Site-specific labeling of cytoplasmic peptide:N-glycanase by N,N'-diacetylchitobiose-related compounds.
Peptide:N-glycanase (PNGase) is the deglycosylating enzyme, which releases N-linked glycan chains from N-linked glycopeptides and glycoproteins. Recent studies have revealed that the cytoplasmic PNGase is involved in the degradation of misfolded/unassembled glycoproteins. This enzyme has a Cys, His, and Asp catalytic triad, which is required for its enzymatic activity and can be inhibited by "free" N-linked glycans. These observations prompted us to investigate the possible use of haloacetamidyl derivatives of N-glycans as potent inhibitors and labeling reagents of this enzyme. Using a cytoplasmic PNGase from budding yeast (Png1), Man9GlcNAc2-iodoacetoamide was shown to be a strong inhibitor of this enzyme. The inhibition was found to be through covalent binding of the carbohydrate to a single Cys residue on Png1, and the binding was highly selective. The mutant enzyme in which Cys191 of the catalytic triad was changed to Ala did not bind to the carbohydrate probe, suggesting that the catalytic Cys is the binding site for this compound. Precise determination of the carbohydrate attachment site by mass spectrometry clearly identified Cys191 as the site of covalent attachment. Molecular modeling of N,N'-diacetylchitobiose (chitobiose) binding to the protein suggests that the carbohydrate binding site is distinct from but adjacent to that of Z-VAD-fmk, a peptide-based inhibitor of this enzyme. These results suggest that cytoplasmic PNGase has a separate binding site for chitobiose and other carbohydrates, and haloacetamide derivatives can irreversibly inhibit that catalytic Cys in a highly specific manner.
Suzuki
Tadashi
T
21st Center of Excellence Program, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan; CREST, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan. Electronic address: tsuzuki@biochem.med.osaka-u.ac.jp.
Hara
Izumi
I
CREST, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan.
Nakano
Miyako
M
21st Center of Excellence Program, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan.
Zhao
Gang
G
Center for Structural Biology, Stony Brook University, Stony Brook, New York 11794-5215; Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York 11794-5215.
Lennarz
William J
WJ
Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York 11794-5215.
Schindelin
Hermann
H
Center for Structural Biology, Stony Brook University, Stony Brook, New York 11794-5215; Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York 11794-5215; Rudolf Virchow Center for Experimental Biomedicine and Institute of Structural Biology, University of Würzburg, 97078 Würzburg, Germany.
Taniguchi
Naoyuki
N
21st Center of Excellence Program, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan; Department of Disease Glycomics, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan.
Totani
Kiichiro
K
CREST, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan; RIKEN, The Institute of Physical and Chemical Research, Wako 351-0198, Japan.
Matsuo
Ichiro
I
CREST, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan; RIKEN, The Institute of Physical and Chemical Research, Wako 351-0198, Japan.
Ito
Yukishige
Y
CREST, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan; RIKEN, The Institute of Physical and Chemical Research, Wako 351-0198, Japan.
D019943:Amino Acid Substitution; D001665:Binding Sites; D002241:Carbohydrates / Q000737:chemistry; D003593:Cytoplasm / Q000201:enzymology; D004187:Disaccharides / Q000737:chemistry / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry; D007460:Iodoacetamide / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics
D002241:Carbohydrates; D004187:Disaccharides; D005656:Fungal Proteins; C032438:chitobiose; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D007460:Iodoacetamide