Fig. 4. Synthesis of CpGV-MMP during infection. (A) Synthesis of CpGV-MMP. Sf9 Granzyme B Inhibitor Z-AAD-CH2Cl were infected at an MOI of 5 PFU/cell with Ac-CpGV-ORF46-PG, AcWT-PG, or mock-infected (m.i.). At the indicated time points, cell pellet (P) and extracellular media (M) were collected and proteins immunoblotted using anti-HA antibody to detect CpGV-MMP. (B) N-glycosylation of CpGV-MMP. Sf9 cells were infected at an MOI of 5 PFU/cell with Ac-CpGV-ORF46-PG or mock-infected (m.i.). Cell pellets (P) and extracellular media (M) were collected at 24 h p.i. and incubated with PNGase F at 37 °C for 4 h. PNGase F-treated (T) and -untreated (UT) samples were then analyzed by immunoblotting using anti-HA antibody.Figure optionsDownload full-size imageDownload high-quality image (193 K)Download as PowerPoint slide
CpGV-MMP migrated as a protein with molecular mass of approximately 72 kDa, higher than the predicted mass of 65 kDa. In silico CpGV-MMP analyses, using NetNGlyc 1.0 (Chuang et al., 2012), indicated eight potential N-glycosylation sites. The increased molecular mass observed in immunoblots may be explained by protein N-glycosylation. In order to confirm N-glycosylation of CpGV-MMP, we treated cell pellets and extracellular media collected from Ac-CpGV-ORF46-PG-infected cells with PNGase F, a glycosidase that cleaves N-linked oligosaccharides from glycoproteins, and analyzed the results by immunoblotting. We observed a significant reduction in molecular mass when both cellular and extracellular CpGV-MMP-containing lysates were treated with PNGase F (Fig. 4B). The molecular mass of PNGase F-treated CpGV-MMP was closer to the predicted mass of 65 kDa. These findings indicate that CpGV-MMP is secreted as an N-glycosylated protein.
CpGV-MMP migrated as a protein with molecular mass of approximately 72 kDa, higher than the predicted mass of 65 kDa. In silico CpGV-MMP analyses, using NetNGlyc 1.0 (Chuang et al., 2012), indicated eight potential N-glycosylation sites. The increased molecular mass observed in immunoblots may be explained by protein N-glycosylation. In order to confirm N-glycosylation of CpGV-MMP, we treated cell pellets and extracellular media collected from Ac-CpGV-ORF46-PG-infected cells with PNGase F, a glycosidase that cleaves N-linked oligosaccharides from glycoproteins, and analyzed the results by immunoblotting. We observed a significant reduction in molecular mass when both cellular and extracellular CpGV-MMP-containing lysates were treated with PNGase F (Fig. 4B). The molecular mass of PNGase F-treated CpGV-MMP was closer to the predicted mass of 65 kDa. These findings indicate that CpGV-MMP is secreted as an N-glycosylated protein.