©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Carbohydrate-binding Property of Peptide:N-Glycanase from Mouse Fibroblast L-929 Cells as Evaluated by Inhibition and Binding Experiments Using Various Oligosaccharides (*)

Tadashi Suzuki (1), Ken Kitajima (1), Yasuo Inoue (1)(§), Sadako Inoue (2)

From the (1)Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Hongo-7, Tokyo 113, Japan and the (2)School of Pharmaceutical Sciences, Showa University, Hatanodai-1, Tokyo 142, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Carbohydrate binding to peptide:N-glycanase from mouse fibroblast L-929 cells (L-929 PNGase) and inhibition by oligosaccharides of its catalytic activity were studied. L-929 PNGase was found to bind strongly with oligosaccharides having triomannosido-N,N`-diacetylchitobiosyl (ManGlcNAc) structure (K= 10 µM). This binding was inhibited by mannotriose (Man; Man13[Man16]Man) but not by N,N`-diacetylchitobiose (GlcNAc; GlcNAc14GlcNAc). Scatchard analysis indicated that there exist two binding sites for Man on a homodimeric form of a 105-kDa subunit. Oligosaccharides having Man GlcNAc structure were also shown to be strong inhibitors for the PNGase-catalyzed reaction (K = 10 µM). The minimum structural requirements for inhibition of the PNGase activity were Man and GlcNAc. Enzyme kinetic studies showed that the mechanism of inhibition by the oligosaccharides and Man fits well with a model wherein two inhibitor binding sites reside on L-929 PNGase. The conformity of K with IC values may be taken as an evidence for inhibition of the catalytic activity by the oligosaccharides and Man through the occupation of the binding sites with these molecules. On the other hand, inhibition by GlcNAc followed the simple competitive mode. Since the minimum substrate for the L-929 PNGase was shown to be Man14GlcNAc14GlcNAc1peptide, GlcNAc may be directly accessible to the catalytic site in competition with substrate. Interestingly, alkylation of -SH group in L-929 PNGase caused complete loss of the catalytic activity, but the carbohydrate binding activity was completely retained, indicating that the catalytic site(s) is discriminated from the carbohydrate-binding sites in the active site of this enzyme.

The carbohydrate-binding property seems to be unique to soluble PNGases from mammals and may be associated not only with regulation of the enzyme activity, but also with receptor and carrier functions for glycoconjugates in certain intracellular processes.


INTRODUCTION

Peptide:N-glycanase (PNGase; ()peptide-N-(N-acetyl--D-glucosaminyl)asparagine amidase, EC 3.5.1.52) is an enzyme catalyzing detachment of N-linked glycan chains from glycopeptides and/or glycoproteins by hydrolyzing the -aspartylglucosaminyl linkage. This enzyme can be classified as a member of ``proximal glycanase,'' which is a collective name of the enzymes that catalyze the cleavage of the linkage between the proximal saccharides and core proteins or ceramides, thereby releasing mono- or oligosaccharides from glycoconjugates (Suzuki et al., 1994a). Biological significance of such enzymes remains unclarified in the context other than catabolism of glycoconjugates, although they are recognized as useful reagents for structural and functional studies of glycoconjugates.

PNGase activities had only been found to occur in a variety of plant and bacterial cells (Takahashi, 1977; Plummer et al., 1984, 1987; Yet and Wold, 1988) until our demonstration of the occurrence of the enzyme in early embryos of Oryzias latipes (Medaka fish) (Seko et al., 1991a, Inoue et al., 1993). Since then, PNGase activities have been shown to be widely distributed among various cells and tissues of animals including mouse, human, and chicken (Suzuki et al., 1993, 1994a, 1994b, 1994c, 1995; Kitajima et al., 1995).

Recently, we have purified to homogeneity a mammalian PNGase from C3H mouse-derived fibroblast L-929 cells (Suzuki et al., 1994c). This enzyme, designated L-929 PNGase, is shown to have several unique properties distinguishable from bacterial PNGase F and plant seed PNGase A. Among these, most prominent is a carbohydrate-binding property, which was revealed by the following observations: (i) L-929 PNGase had relatively low K values for glycopeptide substrates (Suzuki et al., 1994c); (ii) free oligosaccharides formed from substrates inhibited the enzyme activity (Suzuki et al., 1994c); (iii) this enzyme bound to carbohydrate moiety of yeast mannan-conjugated Sepharose 4B (Suzuki et al., 1994d). L-929 PNGase was found to be a homodimer of a 105-kDa subunit in native state (Suzuki et al., 1994c). The fact that this molecular form is larger than PNGase F and A (34.8 and 66.8 kDa, respectively) in size was suggestive of the presence of multifunctional domain(s) other than a catalytic one in the enzyme. In this study, we demonstrate directly the binding of the enzyme with oligosaccharides. The enzyme kinetic study also revealed the mechanism of carbohydrate binding to L-929 PNGase and inhibition by the oligosaccharides of the L-929 PNGase catalysis.


EXPERIMENTAL PROCEDURES

Preparation of Peptide:N-glycanase from L-929 Cells

Cell culture of L-929 cells and purification of L-929 PNGase have been described previously (Suzuki et al., 1994c). The specific activity of the enzyme prepared was 110 milliunits/mg protein. One unit of L-929 PNGase activity was defined as described below. Protein was quantitated by the modified Lowry method (bicinchonic acid, Pierce) with bovine serum albumin (Pierce) as the standard.

Preparation of Glycopeptide Substrate and Related Compounds

For determination of PNGase activity, asialofetuin glycopeptide I, Leu-Asn(GalManGlcNAc)-Asp-Ser-Arg (asialo-fetGP I), was prepared from calf serum fetuin (Nacalai Tesque Co., Japan) as described previously (Suzuki et al., 1994c). Asialo-fetGP I was C-labeled at the amino-terminal residue by reductive methylation as described previously (Seko et al., 1991a). A C-labeling experiment was carried out at the Radioisotope Centre, University of Tokyo. Specific radioactivity for [C]asialo-fetGP I was determined to be 7.0 10 cpm/nmol.

C-Labeled glycoasparagine, [C]Asn(ManGlcNAc) ([C]GP-IVD), derived from ovalbumin and H-labeled oligosaccharide alditol, GalManGlcNAc[H]GlcNAcol ([H]asialo-fetGL), obtained from asialofetuin were prepared as described before (Suzuki et al., 1994d). Specific radioactivity for these compounds were 1.3 10 and 3.4 10 cpm/nmol, respectively.

[C]Asialo-fetGP I (9.8 10 cpm; 140 nmol) was digested sequentially with the following enzymes; jack bean -galactosidase (Seikagaku Kogyo Co.; 0.5 units in 0.5 ml of 50 mM sodium acetate buffer, pH 4.5, 37 °C for 3 h) and jack bean -N-acetylhexosaminidase (Seikagaku Kogyo Co.; 0.5 units in 0.5 ml of 50 mM sodium acetate buffer, pH 4.5, 37 °C for 3 h). Subsequently the digests were separated into two, and one was further digested with the following exoglycosidases: jack bean -mannosidase (Seikagaku Kogyo Co.; 0.5 units in 2.7 ml of 50 mM sodium acetate buffer, pH 4.5, 37 °C for 16 h) and snail -mannosidase (Sigma; 0.4 units in 2.7 ml of 50 mM sodium acetate buffer, pH 4.5, 37 °C for 2 h). The other was digested with endo--N-acetylglucosaminidase D from Diplococcus pneumoniae (Seikagaku Kogyo Co.; 0.05 units in 1.0 ml of 0.1 M sodium citrate-phosphate buffer, pH 6.5, 37 °C for 24 h). Between glycosidase digestions, the digests were desalted by Sephadex G-25 (1.2 100 cm; eluted with water) and a 5.0 10 cpm (7.1 nmol) portion of the respective sample was set aside, and used for determination of substrate specificity of the known PNGases. The purity and structure of each glycopeptide were examined by thin layer chromatography on a silica gel plate (Kiesel gel 60 (Merck); ethyl acetate/pyridine/acetic acid/water = 5:5:1:3 (v/v/v/v); 3 h).

Assay for PNGase Activity

PNGase activity was assayed by the method described previously (Suzuki et al., 1994c). Briefly, the reaction mixture containing the enzyme fraction in a total volume of 10 µl of 100 mM MES buffer (pH 7.0), 0.25 M sucrose and 10 mM DTT together with 210 pmol of [C]asialo-fetGP I (15,000 cpm) was incubated in a polypropylene microtube at 25 °C for 24 h. Reaction products were separated by paper chromatography and paper electrophoresis, followed by quantitation of the radioactivity by a Bio-imaging analyzer (Fujix BAS 2000). Paper electrophoresis was performed on Whatman No. 3MM paper at 13 V/cm for 3 h in pyridine/acetic acid/water (5:0.2:95, v/v/v, pH 6.5). One unit of L-929 PNGase activity was defined as the amount of enzyme that catalyzed the release of 1 µmol of fetuin [C]peptide under the assay conditions.

Inhibitory Effects of Various Compounds on L-929 PNGase Activity

Free oligosaccharide A-1, NeuAc23Gal14GlcNAc12Man16(NeuAc23Gal14GlcNAc12Man13)Man14GlcNAc14GlcNAc (NeuAcGalManGlcNAc), was obtained from unfertilized eggs of a freshwater trout, Plecoglossus altivelis, as described before (Ishii et al. 1989). Free oligosaccharides (U3`-1, NeuAc23-(Gal14)Gal14GlcNAc12Man16[NeuAc23(Gal14)Gal14GlcNAc12Man13]Man14GlcNAc; U3`-2, NeuAc-23(Gal14)Gal14GlcNAc12Man16(3)[NeuAc23Gal14GlcNAc12Man13(6)]Man14GlcNAc; U3`-3, NeuAc23Gal14GlcNAc12Man16[NeuAc23Gal14 GlcNAc12Man13]Man14GlcNAc), were also prepared from unfertilized eggs of a dace, Tribolodon hakonensis, as described previously (Inoue et al., 1989). Asialooligosaccharide (GalManGlcNAc; asialo-fetOS) derived from fetuin was prepared as described previously (Suzuki et al., 1994d). Asialo-fetOS and U3`-3 were treated with 1 M NaBH in 0.5 ml of 0.1 N NaOH at 4 °C for 16 h, and reduction reaction was stopped by adding 660 µl of glacial acetic acid, followed by desalting. Glycoasparagine, Asn(ManGlcNAc), Man16(Man13)Man16(GlcNAc14)(GlcNAc12Man13)Man14-GlcNAc14GlcNAc1Asn) (GP-IIIA), was prepared from ovalbumin as described previously (Nomoto et al., 1992).

The following compounds were tested for their effects on L-929 PNGase activities: Man, GlcNAc, Gal, Man13Man (Dextra Laboratories Ltd., United Kingdom), Man16Man (Dextra Laboratories Ltd.), 13/16 mannotriose (Man or Man13(Man16)Man; Dextra Laboratories Ltd.), mannopentaose (Man13(Man16)Man16(Man13)Man; Dextra Laboratories Ltd.), N,N`-diacetylchitobiose (GlcNAc; Seikagaku Kogyo Co.), GP-IIIA, asialo-fetOS, A-1, U3`-1, U3`-2, U3`-3, reduced asialo-fetOS, and reduced U3`-3. [I] - 1/v plot analysis was carried out by altering the concentration of GlcNAc and Man from 1 to 20 mM. The effect of GlcNAc on PNGase A (Seikagaku Kogyo Co.) and PNGase F (Takara Shuzo Co.) was similarly examined by [I] - 1/v plot analysis. 210 pmol (15,000 cpm) of [C]asialo-fetGP I was incubated with PNGase A (20 microunits) in 10 µl of 50 mM sodium citrate-phosphate buffer (pH 5.0) or PNGase F (500 microunits) in 10 µl of 50 mM sodium phosphate buffer (pH 8.5) at 25 °C for 6 h with 1-25 mM GlcNAc as inhibitors.

K value of GP-IIIA for L-929 PNGase was calculated using [C]fetGP I as a substrate. Enzyme reaction was carried out at various concentration of the substrate (2.8-140 nM) at 25 °C for 16 h. Initial reaction rates were expressed as nanomoles per liter of de-N-glycosylated peptide released per minute under the assay conditions described above.

Assay for Carbohydrate Binding Activity

Binding activity of L-929 PNGase to [C]GP-IVD or [H]asialo-fetGL was assayed as follows. L-929 PNGase (224 nM) was incubated on ice with 50 µl of 100 mM MES buffer (pH 7.0) containing 0.25 M sucrose and 10 mM DTT with 0.15-2.9 nmol of [H]asialo-fetGL or 0.044-1.1 nmol of [C]GP-IVD for 1 h, and an equal volume of saturated ammonium sulfate (pH 7.1) was slowly added to the reaction mixture and left at 4 °C for 16 h. The solution was centrifuged at 17,000 g at 4 °C for 30 min, the precipitate was resuspended in 100 µl of saturated ammonium sulfate (pH 7.1) and centrifuged at 17,000 g at 4 °C for 30 min, and radioactivity in the precipitate was measured by an Aloka liquid scintillation system LSC-700 with ACS-II (Amersham Corp.) as a scintillant. Nonspecific binding of each incubation was estimated by measuring radioactivity in the precipitate with 20-fold excess amount of the unlabeled glycan ligands. The radioactivity accounted for less than 1% of the input counts. The dissociation constant was estimated by Scatchard analysis as described previously (Maynard and Baenziger, 1982).

Effects of Various Compounds on Carbohydrate Binding Activity of L-929 PNGase

Carbohydrate binding activity of L-929 PNGase was measured in the presence of 2 mMN-ethylmaleimide (NEM) or 5 mM EDTA. GlcNAc and Man were also added to the reaction mixture at 2, 4, and 20 mM to determine their effects on carbohydrate binding activity using [H]asialo-fetGL (0.58 nmol) as a ligand.


RESULTS

Inhibitory Effects of Mono- and Oligosaccharides on L-929 PNGase Activity

As shown in , the enzyme activity was most strongly inhibited in the presence of 1 mM each of GP-IIIA, asialo-fetOS, and A-1, which had a triomannosido-N,N`-diacetylchitobiose (ManGlcNAc) structure. These results showed that both complex- and hybrid-type N-glycans can be effective inhibitors.

In order to determine the minimum oligosaccharide structure required for inhibition of the enzyme activity, various mono- and oligosaccharides were tested (). N,N`-Diacetylchitobiose (GlcNAc) inhibited the enzyme activity by 60% at 10 mM, while GlcNAc had no inhibitory effect even at above 10 mM. 13/16-Mannotriose (Man) also exhibited an inhibitory effect on the enzyme activity (76% inhibition at 10 mM). Mannose and two kinds of mannobioses, Man13 Man and Man16Man, did not inhibit at the concentration of 10 mM. These results indicated that two different structural elements, GlcNAc and Man, were required for inhibition of the activity.

For the oligosaccharides containing the triomannosido structure but lacking the N,N`-diacetylchitobiosyl structure, such as U3`-1, U3`-2, and U3`-3, less marked inhibitory effects were observed as compared to GP-IIIA, asialo-fetOS, and A-1. It should be noted that A-1 and U3`-3 only differ from each other in having the N,N`-diacetylchitobiose group and a single N-acetylglucosamine residue at their reducing termini, respectively. Since the extent of inhibition exhibited by these oligosaccharides was similar to that by Man at 1 mM (), the observed inhibitory effects may be attributable to the triomannosido unit in these oligosaccharides. The inhibitory effect of the reduced asialo-fetOS was as effective as that of asialo-fetOS, and contrasted with the less marked effect observed for the reduced U3`-3. These results indicate that the -glycosidic linkage of the penultimate GlcNAc residue in ManGlcNAc structure is important for strong inhibitory effects. Mannopentaose (Man), which contained two triomannosido structural units, had less inhibitory effect on the activity than Man.

Kinetic Studies of Inhibition of L-929 PNGase Activity by Oligosaccharides

The Lineweaver-Burk plots for L-929 PNGase-catalyzed hydrolysis of [C]fetGP I in the presence and absence of GP-IIIA were shown in Fig. 1. Inhibition by GP-IIIA can be plausibly explained if one considers that the enzyme has two binding sites for the inhibitor oligosaccharide and substrate glycopeptide (Fig. S1). The K value for this substrate was determined to be 114 µM. Since a plot of 1/V against the concentration of GP-IIIA was not apparently linear, it is reasonable to envisage k > 0 (see Fig. S1, and Equations 1 and 2, where C = k/k).

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

From the data shown in Fig. 1, K was evaluated to be 4.8 µM for GP-IIIA. The ratio k/k was 0.2.


Figure 1: Lineweaver-Burk plots for the L-929 PNGase-catalyzed N-deglycosylation of fetuin [C]glycopeptide I in the presence and absence of glycoasparagine, GP-IIIA. v, initial reaction rate (nM/min); [S], concentration of fetuin [C]glycopeptide I (µM). Initial reaction rate was determined as described under ``Experimental Procedures'' in the absence () and in the presence of GP-IIIA at 10 µM () or 20 µM ().




Figure S1: Scheme 1Mechanism for inhibition of L-929 PNGase-catalyzed hydrolysis by oligosaccharides. E, enzyme; S, substrate; I, inhibitor; ES, enzyme-substrate complex; EI, enzyme-inhibitor complex I; EI, enzyme-inhibitor complex II; ESI, enzyme-substrate-inhibitor complex; P, products. Equations 1 and 2 are obtained based on the following assumptions: 1) enzyme has two binding sites for inhibitors with an identical inhibition constant (K); 2) formation of ES and ESI complexes are prohibited; 3) products are formed from both ES and ESI complexes, the rate constants of which are k and k, respectively.



When 1/v was plotted against the concentration of inhibitors, Man was clearly distinguished from GlcNAc in the mode of inhibition (Fig. 2). A straight line was obtained for inhibition by GlcNAc. This is consistent with the mechanism in which GlcNAc competes with the substrate for a catalytic site of the enzyme. The inhibition constant for competitive mode (K) was determined to be 5.6 mM by the method of Dixon (Dixon, 1956). On the other hand, Man showed a parabolic curve, which can be approximated as a quadric function by which the mechanism of inhibition can be explained with a scheme shown in Fig. S1. From the data depicted in Fig. 2, IC, the concentration required for 50% inhibition of PNGase activity, was determined to be 4.8 mM.


Figure 2: Inhibitory effects of N,N`-diacetylchitobiose (GlcNAc; GlcNAc14GlcNAc) and 13/16 mannotriose (Man; Man13[Man16]Man) on L-929 PNGase activity. Man () and GlcNAc () were separately added to the reaction mixture at varying concentrations, [I], and the initial reaction rate, v, was measured as described under ``Experimental Procedures.''



Effects of GlcNAcand Manon PNGase A and PNGase F Activities

In order to examine if such inhibitory effects observed for L-929 PNGase by GlcNAc and Man are shared by PNGases A and F, the effect of these oligosaccharides on the latter two PNGases was tested. Neither of these enzyme activities was affected by Man at all concentrations examined (1-10 mM). On the other hand, GlcNAc inhibited these enzyme activities in a concentration-dependent manner, and the IC values were determined to be 1.5 mM for PNGase A and 3.9 mM for PNGase F (Fig. 3), while IC of GlcNAc for L-929 PNGase was 6.9 mM (Fig. 2). These results perhaps indicate that GlcNAc is a common inhibitor for bacterial, plant, and animal PNGases, whereas Man is a unique inhibitor for animal PNGase.


Figure 3: Inhibitory effect of N,N`-diacetylchitobiose (GlcNAc; GlcNAc14GlcNAc) on PNGase A (a) and PNGase F (b).



Binding of L-929 PNGase to Oligosaccharides

In our previous study using yeast mannan-conjugated column, we showed that L-929 PNGase was bound to carbohydrate moiety of yeast mannan and the binding was dissociated in the presence of Man, suggesting that L-929 PNGase had a carbohydrate-binding property. To demonstrate this property more directly, the carbohydrate binding activity was quantitatively evaluated by the methods as described under ``Experimental Procedures.'' H-Labeled complex-type oligosaccharide alditol ([H]asialo-fetGL) derived from asialofetuin and C-labeled high mannose-type glycoasparagine GP-IVD ([C]GP-IVD) prepared from ovalbumin were used as ligand oligosaccharides. Both oligosaccharides were unable to act as substrates, but they were inhibitors for the PNGase activities. As shown in Fig. 4a, [H]asialo-fetGL bound to L-929 PNGase, and the dissociation constant (K) and the number of binding sites (n) on the dimeric enzyme were estimated to be 1.1 10M and 1.6, respectively. The enzyme also bound to [C]GP-IVD with K = 1.1 10M and n = 1.8 (Fig. 4b). These results show that L-929 PNGase can bind both complex- and high mannose-type N-glycan chains with high affinity (K = 10M). Binding with NaBH-reduced U3`-3, which had triomannosyl GlcNAcol structure at the reducing terminal structure, was not observed when 6.3-31 µM oligosaccharide were used, suggesting that the K of the reduced U3`-3 for the enzyme must be extremely large.


Figure 4: Scatchard plots of carbohydrate binding to L-929 PNGase for H-labeled asialo-fetGL (a) and C-labeled GP-IVD (b). [B], concentration of the bound ligand; [F], concentration of free ligand; [E], enzyme concentration.



Characterization of Carbohydrate-binding Properties of L-929 PNGase

L-929 PNGase was suggested to require -SH group(s) for its enzymatic activity because the enzyme was active only in the presence of DTT and inhibited by monoiodoacetic acid (Suzuki et al., 1994c). The effect of NEM on the enzyme activity was examined, and this compound also inhibited the enzyme activity by >96% at 2 mM. However, as shown in , 2 mM NEM-treated L-929 PNGase still retained binding activity with [H]asialo-fetGL, and both K and n values remained almost the same as those of intact L-929 PNGase. Addition of 5 mM EDTA had no effect on the binding activity (), showing that the carbohydrate-binding property of L-929 PNGase is different from that of C-type lectin (Drickamer and Taylor, 1994).

To examine which structural element of the ligand molecules is involved in the binding, carbohydrate-binding experiments were carried out in the presence of Man and GlcNAc (Fig. 5). Binding of L-929 PNGase with [H]asialo-fetGL was inhibited by Man in a concentration-dependent manner. IC of Man was estimated to be as large as 10 mM from the inhibition experiment at the substrate concentration of 5.8 µM (Fig. 5). On the other hand, GlcNAc did not inhibit the binding activity at the range of concentration (2-20 mM) examined.


Figure 5: Effects of Man and GlcNAc on the carbohydrate binding activity of L-929 PNGase. The binding of L-929 PNGase with [H]asialo-fetGL was assayed in the presence of Man (shaded) or GlcNAc (blank) at varying concentrations. Relative binding activity is expressed as the proportion of the amount of bound [H]asialo-fetGL to that in the absence of Man or GlcNAc set equal to 100. The value represents the mean of two independent assays. For details, see ``Experimental Procedures.''



Determination of Glycan Structure Essential for L-929 PNGase Substrates

To determine the minimum glycan structure required as a substrate for L-929 PNGase and other known PNGases A and F, [C]fetGP I and its derivatives, which shared the common peptide sequence, were used to measure susceptibility to these different PNGases. As summarized in I, trisaccharide-[C]fetGP I, Man14GlcNAc14GlcNAc1peptide, was shown to be the minimum substrate for L-929 PNGase. In contrast to this, PNGases A and F were active to N,N`-diacetylchitobiosyl-[C]fetGP I, while GlcNAc-[C]fetGP I was almost inactive to these enzymes. The data shown in I are apparently consistent with the previous observations for PNGase A by Plummer and Tarentino(1981) and for PNGase F by Chu(1986). Pentasaccharide-[C]fetGP I containing ManGlcNAc structure was the best substrate for L-929 PNGase and was hydrolyzed by this enzyme twice as fast as the other truncated asialo-fetGP I. Sialylation of the substrate reduced its susceptibility toward L-929 PNGase, but not the case with other PNGases tested. Thus, L-929 PNGase is distinguished from PNGases A and F in sensitivity to the structure of glycan moieties.


DISCUSSION

The present study revealed that L-929 PNGase activity is strongly inhibited by N-linked oligosaccharide chains having triomannosido-N,N`-diacetylchitobiosyl (ManGlcNAc) unit with Kvalues ranging from 10 to 10M. Although GlcNAc and Man were effective as the minimum structural elements for inhibition of the enzyme activity, inhibition by these oligosaccharides was weaker by 10-fold than oligosaccharides comprising ManGlcNAc structure. Enzyme kinetic studies showed that Man inhibited the enzyme activity by binding at two binding sites on the enzyme (Fig. S1; Fig. 1and 2). On the other hand, binding of GlcNAc followed a simple competitive inhibition mechanism. L-929 PNGase requires a trisaccharide linked to the peptide, i.e. Man14GlcNAc14GlcNAc1peptide, as the minimum carbohydrate unit of the substrate. Thus, GlcNAc was anticipated to be accessible to the catalytic site(s) as an analogue of the minimum structure. It is also noted that GlcNAc, but not Man, was found to act as a competitive inhibitor for bacterial PNGase F and plant seed PNGase A, as well as animal PNGase.

L-929 PNGase was revealed to bind with N-linked oligosaccharide chains with a high affinity (K = 10M) irrespective of the type of N-linked glycan chains, and this binding was inhibited by Man. The reduced U3`-3, which bore triomannosyl structure attached 14 to GlcNAcol instead of GlcNAc14GlcNAcol at the originally reducing terminus, was found to bind only with low affinity as in the case with Man. Neither GlcNAc nor other monosaccharides such as Man and GlcNAc inhibited the carbohydrate binding activity of L-929 PNGase. Thus, it was concluded that ManGlcNAc structure is important for binding with high affinity to the enzyme. The observed mode of inhibition by Man may be compatible with the fact that the L-929 enzyme exists in a dimeric form. Interestingly, the binding constants for [H]asialo-fetGL and [C]GP-IVD, both of which have ManGlcNAc structure, are comparable to the K value (4.8 µM) for GP-IIIA, which also contains the same pentasaccharide structural unit. IC (4.8 mM) of Man for L-929 PNGase-catalyzed reaction is also comparable to the binding constant of Man (10 mM) estimated from the binding inhibition experiments (Fig. 5). These results strongly suggest that the binding of oligosaccharides is directly associated with the inhibition of the enzyme activity.

No change of carbohydrate binding activity of L-929 PNGase occurred when -SH group(s) at or nearby the active site on the molecule were alkylated by 2 mM NEM, which resulted in a complete loss of the enzyme activity. This, together with the results of kinetic and binding experiments described above, led us to the conclusion that carbohydrate-binding sites can be discriminated from the catalytic site(s), although spatial relationship between these two discrete sites on the enzyme still remains to be elucidated.

In this study, we have further validated the hypothesis that L-929 PNGase may possibly have a dual role as an N-deglycosylating enzyme and a carbohydrate-binding protein. The carbohydrate binding activity is unique to soluble PNGase from animal source, and bacterial PNGase F and plant PNGase A have no such activity (Suzuki et al., 1994d). N-Deglycosylation catalyzed by PNGase is thus regarded to represent a basic post-translational modification pathway of certain glycoproteins by altering the physiological and/or physicochemical properties of the target proteins (Ishii et al., 1989; Inoue, 1990; Inoue et al., 1989, 1993; Seko et al., 1989, 1991a, 1991b; Iwasaki et al., 1992; Suzuki et al., 1993, 1994a, 1994b, 1994c, 1994d, 1995; Kitajima et al., 1995). N-Deglycosylation of the target glycoproteins may be regulated by binding of the animal PNGase with glycoproteins or free oligosaccharides. Feedback inhibition is one of such regulation mechanisms. Alternatively, carbohydrate binding activity of the PNGase may be associated with intracellular transport, anchoring to the membrane, and migration of certain glycoproteins in such a manner as ligand-carrier or receptor interaction. In this connection, it is interesting to note that, although L-929 PNGase was shown not to act on native forms of ribonuclease B and ovalbumin (Suzuki et al., 1994c), they appeared to bind with the PNGase and inhibited its activity (data not shown). Determination of intracellular localization of the enzyme and identification of the target glycoproteins are important for elucidation of biological function of the soluble PNGase in cellular processes and this lines of experiments are currently under way in our laboratories.

  
Table: Effects of various mono- and oligosaccharides on L-929 PNGase activity


  
Table: Effects of NEM and EDTA on the carbohydrate binding activity of L-929 PNGase


  
Table: Relative rate of hydrolysis of [C]fetGP I with various glycan structures catalyzed by known PNGases



FOOTNOTES

*
This research was supported in part by grants from the Mizutani Foundation for Glycoscience (to Y. I. (1993-1995) and to K. K. (1995-1996)). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

The abbreviations used are: PNGase, peptide:N-glycanase, peptide-N-(N-acetyl--D-glucosaminyl)asparagine amidase; asialo-fetGL, asialofetuin-derived oligosaccharide alditol or reduced asialo-fetOS, GalManGlcNAcGlcNAcol; asialo-fetOS, asialooligosaccharide derived from fetuin (GalManGlcNAc); DTT, dithiothreitol; fetGP I, fetuin-derived glycopeptide I, Leu-Asn(NeuAcGalManGlcNAc)-Asp-Ser-Arg; GlcNAcol, N-acetylglucosaminitol; GP-IIIA, a glycoasparagine derived from ovalbumin, Asn(ManGlcNAc); GlcNAc, N,N`-diacetylglucosamine; GP-IVD, a glycoasparagine derived from ovalbumin, Asn(ManGlcNAc); L-929 PNGase, PNGase from mouse-derived fibroblast L-929 cells; Man, 13/16 mannotriose, Man13(Man16)Man; ManGlcNAc, triomannosido-N,N`-diacetylchitobiose; MES, 2-(N-morpholino)ethanesulfonic acid; NEM, N-ethylmaleimide.


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