGC​GCT​CTG​AGT​AGT​AAA​TTC 3′

HEK293 A cells and NIH 3T3 cells from the American Type Tissue Collection (ATCC, Manassas, VA, United States) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; ExCellBio, China) supplemented with 10% fetal bovine serum (FBS; ExCellBio, China) and 1% pen/strep (Cellgro, United States) at 37°C in a 5% CO₂ incubator. Cells were passaged one day before they were subjected to various treatments.

Plasmids were transfected into HEK293 A cells with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocols. In brief, plasmids were mixed with Lipofectamine 2000 in Opti-MEM medium and incubated for 5 min at room temperature to allow for complex formation. The complex was then added to cell cultures in a complete culture medium as described above. Cells were incubated for 48 h before further evaluation and/or use in experimental protocols.

Fresh tissues or cultured cells were lysed in radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitor cocktails (ThermoFisher Scientific). Samples were quantitated with a DC™ Protein Assay Kit (Bio-Rad), separated on an 8–12% sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) matrix, and transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad). After blocking with 5% bovine serum albumin (BSA), the membrane was incubated with a primary antibody at 4°C overnight, followed by incubation with horseradish peroxidase (HRP)–conjugated secondary antibody (1:5,000) for 1 h at room temperature. Each step was followed by three washes with Tris-buffered saline buffer with Tween-20 (TBST), after incubation. Primary antibodies included goat anti-PDGF-C (core domain; 1:1,000, R&D Systems, AF1447), rabbit anti-ZsGreen (1:1,000, Takara, 632,474), rabbit anti-p-PDGFRα Tyr762 (1:1,000, Cell Signaling Technology (CST), 24,188), rabbit anti-PDGFRα (1:750, Abcepta, AP7666d), rabbit anti-PDGFRβ (1:1,000, CST, 3,169), rabbit anti-p-PDGFRβ Tyr1009 (1:1,000, CST, 3124s), mouse-anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Beijing Ray Antibody Biotech), and mouse-anti-β-actin (Beijing Ray Antibody Biotech). Immobilon Western Chemiluminescent HRP substrate (Merck Millipore) was used to detect specific antibody binding. These signals were captured using a G-BOX (Syngene) imaging system or a LI-COR Odyssey Sa system (LI-COR Biosciences).

A Native Protein Deglycosylation Kit (Sigma, NDEGLY-1KT) and PNGase F (New England Biolabs) were used to detect protein glycosylation. The Native Protein Deglycosylation Kit was also used to identify the various types of glycosylation. This kit contains three enzymes, including endoglycosidase F3 (removes triantennary and trimannosyl chitobiose core structures), endoglycosidase F2 (removed biantennary structures), and endoglycosidase F1 (targets oligomannose and hybrid structures). The enzymatic assays were conducted according to the product manuals.

HEK293 A cells were transfected using Lipofectamine 2000 (Invitrogen). Two days later, the cells were washed in phosphate buffered saline (PBS) and were maintained in serum-free medium. Conditioned medium (CM) was collected after 6 h in culture. Debris was removed by centrifugation. CMs were concentrated by Amicon Ultra filtration device (Merck Millipore UFC801096) before analysis for PDGF-C expression by Western blot.

NIH 3T3 cells were maintained in a serum-free medium overnight before the addition of CM collected from transfected HEK293 A cells as described above. After 5 min of incubation with CM, NIH 3T3 cells were collected, lysed in RIPA buffer containing protease and phosphatase inhibitor cocktails (ThermoFisher Scientific), and analyzed with anti-p-PDGFRα Tyr762 and anti-p-PDGFRβ Tyr1009 by Western blot as described above.

Immunofluorescence was performed on cryostat-generated sections of spleens from C57BL/6 mice and transfected cells that were plated on glass coverslips. Tissues and cells were washed briefly with PBS and fixed for 15 min in 4% paraformaldehyde. After three washes with PBS, samples were pretreated for 1 h in PBS supplemented with 5% donkey serum and 0.5% Triton X-100. Cells and tissues were then incubated overnight at 4°C with the primary antibody in PBS supplemented with 5% donkey serum and 0.1% Triton X-100 and Alexa Fluor–conjugated secondary antibodies (1:500, Invitrogen) for 1 h. Then, 0.1% DAPI (4′,6-diamidino-2-phenylindole) was applied for 5 min followed by three washes. Primary antibodies included goat anti–PDGF-C core domain (1:100, Abcam, AF1447), mouse anti-ERP72 (1:50, Proteintech, 66,365–1), and sheep anti-TGN46 (1:100, Bio-Rad, AHP500). Cells were observed under a confocal microscope (LSM710, Carl Zeiss).

We identified three potential N-linked glycosylation sites that were conserved in human, rat, and mouse PC-FL that included Asn25, Asn55, and Asn254 (https://services.healthtech.dtu.dk/service.php?NetNGlyc-1.0). No O-linked glycosylation sites were predicted (https://services.healthtech.dtu.dk/service.php?NetOGlyc-4.0). Both Asn25 and Asn55 were located in the CUB domain of PC-FL. Of these, Asn25 immediately followed a predicted 22-amino acid signal peptide. Asn254 was located in the core domain and was situated near the cleavage site for the activation of PDGF-C protein (Figure 1).

To confirm these predictions, we performed a deglycosylation assay. Lysate of mouse spleen tissue were treated with PNGase F and analyzed by Western blotting. The bands were detected with an anti-PDGF-C antibody. We identified two immunoreactive bands in the untreated lysate. The band migrating at ∼50 kDa molecular weight disappeared after PNGase F treatment. This result suggests that PDGF-C is a glycoprotein (Figure 1B).

Next, we subcloned the PC-FL sequence upstream of the IRES in the pAAV-IRES-ZsGreen plasmid to express rhPDGF-C in mammalian cells. We selected HEK293 A cells for the heterologous expression of PDGF-C because we were unable to detect any endogenous PDGF-C expression in these cells in our pilot test. We used a native protein deglycosylation kit to characterize the nature of N-linked glycosylation. We found that the high molecular weight band disappeared in response to the treatment with endoglycosidase F1 but not with endoglycosidases F2 or F3 (Figure 1C). These results suggest that PDGF-C is substituted with high mannose-containing glycoprotein.

We then generated NA mutants to locate the specific glycosylation sites. For this experiment, the three predicted Asn residues were mutated to Ala and evaluated in single, double, or triple combinations. Wild-type and mutant constructs were transfected into HEK293 A cells to evaluate the size of the PDGF-C proteins and their glycosylation state.

As shown in Figure 1D, PDGF-C proteins were detected in lysates from all mutant-transfected cells by Western blotting. Four distinct bands were detected. The wild-type (WT) protein ran as three bands that corresponded to PC-FL with three (3x g), two (2x g), or one (1x g) glycan chains. The apparent molecular weights of the main bands in all three single mutants decreased compared with that of the WT. These results suggest that all three predicted Asn residues are functional glycosylation sites.

Of the single mutants, PDGF-C-N25 A contained two glycans (2x g), whereas the N55 A and N254 A mutants each contained one glycan (1x g) only. As shown, the N55 A and N254 A single mutants displayed the same Western blot pattern as did their corresponding double mutants that also included N25 A (i.e., N25 A + N55 A, and N25 A + N254A, respectively). These results suggest that glycosylation at Asn25 requires concomitant glycosylation at Asn55 or Asn254. Alternatively, glycosylation at Asn55 and Asn254 takes place earlier than that occurring at Asn25.

The main band in the triple NA mutant is considered to be a fully unsubstituted or nude protein that contains no glycan residues. We note detection of a low intensity band suggestive of single glycan-substitution which was detected on a Western blot of the triple NA mutant. This result suggests that PDGF-C may have an additional yet an unidentified glycosylation site. To explore the possibility of one or more additional glycosylation sites, we analyzed lysates from cells transfected with the N55 A + N254 A double mutant and the triple NA mutant with the Native Protein Deglycosylation kit. As shown in Figure 1E, the low intensity bands disappeared after treatment. This result suggests that there may be additional glycosylation sites in PDGF-C.

The results discussed above indicate that both WT and NA mutants of PDGF-C proteins were detected in transfected cell lysates. In its role as a growth factor, PDGF-C is secreted and functions extracellularly to activate PDGFRs. To examine the secretion of immunoreactive PDGF-C, we collected CMs from transfected cells and analyzed protein expression by Western blot. Full-length PDGF-C proteins were detected in CMs from all cultures. Of note, PDGF-C secretion was not prevented in response to mutations introduced at any of the aforementioned glycosylation sites in single, double, or triple form (top panel in Figure 2A). This result indicates that glycosylation is not required for the expression and secretion of PDGF-C protein.

The results in Figure 2A were somewhat surprising because glycosylation is generally considered to be a critical modulator of protein processing. To examine the functional impact of PDGF-C glycosylation, we examined the activity of the secreted form of the protein. Once secreted, full-length PDGF-C is activated via proteolytic removal of the N-terminal CUB domain. The remaining PDGF-C core domain functions as a PDGFR agonist. We evaluated the core domain in CMs from WT and mutant-transfected cells. As shown in the bottom panel of Figure 2A, the core domain was detected in the CMs from both WT and N25 A and N55 A PDGF-C mutants. These results suggested that glycosylation at N25 or N55 is not required for the PDGF-C activation. By contrast, we were unable to detect the core domain in CMs from cells transfected with single, double, or triple mutants that included N254 A. These results suggest that glycosylation at N254 may be required for the activation and generation of full-length PDGF-C protein.

The results presented in Figure 2A suggest that the N254 A mutant form of PDGF-C may be nonfunctional because of the absence of the core domain. To explore the functionality of PDGF-C in the absence of the core domain, we examined receptor activation, specifically in an assay designed to detect phosphorylation of PDGFR-α and PDGFR-β at Tyr762 and Tyr1009, respectively. NIH 3T3 cells were selected for this assay because of their abundant expression of PDGFRs. To perform this experiment, HEK293 A cells were transfected with plasmids carrying WT or mutant forms of PDGF-C. CMs were collected and added to cultured NIH 3T3 cells. After 5 min of exposure to the CMs, NIH 3T3 cells were collected, lysed, and analyzed by Western blot. We found that both PDGFR-α and PDGFR-β were phosphorylated in response to CMs from all HEK293 A cultures transfected with plasmids that maintained an intact Asn254 residue. No receptor activation or phosphorylation was detected in response to CMs from any of the N254 A mutant transfections (Figure 2B). Taken together, these results suggest that glycosylation at Asn254 is required for PDGF-C mediated receptor signaling.

The studies discussed above focused on the detection of PDGF-C protein in cell lysates. We then assessed the impact of aberrant glycosylation patterns on the intracellular distribution of PDGF-C. The physiologic distribution of PDGF-C expression was assessed in mouse spleen tissue; immunostaining revealed that the endogenous PDGF-C was located in the cell cytoplasm (Figure 3A). Next, we evaluated the intracellular localization of PDGF-C in transfected HEK293 A cells. Untransfected HEK293 A cells expressed minimal levels of PDGF-C. Thus, most PDGF-C detected in transfected cells results from heterologous PDGF-C expression. Similar to our findings in spleen cells, immunoreactive PDGF-C (both WT and NA mutants) in transfected HEK293 A cells was also localized in the cell cytoplasm (Figure 3B). Thus, disruption of PDGF-C glycosylation sites has no apparent impact on its intracellular distribution.

To examine the impact of glycosylation on PDGF-C protein processing, we performed immunofluorescence staining that targeted the ER and Golgi apparatus in transfected HEK293 A cells. We found that the WT PDGF-C protein was partially retained in the ER as were all of the PDGF-C proteins with single NA mutations (Figure 4). This result indicates that disruption of PDGF-C glycosylation sites does not affect its progression through the ER.

The staining pattern with respect to the Golgi apparatus was similar in both WT-transfected and untransfected cells. This pattern remained in cells transfected with each of the single NA mutants (Figure 5). These findings combined with results from the aforementioned Western blots suggest that glycosylation is not required for PDGF-C secretion; we deduce that disruption of the PDGF-C glycosylation sites has no impact on its progression through the Golgi apparatus.