Site-specific Labeling of Cytoplasmic Peptide:N-Glycanase by N,N′-Diacetylchitobiose-related Compounds

Tadashi Suzuki,Izumi Hara,Miyako Nakano,Gang Zhao,William J Lennarz,Hermann Schindelin,Naoyuki Taniguchi,Kiichiro Totani,Ichiro Matsuo,Yukishige Ito

doi:10.1074/jbc.m603236200

Abstract

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.

Highlights

The chemical synthesis of high mannose glycans and its derivatives have been reported (20 –23)
Synthesis of GlcNAc2-IAc—Chitobiose Our studies suggest that these haloacetamidyl sugars could [5] (44.0 mg, 0.104 mmol) was subjected to a series of reactions serve as novel, carbohydrate-based potent inhibitors of these enzymes, and provide insight into the reaction mechanism of the cytoplasmic PNGase
Among the oligosaccharide-related compounds synthesized, iodoacetamide derivatives of oligosaccharides may be of particular interest because they can generate a neoglycoprotein through a Cys residue bearing a homogeneous oligosaccharide structure

Summary

Chitobiose Binding to Yeast PNGase

Synthesis of Man9GlcNAc2-IAc (Structure 3 in Fig. 1)—Undecasaccharide 1 (Fig. 1: 4.9 mg, 2.6 ␮mol), synthesized as described previously [20], was dissolved in saturated aqueous NH4HCO3 (1.5 ml) and stirred at 40 °C for 72 h, the mixture was concentrated and lyophilized with H2O (3 times). The residue was purified by Sep-Pak C18 cartridge (5 g, Waters adsorbed and washed with H2O and eluted with H2O/methanol ϭ 20/1), followed by gel filtration (Amersham Biosciences: Superdex 30 (20 mm ϫ 60 cm), H2O) to give 33.9 mg (58%, 2 steps) of GlcNAc2-ClAc: 1H NMR (400 MHz, D2O) ␦ 1.87 (s, 3 H), 1.94 (s, 3 H), 3.30 –3.79 (m, 12 H), 3.98 (m, 2H), 4.47 (d, J ϭ 8.5 Hz, 1H), 4.95 (d, J ϭ 9.8 Hz, 1H); MALDITOF mass m/z calculated for C18H30N3O11ClNa 522.2 (MϩNa)ϩ, found 522.2. 50 ␮M) was reacted with yeast PNGase (70 ␮g in 700 ␮l) for 30 min, and the mixture was ultrafiltrated with Nanosep (10-kDa molecular mass cutoff) to remove unreacted GlcNAc2-IAc, and the final retentate (400 ␮l in 20 mM Tris-HCl (pH 8.0)) was evaporated to dryness. The model with the lowest binding energy was selected and analyzed in PyMol

RESULTS

To get additional insights into the specific binding of

DISCUSSION