| PNGase (Peptide-N~4-(N-acetyl-β-D-glucosaminyl) asparigine amidase; EC 3.5.1.52) is a deglycosylating enzyme, which catalyzes detachment of iV-linked glycan chains from glycopeptides or glycoproteins by hydrolyzing theβ-aspartylglucosaminyl bond. It involves in the proteasome-dependent quality control machinery used to degrade newly synthesized glycoproteins that do not fold correctly in the ER (Endoplasmic Reticulum), and acts upstream of the proteasome. Pnglp has been reported to be present in the ER, microsomes, and cytosol, but mostly in the Cytoplasm. Cytoplasmic PNGase is distributed into two populations: ER-associated and free in the cytosol.Pnglp was supposed to be able to distinguish between native and non-native glycoproteins. This work set up a number of experiments to test how conformation change of glycoprotein substrate such as ribonuclease B (RNase B) affect the deglycosylation activity of peptide: N-glycanase in vitro. In this study, RNase B was denatured by two different ways and measured by circular dichroism. At the same time, the deglycosylation efficiency of recombinant Pnglp from E.coli was investigated with respect to the substrate conformation in vitro. The results confirmed the hypothesis and also indicated that this efficiency was related to the enzymatic activity of the target glycoprotein. The critical point for RNase B deglycosylation is 60-65°C and 40-60 mM DTT.In yeast, peptide: N-glycanase (Pnglp) binds to the 26S proteasome through its interaction with Rad23p. The degradation pathway of glycoprotein is mediated by the Pnglp-Rad23p complex in vivo. In the present study, the complex of Pnglp-Rad23p was purified and characterized in vitro. The in vitro deglycosylation reaction results showed that the Png1p-Rad23p complex exhibited a higher deglycosylation activity than that of Pnglp alone. Meanwhile, Pnglp-Rad23p complex was confirmed to be able to deglycosylate misfolded or denatured glycoproteins but not native glycoproteins. Furthermore, the substrate conformation was able to affect the deglycosylation efficiency. The optimum deglycosylation temperature of Pnglp-Rad23p complex was proved to be 30°C, while the optimum pH is 7.0, which is similar to Pnglp alone, but Pnglp-Rad23p complex shows higher activity and stability than Png1p at wide temperature and pH.In yeast, the Rad23p-binding domain of Png1p consists of four helices (H1, H2, H11, and H12), and the amino-terminal helix adopts an extended conformation. The amino -terminal helix, H1, is extended against the carboxy -terminal helix, H12, at a 45°angle, and Rad23p directly bind to these two helices. In the present study, experiments were carried out to test the activity of Png1p without the amino - terminus (H1, H2) and the carboxy - terminus (H11, H12), and compare deglycosylation the activity between the Png1p mutant and wt-Png1p. The in vitro deglycosylation reaction results showed that the Png1-ΔH1 exhibited a higher deglycosylation activity than that of Png1p, but Pngl-Δ(H1, H12); Pngl-AH12 and Pngl-Δ(H1, H2, H11, H12) have no deglycosylation activity. Meanwhile, Png1-ΔH1 was found not only possess high deglycosylation activity but also act on the native glycoproteins in vitro. Furthermore, the substrate conformation was also able to affect the deglycosylation efficiency. The optimum deglycosylation temperature of Png1-ΔH1 was proved to be 30°C, while the optimum pH is 7.0, which is similar to Png1p. Analyzing and comparing the Dynamics of Png1p and Png1-ΔH1 in water shows that three region in Png1-ΔH1 are more flexible which will affect the enzyme activity and substrate specificity. |
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