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Cleavage of the Xylosyl Serine Linkage between a Core Peptide and a

Glycosaminoglycan Chain by Cellulases

Keiichi Takagaki, Mito Iwafune, Ikuko Kakizaki,

Keinosuke Ishido, Yoji Kato*, and Masahiko Endo

Department of Biochemistry, Hirosaki University School of Medicine,

5 Zaifu-cho, Hirosaki 036-8562 and

*Laboratory of Food Science, Faculty of Education, Hirosaki University,

1 Bunkyo-cho, Hirosaki 036-8560,

Japan

Running title: Release of Glycosaminoglycan Chains by Cellulases

Address for correspondence: Prof. Masahiko Endo, M.D.

Department of Biochemistry,

Hirosaki University School of Medicine,

5 Zaifu-cho, Hirosaki 036-8562,

Japan

Tel: +81-172-39-5015

Fax: +81-172-39-5016

Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on February 27, 2002 as Manuscript M111985200 by guest on June 20, 2018

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SUMMARY

We previously found that endo-β-xylosidase from Patinopecten is an endo-type

glycosidase that cleaves the xylosyl serine linkage between a glycosaminoglycan chain and

its core protein (Takagaki, K., Kon, A., Kawasaki, H., Nakamura, T., Tamura, S., and Endo,

M. (1990) J. Biol. Chem. 265, 854-860). Screening for endo-β-xylosidase activity in several

cellulases detected this activity in the enzymes from Aspergillus niger, Penicillium

funiculosum, Trichoderma reesei, Trichoderma viride and Irpex lacteus. The cellulase derived

from Aspergillus niger was purified and its molecular weight was determined to be 26,000 by

SDS-PAGE. Examination of the specificity of the cellulase revealed that 1) the enzyme acts

on the linkage region (xylosyl serine) between a core peptide and a glycosaminoglycan chain;

2) enzymatic activity is greater with shorter glycosaminoglycan chains; 3) the enzyme readily

hydrolyzes the linkage in glycosaminoglycan-peptides, but intact proteoglycan is cleaved

only slowly; 4) the activity is unaffected by the glycosaminoglycan component (chondroitin

sulfate, dermatan sulfate and heparan sulfate). Judging from these enzymatic characteristics,

this cellulase is different from the endo-β-xylosidase of Patinopecten. We believe that this

cellulase will become a useful tool in the further development of glycotechnology, because,

like the endo-β-xylosidase of Patinopecten, it enables the release of intact

glycosaminoglycans from glycosaminoglycan-peptides.

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INTRODUCTION

Proteoglycans (PGs) are widely distributed in connective tissue and on the cell surface of

mammalian tissues, and are functional materials influencing cell growth, differentiation and

morphogenesis (1, 2). It is known that PGs consist of a core protein linked to

glycosaminoglycan (GAG) chains, and that the GAG chains interact with a number of growth

factors (3) and with important functional proteins such as antithrombin III (4-6) and heparin

cofactor II (7). Although GAG chains are broadly divided into chondroitin sulfate (ChS),

dermatan sulfate (DS), heparan sulfate (HS), heparin, hyaluronic acid and keratan sulfate by

their particular combination of repeating disaccharide units, ChS, DS, HS and heparin are all

attached to a serine residue of a core protein by the linkage region GlcAβ1-3Galβ1-3Galβ1-

4Xylβ1-O-Ser (8). Recently, it has been shown that the linkage region is modified by

phosphate and sulfate groups (9-11). Such diversity of linkage regions is notable, because it

suggests that the modification is a signal involved in the control of GAG biosynthesis (11).

The metabolism of PGs in vivo has not yet been clarified. Matsue and Endo (12)

demonstrated that urinary GAG chains bear in part glucuronic acid, galactose or xylose

residues at their reducing terminals. These observations demonstrate that nonterminal internal

glucuronide, galactoside and xyloside linkages of GAG chains are cleaved in tissues. Indeed,

Takagaki et al. (13, 14) found endo-type glycosidase activities acting on the linkage regions

in rabbit liver, in the form of ChS-degrading endo-β-glucuronidase, endo-β-galactosidase and

endo-β-xylosidase activities. These enzymes appear to act in the initial catabolism of PGs in

animal tissues. One of them, endo-β-xylosidase, was purified from the mid-gut gland of the

mollusk Patinopecten (15). It specifically hydrolyzes the internal xylosyl serine (Xyl-Ser)

linkage between a core protein and a GAG chain (ChS, DS and HS) and is therefore a useful

tool for isolating native GAG chains, including the linkage region, from GAG-peptides.

Cellulases endolytically hydrolyze 1,4-β-glucoside linkages, and are widely distributed

in plants, microorganisms and in particular, fungi. Furthermore, the presence of endogenous

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cellulases in animals has been suggested recently (16-21). There is evidence that exo-type

enzymes recognize both xyloside and glucoside linkages. An exo-xylosidase acting on p-

nitrophenyl β-xylose and p-nitrophenyl β-glucose has been purified from Charonia lampas

(22, 23). Additionally, it was suggested that β-glucosidase purified from Aspergillus sojae has

β-xylosidase activity (24), and a single protein in pig kidney has both β-xylosidase activity

and β-glucosidase activity (25). Xylose and glucose have very similar configurations, with

the C-5 of xylose having a hydrogen atom rather than the hydroxymethyl group of glucose.

Therefore, it is likely that enzymes that recognize glucose will also recognize xylose.

Additionally, it is of interest to determine whether the endo-β-xylosidase derived from

Patinopecten was originally a cellulase, or whether it is a cellulase with endo-β-xylosidase

activity, or whether the two activities represent independent enzymes.

In this paper, we demonstrate that a fungal cellulase is able to hydrolyze the Xyl-Ser

linkage between the core peptide and the GAG chain of GAG-peptides. Although this

cellulase has endo-β-xylosidase activity, it is different from the endo-β-xylosidase of

Patinopecten.

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EXPERIMENTAL PROCEDURES

Materials — Cellulase preparations (EC 3.2.1.4) derived from Aspergillus niger (1.6

units/mg, 1 unit defined as the amount of enzyme that liberates 1 µmol of reducing sugar as

glucose in 1 h at pH 4.5 at 37°C), Penicillium funiculosum (5.1 units/mg, 1 unit defined as the

amount of enzyme that liberates 1 µmol of reducing sugar as glucose in 1 h at pH 5.0 at

37°C), Trichoderma reesei (6.3 units/mg, 1 unit defined as the amount of enzyme that

liberates 1 µmol of reducing sugar as glucose in 1 h at pH 5.0 at 37°C), and Trichoderma

viride (4.0 units/mg, 1 unit defined as the amount of enzyme that liberates 1 µmol of reducing

sugar as glucose in 1 h at pH 5.0 at 37°C) were purchased from Sigma Chem. Co., St. Louis,

MO. A cellulase preparation derived from Eupenicillium sp. (1.0 units/mg, 1 unit defined as

the amount of enzyme that liberates 1 µmol of 4-methylumbelliferone from 4-

methylumbelliferyl-β-lactoside in 1 h at 37°C) was a gift from Dr. Yasushi Mitsuishi,

National Institute of Advanced Industrial Science and Technology, Japan. Cellulase from

Irpex lacteus (1.07 units/mg, 1,000 units defined as the amount of enzyme that completely

destroys 2 sheets of quantitative filter paper 1 cm x 1 cm in 1 min at pH 5.0 at 37°C) was a

gift from Kyowa Hakko Kogyo Co., Tokyo, Japan. Cellulases from Pisum sativum L. and

Populus nigra (2 units/ml and 1 unit/ml, respectively, 1 unit defined as the amount of enzyme

that causes a 1% decrease in viscosity of carboxymethyl-cellulose in 2 h at pH 6.2 at 35°C)

were gifts from Dr. Takahisa Hayashi, Wood Research Institute of Kyoto University, Japan.

Endo-β-xylosidase was purified from Patinopecten as described previously (15). 4-

Methylumbelliferyl GAG (GAG-MU), which is an artificial substrate for endo-β-xylosidase,

was prepared from cultured medium of human skin fibroblasts by the method previously

reported (26). After digestion of GAG-MU with hyaluronidase from Streptomyces

hyalurolyticus (Seikagaku Kogyo Co., Tokyo, Japan), Galβ1-4Xylβ1-MU and Galβ1-

3Galβ1-4Xylβ1-MU were purified by Bio-Gel P-4 (Bio-Rad, Richmond, CA) column

chromatography as described previously (27, 28). To obtain ∆GlcAβ1-3GalNAcβ1-3GlcAβ1-

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3Galβ1-3Galβ1-4Xylβ1-MU and ∆GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-MU, GAG-MU was

digested with chondroitinase ABC and chondroitinase ACII (Seikagaku Kogyo Co.) as

described by Saito et al. (29), and then purified by Sephadex G-50 (Amersham Pharmacia

Biotec AB, Uppsala, Sweden) column chromatography. 4-Methylumbelliferyl-β-cellobioside

(cellobiose-MU) and 4-methylumbelliferyl-β-xylose (Xyl-MU) were purchased from Sigma

Chem. Co.

Chondroitin sulfate proteoglycan (ChS-PG), dermatan sulfate proteoglycan (DS-PG) and

heparan sulfate proteoglycan (HS-PG) were extracted from salmon cartilage, pig skin and

bovine lung, and purified by standard procedures as described by Heinegård and Hascall (30).

These PGs were digested with actinase E (Kaken Pharmaceutical Co., Tokyo, Japan) in 0.1 M

Tris-HCl buffer, pH 8.0, containing 10 mM CaCl2 at 50°C for 24 h. After digestion, the GAG-

peptides (ChS-peptides, DS-peptides and HS-peptides) were separated by DEAE-cellulose

(Whatman Chemical Separation Ltd., Maidstone, United Kingdom) column chromatography

and then purified by Sephacryl S-200 HR (Amersham Pharmacia Biotec AB) column

chromatography. The molecular weights of the peptide moieties of the ChS-peptides, DS-

peptides and HS-peptides were all about 1,000 as estimated from gel-filtration

chromatography based on the method of Ishido et al. (31). The linkage regions of the GAG-

peptides were analyzed for sulfate and phosphate contents based on the method of Takagaki

et al. (28). Sulfate or phosphate were not detected; therefore, it was considered that the GAG-

peptides used in this study did not contain phosphorylated xylose or sulfated galactose. All

other chemicals were obtained from commercial sources.

Fluorescence labeling with 2-aminopyridine — Fluorescence labeling of the reducing

terminal of the oligosaccharides with 2-aminopyridine (PA; Wako Pure Chemical Ind. Co.,

Tokyo, Japan) was carried out as described previously (32).

Assay of endo-β-xylosidase activity and cellulase activity — Endo-β-xylosidase activity was

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assayed as described previously (15). A complete incubation mixture (200 µl), which

contained 2 µM GAG-MU as a substrate, 0.1 M glucono-1,5-lactone (Wako Pure Chem. Co.)

as an inhibitor of exo-glucosidase, 0.1 M sodium acetate buffer, pH 5.0, and enzyme solution,

was incubated at 37°C for 1 h. The reaction was stopped and fluorescence developed by

adding 1 ml of 0.5 M glycine-NaOH buffer, pH 10.4. The fluorescence was measured on a

spectrofluorometer (Hitachi F-4500; Hitachi, Tokyo, Japan) at excitation and emission

wavelengths of 350 and 450 nm.

Cellulase activity was assayed with 2 µM cellobiose-MU as substrate based on the

method of Chernoglazov et al. (33).

One unit was defined as the amount of enzyme that liberated 1 µmol/min of MU from

GAG-MU or cellobiose-MU.

Another assay for cellulase activity using CM-cellulose as a substrate was performed as

described by Hurst et al. (34).

Assay of hydrolysis of natural substrates — Hydrolysis of natural substrates, GAG-peptides

(10 nmol) or intact ChS-PG (10 nmol; the molar amount of ChS-PG was calculated as the

molar amount of the GAG sugar chains), was assayed as described previously (35). Each

GAG-peptide was incubated with the purified cellulase from A. niger at 37°C for 12 h, and

the reducing terminals of the liberated GAG chains were labeled with PA. Then, each GAG-

PA chain was hydrolyzed in 2 M HCl at 100°C for 2 h. PA-xylose was detected by HPLC

with an Ultrasphere ODS column (4.6 mm x 250 mm; Beckman Coulter Inc., Fullerton, CA).

The molar amounts of the GAG-peptides were determined as the molar amount of PA-xylose

(Takara Shuzo, Kyoto, Japan).

Purification of cellulase— The following procedures were conducted at 4°C. First,

commercial cellulase (30 mg of protein) from A. niger was applied to a column of Sephacryl

S-100 HR (1.5 cm x 102 cm; Amersham Pharmacia Biotec AB) equilibrated previously with

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20 mM Tris-HCl buffer, pH 7.0, at a flow rate of 15 ml/h, and the fractions with cellulase

activity and endo-β-xylosidase activity were pooled. Next, the active fractions were applied

to a column of POROS PI (4.6 mm x 100 mm; Applied Biosystems Japan, Tokyo, Japan)

equilibrated previously with 20 mM Tris-HCl buffer, pH 7.0. The column was eluted with a

linear gradient of NaCl from 0 to 1 M containing the above buffer at a flow rate of 10 ml/min.

The fractions with both activities were pooled and, lastly, the pool was subjected to

isoelectric focusing using a Rotofor IEF Cell (Bio-Rad) with ampholyte (Bio-Rad) ranging

from pH 3.0 to 10.0 for 4 h.

High-performance liquid chromatography (HPLC) — A high-performance liquid

chromatograph (Hitachi L-6200) connected to a fluorescence detector (Hitachi F-1050) was

used. MU-derivatives and MU were analyzed on an Ultrasphere ODS column (4.6 mm x 250

mm; Beckman Coulter Inc.) with a linear gradient of acetonitrile from 0% to 30% for 50 min

at a flow rate of 1.0 ml/min at 30°C. For detection, eluates were monitored at excitation and

emission wavelengths of 325 and 380 nm. PA monosaccharides were analyzed on an

Ultrasphere ODS column (4.6 mm x 250 mm; Beckman Coulter Inc.) with 0.25 M sodium

citrate and 1% acetonitrile at a flow rate of 0.5 ml/min at 30°C, as described previously (26).

For detection, eluates were monitored at excitation and emission wavelengths of 320 and 400

nm. PA-glucose, PA-galactose and PA-xylose (Takara Shuzo) were used as standards.

Electrophoresis — Native polyacrylamide gel electrophoresis (PAGE) was done in 7.5%

polyacrylamide gel at 4°C. Protein was stained with Coomassie Brilliant Blue R-250, and

duplicate gels were cut into 5-mm segments. Each segment was extracted with 10 mM Tris-

HCl buffer, pH 7.0, at 4°C for 24 h, and the activity was assayed. SDS-PAGE was carried out

in 10% polyacrylamide gel according to the methods of Weber and Osborn (36). Protein was

stained with Coomassie Brilliant Blue R-250.

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Ion-spray mass spectrometry — Mass spectra were obtained on an API-100 triple-quadruple

mass spectrometer (PE SCIEX, Ontario, Canada) equipped with an atmospheric pressure

ionization source, as described previously (27, 37). The samples were dissolved in 50%

methanol and injected at 2 µl/min with a micro-HPLC syringe pump. In positive-ion mode,

scanning was done from m/z 30 to 250 during the 1-min scan (ten cycles).

Protein assay – Protein was determined by the method of Bradford (38), with bovine serum

albumin as a standard.

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RESULTS

Screening of endo-β-xylosidase activity in cellulases — Endo-β-xylosidase activity, which

hydrolyzed the Xyl-MU linkage of GAG-MU as a substrate, and cellulase activity, which

hydrolyzed cellobiose-MU as a substrate, were assayed in eight cellulase preparations from

five kinds of ascomycetes, one basidiomycetes and two plants, and in endo-β-xylosidase from

Patinopecten mid-gut gland (Fig. 1). Endo-β-xylosidase activity was found in the five

cellulase preparations from Aspergillus niger, Penicillium funiculosum, Trichoderma reesei,

Trichoderma viride and Irpex lacteus. Particularly high endo-β-xylosidase activities were

detected in the cellulases from A. niger and P. funiculosum. These results indicated that some

cellulases have endo-β-xylosidase activity in addition to cellulase activity. Fig. 1-I shows that

the endo-β-xylosidase from Patinopecten appeared to be different from the cellulases, having

much higher endo-β-xylosidase activity and very little cellulase activity.

To study the purity of each cellulase preparation, electrophoresis in 7.5% polyacrylamide

gel was performed and then the gels were stained with Coomassie Brilliant Blue. As a result,

several protein bands were observed in the cellulase preparations, except for that from

Eupenicillium sp. Duplicate gels were cut into 5-mm segments, extracted individually for 24

h with 10 mM Tris-HCl buffer, pH 7.0, at 4°C, and endo-β-xylosidase activity and cellulase

activity were measured in each fraction. These results with native PAGE are summarized in

Fig. 2. In the cellulase preparation from A. niger, endo-β-xylosidase activity was detected in

the same fractions in which cellulase activity was detected (Fig. 2-A). Similar results were

obtained with the preparations from T. reesei, T. viride, and I. lacteus (Fig. 2-C, D and F). In

contrast, the cellulase preparation from Eupenicillium sp. had only cellulase activity but not

endo-β-xylosidase activity (Fig. 2-E). In P. funiculosum, both activities were detected in

segment numbers 4 - 6 and only cellulase activity was detected in segment numbers 7 - 9 (Fig.

2-B). These results suggested that there are two types of cellulase, one having both endo-β-

xylosidase and cellulase activities and one having only cellulase activity.

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Purification of the cellulase from A. niger — In order to verify that a single enzyme protein

has both cellulase and endo-β-xylosidase activities, we performed the following experiment.

Although the β-xylosidase activity in P. funiculosum was higher than that in A. niger (Fig. 1),

the cellulase from A. niger is more widely available commercially. Thus, from the

commercial cellulase preparation the enzyme was purified 5-fold at 11% yield in endo-β-

xylosidase activity and cellulase activity by the sequential use of Sephacryl S-100 HR

column chromatography, POROS PI column chromatography and isoelectric focusing

electrophoresis (Table I). The cellulase exhibited endo-β-xylosidase activity during the course

of purification, and the ratio of each enzyme activity was almost constant. Fig. 3 shows that

native PAGE of this purified enzyme yielded a single band, with an estimated molecular

weight of 26,000 by SDS-PAGE. These results showed that the single protein band has the

activities of endo-β-xylosidase and cellulase. Additionally, the optimal pH for the enzyme

toward CM-cellulose was 3.8 (data not shown). The molecular weight of this enzyme and its

optimal pH toward CM-cellulose corresponded with the properties of cellulase from A. niger,

which has been purified previously (34, 39).

Action of the purified cellulase from A. niger on artificial substrates — To examine the action

of the purified cellulase from A. niger on the artificial substrates GAG-MU and cellobiose-

MU, the enzymatic products were subjected to reverse-phase HPLC (Fig. 4). After incubation

with the purified cellulase, the peaks of GAG-MU and cellobiose-MU had disappeared and

new fluorogenic components were found to be eluted at the position of authentic MU (Fig. 4).

The new fluorogenic components were collected and ion-spray mass spectrometry analysis

was carried out. The spectra of both new fluorogenic components showed two peaks of m/z

[M+H]+ ion and [M+Na]+ ion at m/z 177 [M+H]+ and 199 [M+Na]+, respectively (Fig. 5). The

combined results of HPLC analysis and ion-spray mass spectrometry indicated that this

fluorogenic component was MU, presumably released from the corresponding reducing ends

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of GAG-MU and cellobiose-MU.

To identify the newly exposed reducing terminal sugars of the other incubation products,

they were fluorescence-labeled with PA and hydrolyzed with 2 M HCl at 100°C for 2 h. After

N-acetylation with acetic anhydride, PA-sugars were subjected to reverse-phase HPLC on an

Ultrasphere ODS column as described previously (35). An elution profile is shown in Fig. 6.

The PA-sugar derived from GAG-MU gave a single peak at the position of standard PA-Xyl

(Fig. 6-A). On the other hand, that from cellobiose-MU gave a single peak at the position of

standard PA-glucose (Fig. 6-B). These data proved that xylose and glucose were exposed at

the reducing terminal ends of the sugar chains after digestion of GAG-MU and cellobiose-

MU with the cellulase. Thus, these results showed that the purified cellulase from A. niger

was able to hydrolyze endolytically the 4-methylumbelliferyl-β-xyloside linkage and the 4-

methylumbelliferyl-β-glucoside linkage of GAG-MU and cellobiose-MU, respectively.

Comparison of endo-β-xylosidase activity and cellulase activity of purified enzyme from A.

niger – The optimal pH values, time courses, and effects of metal ions were compared for

both hydrolysis reactions. For both activities, optimal pH was around 5.0 and the activity

curves were similar (Fig. 7-A). In the time course studies, hydrolysis by endo-β-xylosidase

was slower than that by cellulase (data not shown). Neither activity was affected by

monovalent cations or EDTA, with little difference discernible between endo-β-xylosidase

activity and cellulase activity. However endo-β-xylosidase activity was slightly decreased by

bivalent cations compared with cellulase activity (Table II).

Effect of GAG size on the endo-β-xylosidase activity from A. niger — To investigate the effect

of GAG size on the purified cellulase, several MU derivatives were incubated with the

purified cellulase. After incubation, liberated MU was measured by HPLC. Hydrolysis

activity was greater with shorter GAG chains, but Xyl-MU was not hydrolyzed (Table III).

Hydrolysis by the endo-β-xylosidase from Patinopecten, obtained from a previous report (15),

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is also shown. Endo-β-xylosidase from Patinopecten hydrolyzed GAG-MU efficiently, but

oligosaccharides were unable to serve as substrates. We concluded that substrate specificity

for GAG chain length is different between the purified cellulase from A. niger and the endo-

β-xylosidase from Patinopecten.

Activity of the endo-β-xylosidase from A. niger toward natural substrates — The properties of

the endo-β-xylosidase activity from A. niger toward natural substrates were examined.

First, in order to investigate whether the enzyme can act on intact PG, the time course of

hydrolysis of ChS-PG was compared with that of ChS-peptide (Fig. 8). The results showed

that the hydrolysis rate of ChS-PG was small compared with that of ChS-peptide. Therefore,

it is difficult for the enzyme to cleave the Xyl-Ser linkage of PG.

Secondly, the effects of pH and metal ions on the activity toward ChS-peptide were

examined to determine optimal assay conditions. With ChS-peptide as substrate, the optimal

pH was around 5.0 (Fig. 7-B), which corresponded with the optimal pH values toward

cellobiose-MU and GAG-MU (Fig. 7-A). In addition, the effect of metal ions on endo-β-

xylosidase activity toward ChS-peptide was similar to that on activity toward GAG-MU

(Table II).

Thirdly, the effect of GAG components on hydrolysis activity was examined at pH 5.0

without any metal ions. The purified cellulase hydrolyzed the Xylβ1-O-Ser linkage of ChS-

peptide, DS-peptide and HS-peptide. Hydrolysis rates of the GAG-peptides are shown in

Table IV. The Km value for ChS-peptide was estimated to be 0.84 mM by Lineweaver-Burk

plot (data not shown).

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DISCUSSION

PGs are composed of a core protein and a GAG chain whose molecular weight is several

tens of thousands (1, 2). The GAG chains are attached to serine residues of the core protein

via a linkage region, GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser, which is common to ChS, DS,

HS and heparin (8). In this paper, we showed that the cellulase purified from A. niger has

endo-β-xylosidase activity, which cleaves the Xyl-Ser linkage between the core protein and

the GAG chain of GAG-peptides, in addition to containing endo-β-glucosidase. An endo-β-

xylosidase with similar catalytic activity has been purified from Patinopecten and

characterized (15). Examination of the specificity of the cellulase from A. niger revealed that

1) the enzyme acts on the linkage region (Xyl-Ser) between a core peptide and GAG chains;

2) enzymatic activity is greater with shorter GAG chains; 3) the enzyme readily hydrolyzes

the linkage in GAG-peptides, but intact PG is cleaved only slowly; 4) the activity is

unaffected by the GAG component (ChS, DS and HS). This enzyme was similar to endo-β-

xylosidase from Patinopecten in 1), 3) and 4). However, Patinopecten endo-β-xylosidase

catalyzes hydrolysis of long GAG chains, but short GAG chains are hardly cleaved.

Additionally, the amino acid sequence of endo-β-xylosidase from Patinopecten showed no

homology to that of the cellulases (unpublished data). Taken together with the fact that the

endo-β-xylosidase from Patinopecten had little cellulase activity, these observations show

that the Patinopecten enzyme is different from the cellulase purified in this study.

It is known that some glycosidases recognize both xyloside linkages and glucoside

linkages as a result of the similarity of the configurations of these molecules. An exo-type

glycosidase acting on both p-nitrophenyl β-glucoside and p-nitrophenyl β-xyloside has been

purified from Charonia lampas (22, 23). Moreover, it has been suggested that β-glucosidase

purified from Aspergillus sojae has β-xylosidase activity (24), and a single protein in pig

kidney has both β-xylosidase activity and β-glucosidase activity (25). Also, it has been

reported that addition of Xyl-MU to cultured human skin fibroblasts or Chinese hamster

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ovary cells induces SAα2-3Galβ1-4Xylβ1-MU, which is initiated by β-xyloside as a primer,

rather than the expected SAα2-3Galβ1-4Glcβ1-MU, which is initiated by β-glucose as a

primer (40, 41), suggesting that the galactosyltransferases involved in biosynthesis cannot

distinguish between xylose and glucose. Similarly, cellulase, which is a glucosidase, seems

also to show endo-type β-xylosidase activity.

Endo-β-xylosidase activity was compared with endo-β-glucosidase activity in the

purified cellulase from A. niger. The optimal pH was around 5.0 and the activity curves were

similar for both activities. Hydrolysis by endo-β-xylosidase was slower than that by endo-β-

glucosidase (data not shown). The inhibitory effects of metal ions were similar for endo-β-

xylosidase activity and endo-β-glucosidase activity across the range of 11 reagents examined.

Endo-β-xylosidase activity was slightly inhibited by bivalent cations compared with cellulase

activity. GAG-MU was used as a substrate for assay of endo-β-xylosidase activity. GAG

contains a large number of negative charges such as sulfate groups and carboxyl groups (42).

It is known that bivalent cations, for example Ca2+, can form chelates between the negative

charges of GAG (43). Thus, the configuration of the entire GAG may be altered by binding of

cation, which may have affected enzymatic activity.

The properties of endo-β-xylosidase activity toward natural substrates were investigated.

The optimal pH for, and the effect of metal ions on, hydrolysis of ChS-peptide were similar to

those of hydrolysis of GAG-MU. The enzyme hydrolyzed the Xyl-Ser linkages of ChS-

peptide, DS-peptide and HS-peptide, but not ChS-PG. In addition, the hydrolysis of HS-

peptide was faster than that of ChS-peptide and DS-peptide.

Although GAG chains are attached to serine residues of the core protein via the common

linkage region (8), it has been shown recently that the linkage region is modified by

phosphate and sulfate groups (9-11). Additionally, it has been suggested that the linkage

region can act as a recognition signal for sorting in the biosynthesis of different GAG chains

(11). The linkage regions of the GAG-peptides used in this study did not contain

phosphorylated xylose or sulfated galactose. Therefore, it is unclear whether the enzyme can

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act on substrates containing phosphorylated xylose and/or sulfated galactose, and this

requires further investigation.

Endo-β-xylosidase activity was found in cellulase preparations from A. niger, P.

funiculosum, T. reesei, T. viride and I. lacteus, although not in the preparations from

Eupenicillium sp., P. nigra and P. sativum. Consequently, this activity is not specific for the

cellulase from A. niger. Although the presence or absence of endo-β-xylosidase activity may

be related to the different action patterns of cellulases, for example endolytic or exolytic

cleavage, this is not clear at the present time.

The structural diversity of GAG chains, in terms of sugar chain length, sugar chain

composition and position, and degree of sulfation, is well established. However, all GAG

chains are attached to a xylose residue, which forms an O-glycoside linkage (9). A chemical

method, β-elimination (44), is known to release GAG chains, but may cause their

decomposition by the peeling reaction. On the other hand, endo-β-xylosidase is useful for

analysis of GAG chains, since it specifically releases intact GAG chains from GAG-peptides.

Using this enzyme, it was possible to analyze GAG chains from 50 mg wet weight of animal

tissues with the combined use of PA-labeling at the reducing terminal of the released sugar

chains and HPLC (45). Furthermore, this enzyme is useful in the molar quantification of

GAG chains (45), in giving directivity to GAG chains (46), and in preparing artificial

substrates for novel endo-type enzymes (35). Therefore, we believe that cellulases with endo-

β-xylosidase activity will become useful tools for structural and functional analysis.

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35. Takagaki, K., Nakamura, T., Kawasaki, H., Kon, A., Ohishi, S., and Endo, M. (1990) J.

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Footnotes:

This work was supported by Grants-in-Aid (nos. 09358013, 11121203, 11470029,

12680603 and 12793010) for Scientific Research from the Ministry of Education, Culture,

Sports, Science, and Technology of Japan, and grants from the Aomori Support Center for

Industrial Promotion.

The abbreviations used are: PG, proteoglycan; GAG, glycosaminoglycan; ChS,

chondroitin sulfate; DS, dermatan sulfate; HS, heparan sulfate; Ser, serine; MU, 4-

methylumbelliferone; PA, 2-aminopyridine; HPLC, high-performance liquid

chromatography; PAGE, polyacrylamide gel electrophoresis; GlcA, glucuronic acid; Gal,

galactose; Xyl, xylose; ∆GlcA, D-gluco-4-enepyranosyluronic acid; GalNAc, N-

acetylgalactosamine; SA, sialic acid; IdoA, iduronic acid. All sugars mentioned in this paper

are of D configuration except for iduronic acid.

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FIGURE LEGENDS

Fig. 1. Comparison between endo-β-xylosidase activity and cellulase activity in

cellulase preparations from various origins and in endo-β-xylosidase. Each activity was

measured using 1 unit of each enzyme as defined in Materials under “EXPERIMENTAL

PROCEDURES”. Endo-β-xylosidase activity was measured with 2 µM GAG-MU as

substrate (solid bars), and cellulase activity was measured with 2 µM cellobiose-MU as a

substrate (open bars). The cellulase preparations were derived from A, A. niger; B, P.

funiculosum; C, T. reesei; D, T. viride; E, Eupenicillium sp.; F, I. lacteus; G, P. sativum L.; H,

P. nigra; and I, endo-β-xylosidase from Patinopecten.

Fig. 2. Native PAGE of various cellulase preparations. Each cellulase preparation (A, A.

niger; B, P. funiculosum; C, T. reesei; D, T. viride; E, Eupenicillium sp.; F, I. lacteus) was

separated in 7.5% polyacrylamide gel and stained with Coomassie Brilliant Blue. Duplicate

gels were cut into 5-mm segments, and extracted individually for 24 h with 10 mM Tris-HCl

buffer, pH 7.0, at 4°C, and endo-β-xylosidase activity (solid bars) and cellulase activity (open

bars) were measured in each fraction.

Fig. 3. Native PAGE (A) and SDS-PAGE (B) of the purified cellulase from A. niger.

PAGE and SDS-PAGE were performed in 7.5% and 10% polyacrylamide gel, respectively, at

a constant 20 mA. Proteins were stained with Coomassie Brilliant Blue.

Fig. 4. Analysis by HPLC of GAG-MU (A and B) and cellobiose-MU (C and D) before

and after digestion with the purified cellulase from A. niger. MU derivatives were

analyzed by HPLC before digestion (A and C) and after digestion (B and D) with the purified

cellulase from A. niger. An Ultrasphere ODS column (4.6 mm x 250 mm) was used with a

linear gradient of acetonitrile from 0% to 30% for 50 min at a flow rate of 1.0 ml/min at 30°C.

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For detection, eluates were monitored at excitation and emission wavelengths of 325 and 380

nm. The arrow indicates the elution position of authentic MU.

Fig. 5. Ion spray mass spectra of new fluorogenic products obtained from GAG-MU

and cellobiose-MU by cellulase digestion. GAG-MU (A) and cellobiose-MU (B) were

digested with the purified cellulase from A. niger, and positive-mode mass spectra of the

hydrolysates were analyzed. The spectra were acquired after injection of the samples

dissolved in a mobile phase of 50% methanol.

Fig. 6. HPLC chromatograms of the reducing terminal sugars in the hydrolysates

obtained from GAG-MU and cellobiose-MU by cellulase digestion. GAG-MU (A) and

cellobiose-MU (B) were digested with the purified cellulase from A. niger. The hydrolysates

were fluorescence-labeled with PA and hydrolyzed with 2 M HCl at 100°C for 2 h. After N-

acetylation with acetic anhydride, PA-sugars were subjected to reverse-phase HPLC. An

Ultrasphere ODS column (4.6 mm x✖250 mm) was used with 0.25 M sodium citrate and 1%

acetonitrile at a flow rate of 0.5 ml/min at 30°C. For detection, eluates were monitored at

excitation and emission wavelengths of 320 and 400 nm. The arrows indicate the elution

positions of the PA-monosaccharide standards: 1, PA-glucose; 2, PA-galactose; 3, PA-xylose.

Fig. 7. Effect of pH on the purified cellulase from A. niger. GAG-MU (solid symbols)

and cellobiose-MU (open symbols) were incubated with the purified enzyme (A). ChS-

peptide was incubated with the enzyme (B). The assay method for enzyme activity followed

that described in “EXPERIMENTAL PROCEDURES” except for the composition of the

buffer. The following buffers were used: circles, 0.1 M glycine-HCl buffer; triangles, 0.1 M

sodium acetate buffer; diamonds, 0.1 M sodium phosphate buffer; squares, 0.1 M Tris-HCl

buffer.

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Fig. 8. Time courses of the hydrolysis of natural substrates by the purified cellulase

from A. niger. ChS-PG (open circles) and ChS-peptide (solid circles) were incubated with the

purified enzyme as described under “EXPERIMENTAL PROCEDURES”.

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Masahiko EndoKeiichi Takagaki, Mito Iwafune, Ikuko Kakizaki, Keinosuke Ishido, Yoji Kato and

glycosaminoglycan chain by cellulasesThe cleavage of the xylosyl serine linkage between a core peptide and a

published online February 27, 2002J. Biol. Chem. 

  10.1074/jbc.M111985200Access the most updated version of this article at doi:

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