Glycobiology Advance Access originally published online on July 19, 2007
Glycobiology 2007 17(10):1070-1076; doi:10.1093/glycob/cwm079
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Fluorescently labeled inhibitor for profiling cytoplasmic peptide:N-glycanase
2 RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
3 CREST, Japan Science and Technology Agency, Kawaguchi, Saitama 332-1102, Japan
4 Graduate School of Science and Engineering, Saitama University, Saitama 338-8570, Japan
5 21st COE (Center of Excellence) Program, Osaka University Graduate School of Medicine, 2-2 Yamadaoka Suita, Osaka 565-0871, Japan
1 To whom correspondence should be addressed: Tel: +81-48-467-9430; Fax: +81-48-462-4680; e-mail: yukito{at}riken.jp
Received on April 9, 2007; revised on July 4, 2007; accepted on July 15, 2007
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.
Key words: fluorescent / inhibitor / peptide:N-glycanase
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