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

Authors: WANG, Shengjun1; WANG, Peng George; QI, Qingsheng

Source: Acta Biochimica et Biophysica Sinica, Volume 39, Number 1, January 2007 , pp. 8-14(7)

Publisher: Blackwell Publishing

Key:
Free Content - Free Content
New Content - New Content
Subscribed Content - Subscribed Content
Free Trial Content - Free Trial Content

Abstract:

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°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 °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.

Edited by Minghua XU

Keywords: glycanase; ribonuclease B; glycoprotein; deglycosylation; circular dichroism

Document Type: Research article

DOI: 10.1111/j.1745-7270.2007.00244.x

Affiliations: 1: State Key Laboratory of Microbial Technology, Life Science School, Shandong University, Jinan 250100, China

The full text electronic article is available for purchase. You will be able to download the full text electronic article after payment.

$50.16 plus tax      Refund Policy

 

OR

Back to top

Key:
Free Content - Free Content
New Content - New Content
Subscribed Content - Subscribed Content
Free Trial Content - Free Trial Content
Share this item with others: These icons link to social bookmarking sites where readers can share and discover new web pages.
Page Help Click here for Page Help
Shopping cart
Tools
Sign in






Need to register?
Sign up here
Text size: A | A | A | A