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.

Since those discoveries, this enzyme has been widely used as a powerful tool reagent for analyzing the structure and functions of N-linked glycan chains on glycoproteins. The occurrence of a cytoplasmic PNGase activity has been reported in a wide variety of animal origins as well as in yeast (4)(5)(6)(7)(8).
The cytoplasmic PNGase was found to be quite distinct in terms of enzymatic properties from the PNGases of plant and bacterial origin (9). A gene encoding the cytoplasmic enzyme, PNG1, was first identified in S. cerevisiae (10).
Structural elements of the gene are highly conserved throughout eukaryotes (10,11). The protein is categorized as a member of the transglutaminase superfamily (12) and contains a catalytic Cys, His, and Asp triad which is required for enzyme activity (1,13). The cytoplasmic PNGase has been shown to be involved in the degradation of misfolded/unassembled glycoproteins by a process called ER-associated degradation (ERAD) (14)(15)(16)(17)(18)(19).
Previously the chemical synthesis of high-mannose glycans and its derivatives have been reported (20)(21)(22)(23). Among these, the haloacetamidyl-sugar (CHO-XAc) is an attractive compound because it can generate de novo glycoproteins through Cys residues in the target proteins (22,24). It can also serve as a specific inhibitor for carbohydrate-processing enzymes, which harbor a catalytic Cys residue, as is the case with the cytoplasmic PNGase. In this study, we explored this possibility using purified yeast PNGase (Png1) and found that these compounds are very strong inhibitors of this enzyme.
CHO-XAc was found to be covalently linked to this enzyme, and to our surprise the labeling was specific and under experimental conditions only a monovalent binding to the enzyme was observed, strongly suggesting that the labeling is against a  (25,26). Molecular modeling of chitobiose binding to yeast PNGase suggested the binding site for this carbohydrate, which was distinct from that of Z-VAD-fmk (25), a peptide-based inhibitor of cytoplasmic PNGases (18,27). Our studies suggest that these haloacetamidyl sugars could serve as novel, carbohydrate-based potent inhibitors of these enzymes, and also provide insight into the reaction mechanism of the cytoplasmic PNGase.

EXPERIMENTAL PROCEDURES
Materials -All chemicals used in this study were obtained either from Wako Pure Chemical Industries Ltd. (Osaka, Japan), Nacalai Tesque, Inc (Kyoto, Japan) or Sigma-Aldrich Japan (Tokyo, Japan) unless specified otherwise.

Man 9 GlcNAc 2 -IAc acts as a potent inhibitor of Png1
Recently convergent synthesis of high As shown in Figure 2, yeast PNGase (Png1) was strongly inhibited by the addition of

Glycosylated Png1 could be detected with Con
A-staining ( Figure 3B). It is interesting to note that although Png1 contains 14 cysteines, there was only a single reaction product which is ~2 kDa higher than the non-glycosylated form on SDS-PAGE ( Figures 3A, B). with Png1. Figure 4A shows that both compounds were able to bind to Png1, as judged by a shift in the migration pattern (compare lanes 1 and 2, 3 in Figure 4A). Attachment of GlcNAc 2 to Png1 was also confirmed by WGA-staining ( Figure 4B). The reactivity of GlcNAc 2 -IAc was similar to the corresponding BrAc ( Figure 4C) or ClAc ( Figure 4D) derivatives, with slightly faster kinetics for IAc ( Figure 4E).
However, the reactivity was apparently slower than either Man 8 GlcNAc 2 -IAc or Man 9 GlcNAc 2 -IAc, indicating that the Man residues result in a higher affinity to Png1 ( Figure   4E).
To examine the inhibitory effects of haloacetamidyl sugars to Png1, the concentration required for 50% inhibition (IC 50 ) was determined for these compounds using various concentration of the inhibitors. As shown in Table 1  indicating that the assumption was correct. In addition loss of GlcNAc residues from precursor ions was observed, confirming the occurrence of chitobiose on this peptide. On the other hand, when Cys191 was predicted to be C-CAE and Cys195 to be C-AMGN2, only peptides from y3-y8 were observed (i.e. only peptide from 196-200 could be sequenced), strongly indicating that the underlying assumption was incorrect ( Figure 7F). If Cys195 was assumed to be C-CAM following the release of GlcNAc 2 , the sequence was still not readable through Cys195 ( Figure 7G). All of these data clearly identified Cys191 as the sole binding site for GlcNAc 2 -IAc.

Molecular Modeling of the GlcNAc 2 binding site on Png1
To get additional insights into the specific binding of GlcNAc 2 to Png1, molecular modeling was carried out using the AutoDock program (29). Previously the potential    was incubated for the indicated times with these chemicals (50 μM) at 37˚C before assaying the PNGase activity. Enzyme activities for incubation without Man 9 GlcNAc 2 -IAc at indicated time were set to 1.      Hydrogen bonds are shown as dashed lines in red and the active site cysteine is labeled in yellow.