From the
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
(Man
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.
Peptide:N-glycanase (PNGase
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
[
The following compounds were tested for
their effects on L-929 PNGase activities: Man, GlcNAc, Gal,
Man
K
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
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
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy From the data shown in Fig. 1, K
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
The present study revealed that L-929 PNGase activity is
strongly inhibited by N-linked oligosaccharide chains having
triomannosido-N,N`-diacetylchitobiosyl
(Man
L-929 PNGase was revealed to bind with N-linked oligosaccharide chains with a high affinity (K
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.
GlcNAc
) structure (K
=
10 µM).
This binding was inhibited by mannotriose (Man
;
Man
1
3[Man
1
6]Man) but not by N,N`-diacetylchitobiose (GlcNAc
;
GlcNAc
1
4GlcNAc). 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
Man
1
4GlcNAc
1
4GlcNAc
1
peptide,
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.
; (
)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.
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.
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(GalMan
GlcNAc
)-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(Man
GlcNAc
)
([
C]GP-IVD), derived from ovalbumin and
H-labeled oligosaccharide alditol,
Gal
Man
GlcNAc
[
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,
NeuAc2
3Gal
1
4GlcNAc
1
2Man
1
6(NeuAc
2
3Gal
1
4GlcNAc
1
2Man
1
3)Man
1
4GlcNAc
1
4GlcNAc
(NeuAc
Gal
Man
GlcNAc
),
was obtained from unfertilized eggs of a freshwater trout, Plecoglossus altivelis, as described before (Ishii et al. 1989). Free oligosaccharides (U3`-1,
NeuAc
2
3-(Gal
1
4)Gal
1
4GlcNAc
1
2Man
1
6[NeuAc
2
3(Gal
1
4)Gal
1
4GlcNAc
1
2Man
1
3]Man
1
4GlcNAc; U3`-2,
NeuAc
-2
3(Gal
1
4)Gal
1
4GlcNAc
1
2Man
1
6(3)[NeuAc
2
3Gal
1
4GlcNAc
1
2Man
1
3(6)]Man
1
4GlcNAc; U3`-3,
NeuAc
2
3Gal
1
4GlcNAc
1
2Man
1
6[NeuAc
2
3Gal
1
4
GlcNAc
1
2Man
1
3]Man
1
4GlcNAc), were
also prepared from unfertilized eggs of a dace, Tribolodon
hakonensis, as described previously (Inoue et al., 1989).
Asialooligosaccharide
(Gal
Man
GlcNAc
; 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(Man
GlcNAc
),
Man
1
6(Man
1
3)Man
1
6(GlcNAc
1
4)(GlcNAc
1
2Man
1
3)Man
1
4-GlcNAc
1
4GlcNAc
1
Asn)
(GP-IIIA), was prepared from ovalbumin as described previously (Nomoto et al., 1992).
1
3Man (Dextra Laboratories Ltd., United Kingdom),
Man
1
6Man (Dextra Laboratories Ltd.),
1
3/
1
6 mannotriose (Man
or
Man
1
3(Man
1
6)Man; Dextra Laboratories Ltd.),
mannopentaose
(Man
1
3(Man
1
6)Man
1
6(Man
1
3)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.
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.
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.
) inhibited the
enzyme activity by 60% at 10 mM, while GlcNAc had no
inhibitory effect even at above 10 mM.
1
3/
1
6-Mannotriose (Man
) also
exhibited an inhibitory effect on the enzyme activity (76% inhibition
at 10 mM). Mannose and two kinds of mannobioses,
Man
1
3 Man and Man
1
6Man, 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.
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
Man
GlcNAc
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
).
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;
GlcNAc
1
4GlcNAc) and
1
3/
1
6 mannotriose
(Man
; Man
1
3[Man
1
6]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 GlcNAc
In order to
examine if such inhibitory effects observed for L-929 PNGase by
GlcNAcand Man
on PNGase A and PNGase F Activities
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;
GlcNAc
1
4GlcNAc) 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
10
M and 1.6, respectively.
The enzyme also bound to [
C]GP-IVD with K
= 1.1
10
M 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
=
10
M). Binding with
NaB
H
-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).
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,
Man
1
4GlcNAc
1
4GlcNAc
1
peptide, 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
Man
GlcNAc
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.
GlcNAc
) unit with K
values ranging from 10
to 10
M. 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
Man
GlcNAc
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. Man
1
4GlcNAc
1
4GlcNAc
1
peptide, 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.
=
10
M) 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
1
4 to
GlcNAcol instead of GlcNAc
1
4GlcNAcol 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
Man
GlcNAc
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
Man
GlcNAc
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.
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
-(N-acetyl-
-D-glucosaminyl)asparagine
amidase; asialo-fetGL, asialofetuin-derived oligosaccharide alditol or
reduced asialo-fetOS,
Gal
Man
GlcNAc
GlcNAcol; asialo-fetOS,
asialooligosaccharide derived from fetuin
(Gal
Man
GlcNAc
); DTT,
dithiothreitol; fetGP I, fetuin-derived glycopeptide I,
Leu-Asn(NeuAc
Gal
Man
GlcNAc
)-Asp-Ser-Arg;
GlcNAcol, N-acetylglucosaminitol; GP-IIIA, a glycoasparagine
derived from ovalbumin, Asn(Man
GlcNAc
);
GlcNAc
, N,N`-diacetylglucosamine; GP-IVD, a
glycoasparagine derived from ovalbumin,
Asn(Man
GlcNAc
); L-929 PNGase, PNGase from
mouse-derived fibroblast L-929 cells; Man
,
1
3/
1
6 mannotriose,
Man
1
3(Man
1
6)Man;
Man
GlcNAc
,
triomannosido-N,N`-diacetylchitobiose; MES,
2-(N-morpholino)ethanesulfonic acid; NEM, N-ethylmaleimide.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.