Influence of substrate conformation on the deglycosylation of ribonuclease B by recombinant yeast peptide:N-glycanase.
Peptide:N-glycanase has been thought to be responsible for proteasome-dependent degradation of misfolded glycoproteins translocated from the endoplasmic reticulum (ER) to the cytosol. Therefore, the enzyme was supposed to be able to distinguish between native and non-native glycoproteins. In the present study, a recombinant, yeast peptide:N-glycanase, Png1p, was expressed in Escherichia coli as inclusion bodies and was purified, refolded and characterized. The results showed that the recombinant enzyme has a broad pH range adaptation, from pH 4.0 to pH 10.0, and has an optimum temperature of 30 degrees C. This enzyme is a zinc metalloenzyme. Its activity was abolished with the addition of EDTA and not restored by adding metal ions. Furthermore, the deglycosylation efficiency of recombinant Png1p from E. coli was investigated with respect to the substrate conformation in vitro. When ribonuclease B (RNase B) was denatured at 60-65 degrees C or by 40-60 mM dithiothreitol, indicated by its obvious structural change and sharpest activity change, its deglycosylation by Png1p was most prominent. The deglycosylation efficiency of RNase B by Png1p was found to be related to its structural conformation and enzymatic activity.
Wang
Shengjun
S
State Key Laboratory of Microbial Technology, Life Science School, Shandong University, Jinan 250100, China.
Wang
Peng George
PG
Qi
Qingsheng
Q
D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism*; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D012260:Ribonucleases; C029600:ribonuclease B; C468820:PNG1 protein, S cerevisiae; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase