Fluorescently labeled inhibitor for profiling cytoplasmic peptide:N-glycanase.
Cytoplasmic peptide:N-glycanase (PNGase) is an enzyme that removes N-glycans from misfolded glycoproteins. The function of cytoplasmic PNGase plays a significant role in the degradation of misfolded glycoproteins, which is critical for cell viability. Recently, we reported that haloacetoamidyl derivatives of high-mannose-type oligosaccharides selectively modify the catalytic cysteine of cytoplasmic PNGase and serve as its specific inhibitor. Interestingly, a drastically simplified chloroacetamidyl chitobiose derivative [(GlcNAc)(2)-ClAc] was also reactive to PNGase. In our work, it was conjugated to a hydrophobic fluorophore in order to render (GlcNAc)(2)-ClAc cells permeable. We demonstrated that this compound [BODIPY-(GlcNAc)(2)-ClAc] specifically binds to cytoplasmic PNGase from budding yeast (Png1). To date, only Z-VAD-fmk is known as an inhibitor of PNGase. BODIPY-(GlcNAc)(2)-ClAc and Z-VAD-fmk share the same binding site on Png1, while BODIPY-(GlcNAc)(2)-ClAc has markedly stronger inhibitory activity. The functional analysis of PNGase using Z-VAD-fmk should be carefully interpreted because of its intrinsic property as a caspase inhibitor. In sharp contrast, chloroacetamidyl chitobiose was not reactive to caspase. In addition, BODIPY-(GlcNAc)(2)-ClAc did not bind either chitobiose-binding lectins or PNGase from other sources. Moreover, fluorescent microscopy clearly showed that BODIPY-(GlcNAc)(2)-ClAc was efficiently introduced into cells. These results suggest that this compound could be an in vivo inhibitor of cytoplasmic PNGase.
Hagihara
Shinya
S
RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
Miyazaki
Ayako
A
Matsuo
Ichiro
I
Tatami
Atsushi
A
Suzuki
Tadashi
T
Ito
Yukishige
Y
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D001665:Binding Sites; D001896:Boron Compounds / Q000737:chemistry / Q000378:metabolism; D002240:Carbohydrate Sequence; D061945:Caspase Inhibitors; D002463:Cell Membrane Permeability; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003593:Cytoplasm / Q000201:enzymology*; D004187:Disaccharides / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005456:Fluorescent Dyes; D008358:Mannose / Q000302:isolation & purification; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000494:pharmacology; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D004718:Saccharomycetales / Q000201:enzymology
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D000590:Amino Acid Chloromethyl Ketones; D001896:Boron Compounds; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D004187:Disaccharides; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D009844:Oligosaccharides; D029701:Saccharomyces cerevisiae Proteins; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C032438:chitobiose; C468820:PNG1 protein, S cerevisiae; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D008358:Mannose