Influence of Substrate Conformation on the Deglycosylation of Ribonuclease B by Recombinant Yeast Peptide:N-glycanase  

作　　者：Shengjun WANG Peng George WANG and Qingsheng QI State Key Laboratory of Microbial Technology,Life Science Schooll,Shandong University,Jinan 250100,China 

出　　处：《Acta Biochimica et Biophysica Sinica》2007年第1期8-14,共7页生物化学与生物物理学报（英文版）

基　　金：This work was supported by a grant from National Natural Science Foundation of China(No. 30470399)

摘　　要：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, Pnglp, 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 ℃. 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 Pnglp from E. coli was investigated with respect to the substrate conformation in vitro. When ribonuclease B （RNase B） was denatured at 60-65 ℃ or by 40-60 mM dithiothreitol, indicated by its obvious structural change and sharpest activity change, its deglycosylation by Pnglp was most prominent. The deglycosylation efficiency of RNase B by Pnglp was found to be related to its structural conformation and enzymatic activity.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, Pnglp, 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 ℃. 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 Pnglp from E. coli was investigated with respect to the substrate conformation in vitro. When ribonuclease B （RNase B） was denatured at 60-65 ℃ or by 40-60 mM dithiothreitol, indicated by its obvious structural change and sharpest activity change, its deglycosylation by Pnglp was most prominent. The deglycosylation efficiency of RNase B by Pnglp was found to be related to its structural conformation and enzymatic activity.

关 键 词：glycanase ribonuclease B GLYCOPROTEIN DEGLYCOSYLATION circular dichroism 

分 类 号：Q78[生物学—分子生物学]