Using a small molecule inhibitor of peptide: N-glycanase to probe its role in glycoprotein turnover.
Peptide:N-glycanase (PNGase) is ostensibly the sole enzyme responsible for deglycosylation of unfolded N-linked glycoproteins dislocated from the ER to the cytosol. Here we show the pan-caspase inhibitor, Z-VAD-fmk, to be an active site-directed irreversible inhibitor of yeast and mammalian PNGase at concentrations below those used to inhibit caspases in vivo. Through chemical synthesis we determined that the P1 residue, electrophile position, and leaving group are important structural parameters for PNGase inhibition. We show that Z-VAD-fmk inhibits PNGase in living cells and that degradation of class I MHC heavy chains and TCRalpha, in an identical cellular setting, is markedly different. Remarkably, proteasome-mediated turnover of class I MHC heavy chains proceeds even when PNGase is completely inhibited, suggesting that the function of PNGase may be to facilitate more efficient proteasomal proteolysis of N-linked glycoproteins through glycan removal.
Misaghi
Shahram
S
Department of Pathology, Harvard Medical School, Boston, MA 02115, USA.
Pacold
Michael E
ME
Blom
Daniël
D
Ploegh
Hidde L
HL
Korbel
Gregory Alan
GA
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D061945:Caspase Inhibitors; D045744:Cell Line, Tumor; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D006023:Glycoproteins / Q000378:metabolism*; D015395:Histocompatibility Antigens Class I / Q000187:drug effects; D006801:Humans; D051379:Mice; D015394:Molecular Structure; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000502:physiology*; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000187:drug effects; D013329:Structure-Activity Relationship
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D006023:Glycoproteins; D015395:Histocompatibility Antigens Class I; D016693:Receptors, Antigen, T-Cell, alpha-beta; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C476093:z-Val-Ala-Asp(Ome)-fluoromethylketone; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase