Abstract
Peptide:N-glycanase catalyzes the detachment of N-linked glycan chains from glycopeptides or glycoproteins by hydrolyzing the β-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-ΔH1) 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-ΔH1 is more flexible than wild type Png1p. The efficient deglycosylation of Png1p-ΔH1 towards native and non-native glycoproteins offers a potential biotechnological application.
Highlights
In eukaryotes, newly synthesized proteins, which are destined for the secretory pathway, are subjected to a quality control system [1,2]
Rad23p contains four structural domains connected by long unstructured flexible linker regions: an ubiquitin-like domain (UBL) at the N terminus that interacts with catalytically active proteasomes, two ubiquitin (Ub)-associated (UBA) sequences that bind Ub and a XPC binding (XPCB) domain that mainly mediates the interaction with PNGase
The Png1pRad23p interaction facilitates the direct transfer of deglycosylated ER-associated degradation’’ (ERAD) substrates to the proteasome, which bind to the UBL domain of Rad23p
Summary
Newly synthesized proteins, which are destined for the secretory pathway, are subjected to a quality control system [1,2]. In this control system, the proteins that fail to fold correctly are retained in the ER and subsequently degraded by a mechanism known as ‘‘ER-associated degradation’’ (ERAD) [3]. Rad23p contains four structural domains connected by long unstructured flexible linker regions: an ubiquitin-like domain (UBL) at the N terminus that interacts with catalytically active proteasomes, two ubiquitin (Ub)-associated (UBA) sequences that bind Ub and a XPC binding (XPCB) domain that mainly mediates the interaction with PNGase. Is there any structural change to PNGase upon binding Rad23p and what role does this interaction have to PNGase activity?
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