A cytoplasmic peptide: N-glycanase.
A cytoplasmic peptide:N-glycanase (PNGase) has been implicated in the proteasomal degradation of aberrant glycoproteins synthesized in the endoplasmic reticulum. The reaction is believed to be important for subsequent proteolysis by the proteasome since bulky N-glycan chains on misfolded glycoproteins may impair their efficient entry into the interior of the cylinder-shaped 20S proteasome, where the active sites of the proteases reside. The deglycosylation reaction by PNGase brings about two major changes on substrate proteins; one is a removal of N-glycan chains, and the other is the introduction of negative charge(s) into the core peptide by converting glycosylated asparagine residue(s) into aspartic acid residue(s). Therefore, PNGase action can be accurately monitored by detecting both changes using two different methods; that is, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for deglycosylation and isoelectric focusing for detection of introduction of negative charge(s) into core proteins. This chapter will describe the simple in vivo as well as in vitro assay method to detect PNGase activity.
Tanabe
Kaori
K
Department of Biochemistry, Osaka University, Graduate School of Medicine, Japan.
Lennarz
William J
WJ
Suzuki
Tadashi
T
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D003513:Cycloheximide / Q000378:metabolism; D003593:Cytoplasm / Q000201:enzymology*; D006801:Humans; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D011500:Protein Synthesis Inhibitors / Q000378:metabolism
D010455:Peptides; D011500:Protein Synthesis Inhibitors; D003513:Cycloheximide; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase