N-terminal deletion of peptide:N-glycanase results in enhanced deglycosylation activity

• Published in: PloS one 2009-12, Vol.4 (12), p.e8335
• Main Authors: Wang, Shengjun, Xin, Fengxue, Liu, Xiaoyue, Wang, Yuxiao, An, Zhenyi, Qi, Qingsheng, Wang, Peng George
• Format: Article
• Language: English
• Subjects: Amino acids; Analysis; Binding sites; Biochemistry/Biocatalysis; Biochemistry/Protein Chemistry; Biodegradation; Biotechnology; Biotechnology/Biocatalysis; Capillary electrophoresis; Chromatography; Deglycosylation; Deletion mutant; DNA-Binding Proteins - metabolism; E coli; Enzymes; Escherichia coli; Glycan; Glycopeptides; Glycoprotein degradation; Glycoproteins; Glycosylation; Helices; Hydrogen-Ion Concentration; In vivo methods and tests; Kinetics; Laboratories; Mass spectrometry; Models, Molecular; Mutants; N-glycanase; N-Terminus; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase - chemistry; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase - genetics; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase - metabolism; Peptides; Protein Binding; Protein Structure, Secondary; Proteins; Ribonucleases - metabolism; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Scientific imaging; Sequence Deletion; Structure-Activity Relationship; Studies; Surface Plasmon Resonance; Yeast
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0008335

Peptide:N-glycanase catalyzes the detachment of N-linked glycan chains from glycopeptides or glycoproteins by hydrolyzing the beta-aspartylglucosaminyl bond. Peptide:N-glycanase in yeast binds to Rad23p through its N-terminus. In this study, the complex formed between Peptide:N-glycanase and Rad23p was found to exhibit enhanced deglycosylation activity, which suggests an important role for this enzyme in the misfolded glycoprotein degradation pathway in vivo. To investigate the role of this enzyme in this pathway, we made stepwise deletions of the N-terminal helices of peptide:N-glycanase. Enzymatic analysis of the deletion mutants showed that deletion of the N-terminal H1 helix (Png1p-DeltaH1) enhanced the deglycosylation activity of N-glycanase towards denatured glycoproteins. In addition, this mutant exhibited high deglycosylation activity towards native glycoproteins. Dynamic simulations of the wild type and N-terminal H1 deletion mutant implied that Png1p-DeltaH1 is more flexible than wild type Png1p. The efficient deglycosylation of Png1p-DeltaH1 towards native and non-native glycoproteins offers a potential biotechnological application.