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Novel β-D-Galactofuranose-containing High-mannoseType Oligosaccharides in Ascorbate Oxidase fromAcremonium sp. HI-25Ohta Masayaa, Emi Shinichia, Iwamoto Hiroyukiab, Hirose Junzoab, Hiromi Keitaroab, ItohHomareabc, Shin Takashiabc, Murao Sawaoabc & Matsuura Fumitoa

a Department of Biotechnology, Fukuyama University, Fukuyama, Hiroshima 729-02,Japanb Department of Food Science and Technology, Faculty of Engineering, FukuyamaUniversity, Fukuyama, Hiroshima 729-02, Japanc Department of Applied Microbial Technology, The Kumamoto Institute of Technology,Ikeda 4–22–1, Kumamoto 860, JapanPublished online: 12 Jun 2014.

To cite this article: Ohta Masaya, Emi Shinichi, Iwamoto Hiroyuki, Hirose Junzo, Hiromi Keitaro, Itoh Homare,Shin Takashi, Murao Sawao & Matsuura Fumito (1996) Novel β-D-Galactofuranose-containing High-mannose TypeOligosaccharides in Ascorbate Oxidase from Acremonium sp. HI-25, Bioscience, Biotechnology, and Biochemistry, 60:7,1123-1130, DOI: 10.1271/bbb.60.1123

To link to this article: http://dx.doi.org/10.1271/bbb.60.1123

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Biosci. Biotech. BiocllclII., 60 (7), 1123 I 130, 1996

Novel fJ-o-Galactofuranose-containing High-mannose Type Oligosaccharides in Ascorbate Oxidase from Acremonium sp. HI-25

Masaya OHTA, Shinichi EMI, Hiroyuki IWAMOTO,* Junzo HIROSE,* Keitaro HIROMI,* Homare IToH,** Takashi SHIN,** Sawao MURAO,** and Fumito MATSUURAt

Department of Biotechnology. and * Department (~l Food Science and Technology. Faculty (~l Engineering, Fukuyama (Jllirersity, Fukuyama, Hiroshima 729--02, Japan ** Department (~l Applied Alicrohial Technology. The Kumamoto Institute q( Technology, Ikeda 4--22--/, Kumamoto 86(), Japan Received January 16, 1996

Ascorbate oxidase from the fungus Acremonium sp. HI-25 is a copper-containing glycoprotein that catalyzes the oxidation of ascorbic acid to dehydroascorbic acid. Monosaccharide composition analysis showed that the enzyme contains exclusively N-linked oligosaccharide chains. Following liberation by hydrazinolysis/re-N-acetylation, and fractionation by HPLC on anion exchange, Amide-80 and/or octa­decyl silica columns after derivatization with p-aminobenzoic ethyl ester, the structures of the twelve major neutral oligosaccharides were identified by F AB-MS, 400 MHz 1 H-NMR, methylation analysis, mild acid hydrolysis, and/or sequential exoglycosidase digestions. A cremonium sp. ascorbate oxidase was found to consist of high-mannose type oligosaccharides (76.3 41., ) having 4 to 9 mannose residues and a series of novel o-galactofuranose-containing high-mannose type oligosaccharides (18.6%) with the following structure.

Gal/pI 1 ± Man~1 ~ 2Man~1 ~ 6 2

3Man~1 ~ 6 Man~1 /' 3Man/H ~4GIcNAcpl ~4GIcNAc

(Man~ I ~ 2)() _ 2 Man~ I /'

Ke)' words: ascorbate oxidase; Acremolliul11; N-linked oligosaccharide; galactofuranose-containing oligosaccharide

Ascorbate oxidase [EC 1.1 0.3.3J (AOD), which catalyzes the oxidation of ascorbic acid to dehydroascorbic acid, is an enzyme widely found in higher plants, such as squash I)

and cucumber. 2) Ascorbate oxidase is used as an analytical reagent for clinical and food analyses of ascorbate. How­ever, since ascorbate oxidase from cucumber, which is most commonly used as an analytical reagent, is unstable, a more stable ascorbate oxidase is required.

Efforts to find microorganisms producing ascorbate ox­idase have been made. 3

- 5) Although the occurrence of ascorbate oxidase in microorganisms has been reported, i.e., in Alyrothecium l'eITlICaria 3A

) and Aerohacter aerogenes, 5)

the enzyme has not been purified or characterized. Recently, M unto el al. O

) succeeded in isolating a strain, identified as Acremoniul1l sp. HI-25, producing an ascorbate oxidase. and purified it with no laccase activity. The ascorbate ox­idase from AcremOlliul1l sp. is a monomeric glycoprotein with a molecular mass of 80 kDa containing four copper atoms per molecule. 6

) while the ascorbate oxidases from cucumber and squash are dimeric glycoproteins with mo­lecular masses of 130--140 kDa and containing eight copper atoms per molecule. 7

,8) Hirose et al. 9) reported that the coordination structures of types I and 2 copper atoms in ascorbate oxidase from Acre1110nium sp. are very similar to

t To \vhom correspondence should be addressed.

those in the cucumber enzyme. Furthermore, they have shown that the apparent Km and kcat for ascorbic acid of the ascorbate oxidase from Acremoniul11 sp, are closely similar to those of the monomeric unit of the ascorbate oxidase isolated from cucumber.

The primary structures of the ascorbate oxidases from cucumber and squash have been identified by eDNA clon­ing,7,lO) The amino acid sequences deduced from the nu­cleotide sequences of the cDNAs for the squash and cu­cumber enzymes had 80% identity, with four and three pos­sible N-glycosylation sites, respectively, D'Andrea et al. 81

analyzed the primary structures of the N-linked sugar chains of ascorbate oxidase of squash to be Man:(1 ~6(Xyl/3I ~ 2)(Man:(1 ~3)ManIJl ~4GIcNAc{J~4GIcNAc. However, no report is available on the primary structure of Acre­l11oniul11 sp, ascorbate oxidase. Glycosylation is knO\vn to be required for stability, catalytic activity, secretion, and so on, Thus, identification of the sugar chain structures as well as the amino acid sequences of microorganism ascorbate oxidases may be valuable.

In this paper, we report the structures of N-linked oligo­saccharides of the ascorbate oxidase from Acremoniul11 sp, and the presence of novel galactofuranose-containing high­mannose type oligosaccharides.

Ahhreriatiofl.l: AOO, ascorbate oxidase; ABEE, p-aminohenzoic acid ethyl ester; Man, man nose: GkNAc, lV-acetylglucosamine: Hex, hexose: HexNAc. iV-acetylhexosamine: Gall D-galactofuranose: 20-COSY, tw'o-dimcnsional chemical-shift correlated spectroscopy.

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1124 M. OUTA et al.

Materials and Methods Ascorhate oxidase. AOD was isolated from culture broth of Acremonium

sp. HI-25 by the method of Murao et 01. 6)

Chel11 ica !.I' and ('11::.1'111('.1. L-( + )-2-0ctanol and n-galactose were pur­chased from Wako Pure Chemical Industries Ltd .. Japan. L-Galactose was purchased from Sigma Chemical Co .. U,S.A. StrcptomyC£'s gricclls endo-/i-iV-acetylglucosaminidase H was obtained from Seikagaku Cor­poration, Japan. Jack bean meal l-mannosidase. fJ-galactosidase. and /i­iV-acetylhexosaminidase were prepared by modifications of the published methods. III A.lpergillus .I'aitoi l-mannosidase I \vas purified by the method of Amano and Kobata. III Methyl-galactoside of the furanose form \vas prepared by heating D-galactose \\'ith 0.015°!) methanolic HCI at gO C for 6 h. Unless specified. all other chemicals and chromatographic solvents were purchased from Wako Pure Chemical Industries.

Standard oligosaccharides. The following standard oligosaccharides tagged with p-aminobenzoic ethyl ester (ABEE) were prepared as describ­ed previouslyIJ~151: ManfJl~4GIcNAcfJl~4GlcNAc-ABEE (Man l-GIcNAc 2 -ABEE). Man,d ~6Man/JI->4GIcNAc/JI ~4GIcNAc-ABEE (Man 2 GIcNAc l -ABEE). Man,ll ~6(Man:xl-d)ManfJl ~4GIcNAc­/JI->4GIcNAc-ABEE (Man3GIcNAcrABEE). ManIXI->6(ManIXI->3)­Manll ~6(ManIXI->3)ManfJl ~4GlcNAcfJl->4GIcNAc-ABEE (Man s-GIc:-.JAc 2-ABEE). Man,ll ->6(ManIXI--.3)ManlXl ~6(ManlXl ~2ManIXI--. 3)Man/JI ->4GIcNAcfJl ~4GIcNAc-ABEE (Man{,GIcNAc 2 -ABEE), and (Manxl->2)1 ~ 3Man6GlcNAcrABEE (Man7~()GIcNAc2-ABEE). Man­ll->3ManIXI->6(Manll ->3)Man/JI->4GIcNAc/JI->4GIcNAc-ABEE (Man4GIcNAc2-ABEE) was obtained with recombinant human IX-galac­tosidase A (M, Ohta ('t al., manuscript submitted).

The follmving high-mannose type oligosaccharides having a single iV­acetylglucosamine residue at their reducing end were isolated from urine of patients with IX-mannosidosis. 161 and their structures were analyzed by I H-NMR. ManIXI--.3Manll ~6(Manll->3)ManfJl--.4GIcNAc-ABEE (Man 4GIcNAc l-ABEE). ManlXl ~6(Manll->3)Man'll ~6(ManlXl --.3)­Man/JI ~4GIcNAc-ABEE (Man,GIcNAcl-ABEE). Manll ~2Manll-> 6(Manll ~3)Manll ~6(Man:xI->3)Man/JI ~4GIcNAc-ABEE (Man 6-

GIcNAcl-I-ABEE). ManIXI->6(ManIXI ->3)Manll --.6(ManIXI->2Man­IXI->3)Man/JI --.4GIcNAc-ABEE (Man/lGIcNAc l-II-ABEE). ManlXl ~6-(Manxl ~2Manll->3)Manxl->6(Manxl ~3)Man/JI --.4GIcNAc-ABEE (Man6GIcNAc l-III-ABEE). Manll--.2Manll->6(ManIXI--.3)Manll ~ 6(Manll->2ManIXI->3)Man/i1->4GIcNAc-ABEE (Man 7GIcNAc l-I­ABEE). Manxl->6(ManlXl ~3)ManIXI->6(Manll->2ManlXl ~2ManIXI--. 3)Man/JI ~4GlcNAc-ABEE (Man.,GIcNAcl-II-ABEE). ManIXI--.6(Man­xl ~2Man:xI->3)Manxl ~6(Man:xI--.2Manxl--.3)Man/J1->4GIcNAc­ABEE (Man.,GIcNAc l-I1I-ABEE). Manxl--.2Manll->6(Manll->3)­Manll--.6(Manxl->2Manxl->2Manxl->3)MantJl->4GIcNAc-ABEE (MansGIcNAcl-I-ABEE). Man'll--.2Manxl->6(Manxl--.2Manll->3)­ManlXI ->6(Man:xI->2Manll ->3)Man/JI ->4GIcNAc-ABEE (Man H-

GIcNAc l-II-ABEE). ManIXI--.6(ManIXI--.2Manll--.3)ManlXl --.6(Man­IXI ->2Mam:1 ->2ManIXI ->3)Man/JI--.4GIcNAc-ABEE (Man 8 GIcNAc l-I1I-ABEE). and Man'll ->2Man:x1 ->6(ManIXI->2Man'll->3)Manll->6-(Manll --.2ManIXI->2Manll->3)Man/JI ->4GIcNAc-ABEE (Man 4 -

GIcNAcl-ABEE).

Liheration oj' ,I\/-linked oligosaccharidcs and preparation oj' A BEE deriwl­til'es. lV-Linked oligosaccharides were liberated from 50 mg of AOD by hydrazinolysisire-jV-acetylation as described. I i) The liberated oligosac­charides were tagged with ABEE by reductive amination. and then purified by chromatography first on a PRE-SEP CI8 cartridge (Tessek. U.S.A.). followed by on a Bio-Gel P-4 column (200-400 mesh. 1.0 x 45 cm). as described previously.14.1"1

I/PLC oj' ABEE-o/igosaccharides. HPLC \vas done with a Shimadzu LC 6A HPLC system. Anion-exchange HPLC was done on a TSKgel DEAE-5PW column (cjJ 0.75 x 7.5cm. Tosoh Co., Japan) as described previously. I 8) Fractionation of neutral ABEE-oligosaccharides was done either on a TSKgel Amide-XO column (cjJ 0.46 x 25cm. Tosoh Co.) or a Wakosil 5C18-200 column (cjJ 0.4 x 25 cm. Wako Pure Chemical Industries) under the reported conditions. 14, 151 T .... 'o-dimensional mapping analysis (20-HPLC) of the individual ABEE-oligosaccharides was done as de­scribed previously. I 5. I'll

Monosaccharide analysis. Monosaccharide compositions were analyzed by gas liquid chromatography (GLC) of trimethylsilyl methyl glycosides

obtained by methanolysis of native AOD in 1.5 M methanolic HCI. 20) The absolute configurations of monosaccharides were identified by GLC of trimethylsilyl L-2-octyl glycosides according to the method of Takano et al. 211 G LC of the trimethylsilyl methyl glycosides and trimethylsilyl L-2-octyl glycosides was done on a OB-5 fused silica capillary column (0.32 mm x 25 m, J & W Scientific. U.S.A.) in a Hewlett-Packard 5840A gas chromatograph. The column temperature was programmed from 50C to 170 C at 20 Cmin. and then to 250C at J Cimino

Methylation analysis. ABEE-oligosaccharides were permethylated by the method of Ciucanu and Kerek. 22

) Each methylated sample was hy­drolyzed. reduced. and acetylated by the method of Liang el al.. 23 ) and the resulting partially methylated alditol acetates were analyzed by gas chromatography-mass spectrometry (GCiMS QP-I 000. Shimadzu Corp .. Japan) as previously described. 141 Authentic IA-di-O-acetyl-2.3.5.6-tetra­O-methylgalactitol was prepared from methyl-galactofuranoside in a sim­ilar manner to that described above.

Digestion with en:ymes. Digestion of ABEE-oligosaccharides with jack bean l-mannosidase, jack bean fJ-galactosidase. jack bean fJ-iV-acetyl­hexosaminidase. and A, saitoi rx-mannosidase L respectively, was done as described, 141 Digestion with endo-fJ-lV-acetylglucosaminidase H was done as described by Mizuochi el 01. 24)

Partial acid hydrolysis. Mild acid hydrolysis of ABEE-oligosaccharides. to hydrolyze galactofuranoside linkages but not hexopyranosidic linkages. was done in 0.01 N HCI (100e. 30 min). Under these conditions. about 60(~/o of the galactofuranoside (Gal/) residues were selectively hydrolyzed without degradation of the ester linkages in ABEE. Treatment with 0.02 N

HCI at 100C for 150 min was also done to hydrolyze galactofuranosidic linkages in free N-linked oligosaccharides. which were prepared from AOD by hydrazinolysis. 251 The reaction mixture after mild acid hydrolysis was evaporated to dryness under reduced pressure and then the residue was repeatedly evaporated with water. The residue was then tagged with ABEE and analyzed by 2D-HPLC.

Analysis of the isomeric structures (~lhigh-m(lnnose Iype oligvsacc!llIridi's. We recently found that separation of the isomeric structures of high­mannose type oligosaccharides with the same number of mannose residues can be done by 20-HPLC analysis of ABEE derivatives of oligosac­charides with a single GIcNAc residue at their reducing end (unpublished results). Thus. in this study, ABEE-derivatives of high-man nose type oligosaccharides having a chitobiose structure at their reducing terminus were first digested with endo-/J-N-acetylglucosaminidase H. The resulting counterparts having a single GIcNAc residue at the reducing terminus were analyzed by 2D-HPLC after ABEE derivatization.

40() Mil: III-,VM R ,Ipectrometry. ABEE-oligosaccharides were repeat­edly exchanged in 2H 20 (99.96% atom% 2H). with intermediate lyophi­lization. The I H-NMR spectra were recorded with a Bruker AM 400 spectrometer operating at 400 MHz. with a probe temperature of 40C. Chemical shifts (<5) are expressed in ppm relative to internal acetone in 2H zO (<5=2.221 ppm).

Fast-alom homhardment mass ,Ipeclrometry. Positive ion fast-atom bom­bardment mass spectra (FAB-MS) were obtained with a JEOL JMS-HX 100 mass spectrometer as described elsewhere. 141

Results Monosaccharide analysis

Monosaccharide analysis of AOD showed that the enzyme contained man nose, galactose, and N-acetylglucosamine in a molar ratio of 7.2: 3,4 : 1.0, suggesting that the enzyme consists of high-mannose type and unique galactose-con­taining high-mannose type oligosaccharides. On the basis of the carbohydrate content (10 % (w/w)) and the molecular mass of the enzyme, it was estimated that AOD had four sugar chains per molecule.

Pattern of N-Iinked oligosaccharides in A OD Oligosaccharides were liberated from AOD by hydra­

zinolysis.ire-N-acetylation and then labeled with ABEE.

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N-Linked Sugar Chains of Acremonium sp. Ascorbate Oxidase 1125

Upon anion-exchange HPLC on a OEAE-5PW column, the ABEE-oligosaccharides were separated into neutral (frac­tion ~h and acidic fractions (fraction AI) in the molar ratio of 88.5 : 11.5 (Fig. 1). The elution position of AI cor­responded to monophosphoryl high-mannose type oligo­saccharides. However, no further structural analysis of this fraction was done due to the limited amounts of the sample. In this study, structural analysis of the oligo saccharides in fraction N was done. Upon size fractionation HPLC on an Amide-80 column, fraction N gave rise to twelve peaks, denoted as Na-NI, as shown in Fig. 2. The relative pro­portions of the fractions were: Na, 13.1 %; Nb, 2.4%; Nc, 4.9%; Nd, 2.1 %; Ne, 29.5%; Nf, to.3 % ; Ng, 16.0%; Nh, 2.5%; Ni, 7.0%; Nj, 1.4%; Nk, 7.1 %; and NI, 3.6%. Monosaccharide analysis of each fraction showed that Na, Ne, Ng, Ni, Nk, and NI consisted of only man nose and N-acetylglucosamine, and fractions Nc, Nf, Nh, and Nj of

200 E

N ~ c:: .§. v 0 <t M .' 0 - 150 a..

N ctI :x: UJ nI

Z u '0 z .' .' <{ 100 z CD .' 0 a:

AI ~ 0 a: en I-CD 50 z <{ w > u

I~--~'" z

:::J 0 U

I I

0 10 20 30 40 50 RETENTION TIME (min)

Fig. 1. Ion-exchange HPLC of ABEE-oligosaccharides Obtained from AOO.

!\'-Linked oligosaccharides were released from AOD by hydrazinolysis and then tagged with ABEE. The ABEE-oligosaccharides were put on a column (0.75 x 7.5cm) of TSKgel DEAE-5PW. The column was eluted isocratically with 10m\t NaH zP04

for 10 min. and then with a linear gradient from the same butfer composition to 170 m'<1 NaH zP04 over 40 min at a flow rate 0.5 ml.min. The fractions indicated b) the bars v.cre pooled. The elution positions of fractions :'I and AI corresponded to those of the ABEE derivatives of neutral and monophosphoryl oligosaccharides. respectively.

E r::: v o M -ItS

4 5 6 i ~ ~

e

a 9

7 9 ~ ~

UJ U z <2: CO a: o en CO <2: ~ ~}~----I-------J

~\k I 1.

o 10 20 30 40 50 RETENTION TIME (min)

Fig. 2. Amide-80 HPLC Analysis of the Neutral ABEE-oligosaccharides in Fraction N.

Fraction N in Fig. I was further separated by HPLC on a TSKgel Amide-80 column (0.46 x 25 cm) equilibrated with a 80: 20 mixture of solvent A (acetonitrile water (9: I» and solvent B (acetonitrile water (I: 9». After injection of the sample. elution was done with a linear gradient to a ratio of solvent A: B of 50: 50 over 60 min at a flow rate of 0.8 m1.imin at 40 C. Twelve fractions (Na NI) were collected. Arrows ( i ) indicate the elution positions of standard ABEE-oligosaccharides; I. Man 1 GkNAcz-ABEE; 2. ManzGk:-.lAcrABEE; 3, Man,GkNAcz-ABEE; 4. Man4 GkNAcz-ABEE; 5. Man,GlcNAc 2-ABEE; 6. Man(,GlcNAc 2-ABEE; 7. ManoGkNAcz-ABEE; S. ManHGkNAcz-ABEE: and 9. Man'lGlcNAcrABEE.

galactose, mannose, and N-acetylglucosamine, suggesting that the former group was of the high-mannose type and the latter of a galactose-containing high-mannose type.

Struclures of high-mannose t.lpe oligos([ccharide,\' Upon two-dimensional mapping analysis, fractions Na,

Ne, Ng, Ni, Nk, and NI were eluted at the positions of standard Man4 G1cNAc 2-ABEE, Man sG1cNAc 2-ABEE, Man6G1cNAc2-ABEE, Man7GlcNAcrABEE, Man H-

GlcNAc2-ABEE, and Man q GlcNAc2-ABEE, respectively (Fig. 3). These oligosaccharides were further characterized by sequential exoglycosidase digestion coupled with 20-HPLC analysis. When fractions Ng, Ni, Nk, and NI were digested with A. saitoi ~-mannosidase I, they each gave a single peak, with the same mobility as Ne, that comigrated with standard Man sGlcNAc2-ABEE (Fig. 3). Subsequent treatment of the product and Ne with jack bean ~-man­nosidase produced Man 1GlcNAc2-ABEE. ABEE-oligosac­charide Na was stable as to A. saitoi ~-mannosidase I digestion, but converted to Man 1GlcNAc2-ABEE on jack bean~-mannosidase digestion. On the basis of these re­sults together with their behavior on 20-HPLC analysis, these oligosaccharides were established to be typical high­mannose type oligosaccharides with four to nine mannose residues, as shown in Table II.

Further characterization of the predominant oligosac­charides, Na, Ne, and Ng, was done by 400 MHz 1 H­NMR analysis. The chemical shift values of structural reporter-group protons (Table I) were compared with those previously reported. 26

.27 ) Each fraction was found to con-

11

10 (g). NI ., E 9 (f),.'..

Nk ' .. u ' . It)

8 (e), Ni<:::':. C\I )(

CDUl v:!:: 7 (d). Ng .:::::.

'r::: 8.:::l ' .......

0<11 6 COUl '0 :2g

5 E-<{~ Qj 4 Cl

::.:: en 3 t-(a)

2

1 0.5 0.7 0.9 1.1 1.3 1.5

Relative retention time as to Glc-ABEE Wakosil 5C18-200 (0.4 x 25 cm)

Fig. 3. TViO-Dimensional Mapping Analysis of the High-mannosc Type ABEE-oligosaccharides and Their Digestion Products with Various Gly­cosidases.

Each ABEE-oligosaccharide was analyzed by HPLC on TSKgel Amide-XO and Wakosil 5CIX-200 columns. The elution positions of ABEE-oligosaccharides from the Amide-SO column are expressed as the numbers of glucose units. and from the ODS column as the relative retention times as to glucose-ABEE. Amide-HPLC was done as in Fig. 2. The Wakosil 5CIS-200 (0,4 x 25cm) column was eluted with a linear gradient from 9°·() acetonitrile in 100 mM acetic acid to II () () acetonitrile in 100mM acetic acid over 60min at a flow rate of O.8mlmin at 40 C. Arrows (---.) and ( .) indicate A. sai/oi :t-mannosidase I and jack hean J(-mannosidase digestion. respectively. a. b. c. d. e. f. and g indicate the elution positions of authentic ABEl'­oligosaccharides Man I GlcNAc c-ABEE. Man .. GlcNAc c-ABEE. Man,GlcNAc 2 -

ABEE. Man h GlcNAc 2-ABEE. Man,GlcNAc 2-ABEE. Man HGlcNAc 2 -ABEE. and Man q GlcNAcz-ABEF. respectively.

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Table I. I H-Chemical Shifts of Structural-reporter-groups for the ABEE-derivatives of Oligosaccharides

Chemical shifts (ppm) in

Reporter group

Residue Na u Ne

Man 4 GN 2-ABEE Man,GNrABEE l'g

Man h GN 2 -ABEE

Nf GalfMansGN 2-

ABEE

Peak X GalfMan 2GN 1-

ABEE

H-I

H-2

GIcNAc-2 Man-3 Man-4 Man-4' Man-A Man-B Man-C

Galf GIcNAc-1 Man-3 Man-4 Man-4' Man-A Man-B Man-C

Galf NAc GIcNAc-1

GIcNAc-2

U See Fig. 2.

NO" 4.760 5.109 4.X97 5.109

4.333 4.215 4.074 4.127 4.074

1.919

2.056

NO 4.765 5.105 4.X70 5.096 4.90X

4.332 4.224 4.075 4.140 4.064 3.9XX

1.91 g

2.054

NO NO 4.756 4.762 5.341 5.106 4.X71 5.035 5.096 5.150 4.90X 4.919 5.055

5.120 4.331 4.333 4.215 4.225 4.116 4.075 4.140 4.137 4.067 4.065 3.9XX 3.9XX 4.067

4.105 1.917 1.919

2.052 2.052

NO 4.751

5.045

5.105 4.332 4.073

4.062

4.136 1.917

2.053

4.625 4.752

4.915

4.332 4.067

3.970

1.920

2.053

h The reference compound obtained from bovine fJ-mannosidosis kidney.l.ll , Value could not be determined.

tain a single component based on the relative intensities of anomeric proton signals (data not shown). The assign­ments given in Table I are based on comparison with appropriate oligosaccharides. t4.26.27) The structural re­porter-group signals of oligosaccharide Ne completely match those of the corresponding residues in Man:x 1-+6-(Man,:x1-+3)Man:xI-+6(Man:xI-+3)ManfJl -+4GIcNAc­fJl-+4GlcNAc-ABEE. In the spectrum ofNa. the H-I signal for Man-4' showed a significant downfield shift and H-2 for Man-4' an upfield shift as compared to those for the corresponding residues in Ne, indicating the deletion of a Maned -+6 (Man-B) residue from Ne. Thus, oligosaccharide Na was identified as Man:xI-+3Man:xI-+6(Man:xI-+3)Man­{JI-+4GlcNAcfJl-+4GlcNAc-ABEE (Table II). As to oligo­saccharide Ng, the H-I and H-2 signals for Man-4 under­went downfield shifts characteristic of the introduction of a Man-C into the :x-( 1-+ 2)-linkage at the C -2 position of oligosaccharide Ne. Furthermore, Man-C is characterized by its H-I «)=5.055ppm) and H-2 «5=4.067ppm) signals. Thus, the structure of Ng was deduced to be as shown in Table II.

Since the amounts of Ni, Nk, and Nt were too low for t H-NMR, their structures were further analyzed by the 2D-HPLC technique after their conversion to ABEE-oligo­saccharides with a single GlcNAc residue at their reducing end. The endo-fJ-N-acetylglucosaminidase H digest of frac­tion Ni gave two peaks corresponding to Man:x 1-+ 2Man­:x1-+6(Man:xI-+3)Man:xI-+6(Man:xI-+2Man:xI-+3)Man­f31-+4GlcNAc-ABEE (Man 7GlcNAc t-I-ABEE) and Man­:x1-+6(Man:xI-+3)Man:xI-+6(Man:xI-+2Man:xI-+2Man­:x1-+3)ManfJI-+4GlcNAc-ABEE (Man 7GlcNAc t -III­ABEE) obtained from :x-mannosidosis urine (data not shown) in a molar ratio of approximately 2: 1 (Table II). The endo-fJ-N-acetylglucosaminidase H digests of Nk and Nt each gave a single peak corresponding to Man:xI-+2-

Man:xI-+6(Man:xI-+3)Man,:x1-+6(Man,:x1-+2Man:xI-+2-Man:xI-+3)ManfJI -+4GlcNAc-ABEE and Man:xI-+2-Man:xI-+6(Man,:x1-+2Man:xI-+3)Man:xI-+6(Man,:x1-+2-Man:xI-+2Man:xI-+3)ManfJI-+4GIcNAc-ABEE, respec­tively. These results established the structures of fractions Nk and Nt to be as shown in Table II.

Structures (~l galactose-containing high-mannose type oligo­saccharides Positive ion FAB-MS of intact Nc, Nf, Nh, and Nj,

which contained galactose in addition to mannose and N­acetylglucosamine. had [M + Na] 1- ions at mi~ 1406. cor­responding to HexsHexNAc2-ABEE, m/~ 1568, Hex 6 Hex­NAc2-ABEE, m//~ 1730, Hex7HexNAcrABEE, and m/~ 1892. HexsHexNAc 2-ABEE. respectively. When Nc, Nf. Nh, and Nj were digested \vith jack bean :x-mannosidase. they released 2, 3. 4. and 5 mannose residues. respectively. and gave an ABEE-oligosaccharide with the same mobility (oligosaccharide X; Fig. 4). These results suggested that the galactose-containing oligosaccharides had a common core structure. Positive ion FAB-MS and monosaccharide analysis indicated that oligosaccharide X had the composi­tion, Galt Man 2GlcNAc-GlcNAc-ABEE (data not shown). Methylation analysis showed 2,3,5,6-tetra-O-methvl­galactitol, 3,4,6-tri-O-methylmannitol, 2,3,4-tri-O-meth'yl­mannitol, and 3,6-di-O-methyl-2-N-methylacetamido-2-deoxyglucitol in a molar ratio of I : I : 1 : I, suggesting that the galactose residue was of the furanose form and linked to the C-2 position of the nonreducing terminal mannose residue of Man:xI-+6Manf3I-+4GlcNAcf3I-+4GlcNAc­ABEE. To examine the presence of a hexofuranosyllinkage. which is more acid-labile than a hexopyranose linkage, oligosaccharide X was hydrolyzed with mild acid and then the hydrolyzate was analyzed by HPLC. Since mild acid hy­drolysis under the reported conditions (0.02 N HCI, 100 C,

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N-Linked Sugar Chains of Acremonium sp. Ascorbate Oxidase 1127

10

9

E 8 (J

In N 7 )(

co 1/1 ..:r:'!::

e.§6 OQl co 1/1

.g8 5 '- ::J e-<"4

2

1 0.5

Nj

(a)

0.7 0.9 1.1 1.3

Relative retention time as to Gle-ABEE Wakosil 5C18-200 (0.4 x 25 em)

1.5

Fig. 4. Two-Dimensional Mapping Analysis of the Monogalactosylated High-mannose Type ABEE-oligosaccharides and Their Products Obtained on cx-Mannosidase Digestion and Mild Acid Hydrolysis.

Each ABEE-oligosaccharide was analyzed by HPLC on TSKgel Amide-80 and Wakosil 5C18-20 columns. under the conditions given in Fig. 3. Arrows ( .) and (=) indicate jack bean :x-mannosidase digestion and mild acid hvdrolvsis (0.01 N

HC!. 100 C. 30min). respectively. a. b. c. d. e. and f indicate the ~lutio;l positions or authentic ABEE-oligosaccharides Man1GlcNAI.:;!-ABEE. Man1GkNAI.:;!-ABEE. \1an~GkNAI.:;!-ABEE. Man,GkNAI.::-ABEE. Man"GkNAc 2-ABEE. and Man e-GkNAI.: 2-ABEE. respedively.

150 min)2S) cleaved the ester linkage present in ABEE as well as the galactofuranose linkages, hydrolysis under milder conditions (0.01 N HC!, 1 OOJC, 30 min) was done for ABEE­oligosaccharide X. As shown in Fig. 4, approximately 60% of X was converted to an ABEE-oligosaccharide comigrat­ing with authentic Man:xI-6Manpl-4GlcNAcpl-4Glc­NAc-ABEE.

In a separate experiment, free oligosaccharides prepared from AOD by hydrazinolysis were hydrolyzed with mild acid under the reported conditions (0.02 N HCL 100'lC, 150 min), and the hydrolyzate was derivatized with ABEE before HPLC analysis. Peaks Nc, Nf, Nh, and Nj in Fig. 2 were completely converted to Na, Ne, Ng, and Ni, re­spectively. It was, therefore, concluded that galactose was in the furanose form.

To identify the absolute configuration, free galactose obtained on mild acid hydrolysis was analyzed by GLC as the trimethylsilyl L-2-octyl glycoside. It gave a single peak coeluting with standard trimethylsilyl L-2-octyl-D-galacto­side, indicating the configuration of the galactose to be D.

To identify the anomeric configuration of the galacto­furanse residue, oligosaccharide X was analyzed by 1 H­NMR (Fig. 5). Relevant H-l and H-2 chemical shifts for this oligosaccharide are listed in Table I together with the reference compound. The spectrum contained three anomeric proton signals of equal intensity, pointing to a single component. The H-I signal for GlcNAc-l was not recognizable due to the overlapping with H 20. The H-l signal at c5=4.751 ppm and the H-2 signal at c5=4.073 ppm are indicative of the presence of f3-linked Man (Man-3) substituted at its C-6 position (Table I). The signals at c5 = 5.045 ppm (11.2 < 1.8 Hz) and c5 = 5.105 ppm (11.2 = 2.0 Hz) can therefore be ascribed to H-I of either Man-4' or Gall

5.50

Gal( ~ 1 .J, 2

Ma;<l\':,1 S Man~1-4GlcNAc~1-4GlcNAc _ AIlEE 3

H-1

Galt 4' \ I

3

\

H-2

3- 4' 1, CH3 I

iii

4.00

i I 3.50 ppm

3.50 ppm

Fig. 5. Structural-reporter-group Regions of 400 MHz 1 H-NMR Spectra of the ABEE-oligosaccharides in Peaks Nf and X.

Monosal.:l.:haride residues are numbered the same as those in Table I. Spectra were recorded in 2H 1 0 solution at 40 C. Chemical shifts are expressed in ppm with referenl.:e to internal al.:etone (c5 = 2.221 ppm).

2D-COSY NMR analysis of oligosaccharide X showed a cross-peak between the signals at c5 = 5.045 ppm and c5 = 4.062 ppm. It had been reported that C-2 substitution of the non reducing terminal mannose residue (Man-A) of Man 6 _ 8GlcNAc2 with et-galactofuranose or et-mannose gave rise to a large downfield shift (,1c5 = 0.42 ppm) of the H-l for a mannose residue. 26 - 29) However. no such large shift was observed on the C-2 substitution of the non­reducing terminal man nose residue (Man-4') of Man 2-GlcNAc2 by a galactofuranose residue (Table I), suggesting the anomeric form of Galfto be {J. Substitution at C-2 of Man-4 or 4' by a f3-GlcNAc residue in complex type oligo­saccharides causes only small increases in the shifts for their H-l atoms (,1c5=0.01 1-0.015 ppm), but significant increases for their H-2 atoms (,1c5 = 0.12-0.14 ppm). 30) In this in­stance, the signals at c5 = 5.045 ppm and c5 = 4.062 ppm are tentatively assigned to H-I and H-2 for Man-4' substituted at C-2 by f3-GalJ: respectively, as compared to those for the corresponding residues in Man 2GlcNAc2. The signal at c5 = 5.105 ppm can. therefore, be assigned to H-I for the Galf residue. The coupling constant value of H-l for the Galf residue (11.2 = 2.0 Hz) is close to that of methyl {1-galactofuranoside (11.2 = 2.0 Hz), but different from those of methyl :x-galactofuranoside (J 1.2 = 3.7 Hz) and galacto­furanose residues linked to Man-A in various high-mannose type oligosaccharides (11.2 =4.7 HZ).28.29, Taking these ob­servations into account, the anomeric configuration of ga­lactofuranose is proposed to be {1. However. because the

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1128 M. OInA 1.'1 al.

galactofuranose residue may occur in several possible ring conformations. this assignment is tentative in the absence of an authentic standard. On the basis of these results. the common core oligosaccharide present in all galactose­containing oligosaccharides was characterized as Galfpl-2Man~I-6Man/Jl-4GIcNAc/Jl-4GIcNAc-ABEE.

The most prominent oligosaccharide, Nf (GalfMansGlc­NAc2), was resistant to A. sailoi ~-mannosidase I diges­tion. Upon mild acid hydrolysis, this oligosaccharide pro­duced an ABEE-oligosaccharide comigrating with authentic Man sGlcNAc 2-ABEE (Fig. 4), with the release of galac­tofuranose. The degalactosylated oligosaccharide released four mannose residues on digestion with jack bean~-man­nosidase to produce Man IGlcNAc2-ABEE, confirming that the degalactosylated oligosaccharide was a high-man nose type oligosaccharide with five mannose residues. Methyla­tion analysis of Nf showed the presence of 3 mol of 2.3,4.6-tetra-O-methylmannitoL I mol of 2,3,5,6-tetra-0-methyl­glactitol, I mol of 2,4-di-0-methylmannitoL I mol of 4-mono-O-methylmannitol, and I mol of 3.6-di-0-methyl-2-N-methylacetamido-2-deoxyglucitol. From these results to­gether with the results for oligosaccharide X, the structure ofNfwas proposed to be Man,~1-6(Man~I-3)(Galf/Jl-2)Man~I-6(Man~I-3)Man/JI -4GlcNAc/JI -4Glc­NAc-ABEE. 1 H-NMR of Nf is shown in Fig. 5. Six an­omeric proton signals of equal intensity were observed. The three anomeric proton signals at b = 4.762 ppm, b =

5.106ppm, and b=4.919ppm can be assigned to H-I of Man-3, Man-4, and Man-B, respectively, as compared to those of authentic MansGlcNAc2-ABEE. The signal at () = 5.035 ppm is assigned to H-I of Man-4' as compared to that of GalfMan2GlcNAc2-ABEE. 2D-COSY NMR show­ed a cross-peak between the signals at () = 5.035 ppm and b = 4.137 ppm, as in the case of oligosaccharide X. The down field shift (Jb = 0.075 ppm) of the H-2 signal for Man-4' in comparison to that of GalfPI-2Man~I-6Man/Jl-4GlcNAc/3I -4GlcNAc-ABEE is indicative of disubstitu­tion of this residue at 0-3 and 0-6 by Man-A and Man-B. respectively.2h) And the presence of a fJ-( 1-2)-linked D­

galactofuranose residue is indicated by the broad doublet signal at c5 = 5.120 ppm with the coupling constant, J 1.2 = 1.88 Hz. Another unusual signal at b = 5.150 ppm, thus, can be assigned to H-I of Man-A. Combining these results, it can be concluded that the structure of fraction Nf is as shown in Table II.

The oligosaccharide in peak Nc was also resistant to A. saitoi:x-mannosidase I. When Nc was hydrolyzed with mild acid, the ABEE-oligosaccharide showed a peak shift corresponding to the release of one galactose residue to yield an ABEE-oligosaccharide comigrating with authentic Man4 GlcN Ac2-ABEE (Fig. 4). From these results, the structure of Nc was proposed to be as in Table II.

The ABEE-oligosaccharide in peak Nh was converted to an ABEE-oligosaccharide with the same mobility as GalfMan sGlcNAc2-ABEE (oligosaccharide Nf) on diges­tion with A. sailoi:x-mannosidase I (Fig. 6). That the prod­uct was actually GalfMan sGlcNAc 2-ABEE was demon­strated by similar experiments to those for structural anal­ysis of Nf described above. To locate the ~-( 1-2)-linked mannose residue, degalactosylated Nh obtained on mild acid hydrolysis was digested with endo-/J-N-acetylglucos­aminidase H. followed by 2D-HPLC analysis. The endo-/J-

2 3 5 6 7 8 9

Nh ~ ~ ~ ~ ~ ~ ~

E ij s::

I ~ 0 ~

M 1\ II - 1\ co ,I

W I I

0 _______ f \ -- J=~~ z

<t I m a:

Nj ij 0 C/) m

~ C I f

<t II

> ~ l_ --- _~iL ____ 11,. =>

~_) ~~,-",,,-..f! \ ____

o 10 20 30 40 50 RETENTION TIME (min)

Fig. 6. Amide-80 HPLC Analysis of A. sailoi:x-Mannosidase I Digests of the ABEE-oligosaccharides in Peaks Nh and Nj.

The digest of each fraction was subjected to HPLC on a TSKgel Amide-XO column under the conditions given in Fig. 2. When the hatched peak material was subjected to jack bean :x-mannosidase digestion. a peak corresponding to Gal/In -+2Man:xl-+ hMan/fl --.4GkNAcIJI -+4GkNAc-ABEE appeared (sho",o'!1 by a dolted line). Arrows ( i ) indicate the same as in Fig. 2. Open arrows ( U ) indicate the elution positions of untreated ABEE-oligosaccharides.

N-acetylglucosaminidase H digest gave a peak at the same position as authentic Man~I-6(Man~I-3)Man,~1-6-(Man~I-2Man~I-3)Manpl-4G1cNAc-ABEE (Man 6 -

GlcNAc ,-II-ABEE) (data not shown). These results were consistent with the proposed structure shown in Table II.

When the ABEE-oligosaccharide in peak Nj was digested with A. saitoi :x-mannosidase I, 50% of the fraction was converted to GalfMansGlcNAC 2-ABEE with the release of two mannose residues, the other 50% remaining un­changed (Fig. 6). The endo-fJ-N-acetylglucosaminidase H digest of degalactosylated Nj gave two peaks corresponding to Man 7GlcNAc t -I-ABEE and Man 7GlcNAc t -I1I-ABEE in a molar ratio of 2: I. These results suggested that this fraction contained the two isomers shown in Table II and a small amount of uncharacterized oligosaccharides.

Discussion This study established the structures of N-linked 01-

igosaccharides derived from AOD from a fungal strain, ACrel110llilllll sp. HI-25. AOD was found to only have N-linked oligosaccharide chains. Oligosaccharides were lib­erated from the enzyme by hydrazinolysis and then tagged with ABEE. Among the total ABEE-derivatized oligosac­charides from AOD, approximately 90% were neutral. The neutral oligosaccharides were resolved into twelve subfrac­tions on normal-phase HPLC, the structures of which were identified by FAB-MS, 2D-HPLC analysis, t H-NMR. methylation analysis, mild acid hydrolysis, and/or exoglyco­sidase digestion. In Table II, the structures are compiled, together with their relative amounts. The results obtained in this study show that AOD from Acremolliul11 sp. HI-25 contains two types of oligosaccharides, a series of high­mannose type oligosaccharides, Man4 _ gGlcNAc2, (76.3%), and a series of fJ-galactofuranose-containing high-mannose type oligosaccharides, GalfMan 4 _ 7GlcNAcz (18.6%). Galactofuranose was linked to Man-4' in various high­mannosc type oligosaccharides via a /J-( 1-2)-linkage.

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lV-Linked Sugar Chains of Acremonium sp. Ascorbate Oxidase 1129

Table II. Proposed Structures of Neutral lV-Linked Oligosaccharides of Acremoniu11l sp. Ascorbate Oxidase

Fraction No." Structure Relative amount (%)

~a

,Man:xl ---'6 Man:xl /' - /' 3Man/J1 ..... 4GIcNAc/J1 ..... 4GIcNAc

Man:xl 13.1

Gal/fJl 1 2

Nc Man:xl -" 3Man:x1 "'6

Man:xl -" 3Man/J1 ..... 4GIcNAc/JI ..... 4GlcNAc 4.9

Maml "'6

Man:xl -" 3Man :x1 "'6

,Man/JI ..... 4GIcNAc/JI ..... 4GIcNAc Man:xl /f -

29.5

GalffJl

Nf Man:xl 1 '" 6 2

Maned -" 3Man :x1 "'6

Maned -" 3 Man/Jl ..... 4GlcNAcfJl ..... 4GlcNAc

10.3

Ng

Man:xl ---'6

Man:xl /f 3Man:x1 "'6

Man:xl ..... 2Man:x1 -" 3 Man/JI ..... 4GIcNAc/Jl ..... 4GIcNAc

16.0

GalflJI

Man~l 1

'" 6 2

Man:xl -" 3Man:xl ---'6

Man:xl ..... 2Man:x1 -" 3 Man/Jl ..... 4GIcNAc/JI ..... 4GIcNAc

2.5

Man:xl ..... 2Man:x1 "'6

Man:xl -" 3Man:x1 "'6

-" 3Man/J1 ..... 4GIcNAc/J1 ..... 4GIcNAc Maml ..... 2Man:x1

4.7

Man:xl "'6 1Man:xl ---'6

Man:xl -" - ----' 3Man/JI ..... 4GlcNAcfJl ..... 4GIcNAc Man:xl -+ 2Man,:x1 ..... 2Man:xl

2.3

GalJ/iI

Man~1 -+ 2Man:x1 1

---. 6 2

Man:xl -" 3Man :x1 "'6

-" 3Man/JI ..... 4GIcNAcIJI ..... 4GIcNAc Man:xl ..... 2Man:x1

0.6

Nj

Gal/IJI

Man:xl 1 '"

, 6 -

Man:xl -" 3Man :x1 "'6

Man~l ..... 2Man:xl ..... 2Man:x1 -" 3Man/il ..... 4GIcNAc[JI ..... 4GIcNAc

0.3

Man:xl -+ 2Man:x1 "'6

3Man ,:x1 "'6 Maml -" - -" 3Man,BI-+4GIcNAc/J1-+4GIcNAc

Man:xl -+ 2Man:x1 -+ 2Man,:x1

Nk 7.1

Man:xl -+ 2Man:x1 "'6

Man:xl -+ 2Man:x1 A 3Man :x1 "'6

Man:xl -+ 2Man~1 ..... 2Man:x1 A 3ManfJI ..... 4GlcNAc/Jl-+4GIcNAc NI 3.6

II See Fig. 2.

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1130 M. OHTA et al.

The presence of o-galactose of the furanose form is highly unusual. o-Galactose found in mammalian glycoproteins is in the pyranose form and present in complex type oligo­saccharides with the sequence of Gal-GlcNAc-Man-. In contrast, o-galactose residues in AOD are of the furanose form and present in high-mannose type oligosaccharides.

The presence of galactofuranose in N-linked oligosac­charides was first reported in protozoa, Crithidiafasciculata and Crithidia harnw.'W,2S) and trypanosomatid flagellates. 3') In these protozoa, o-galactofuranose was found to be link­ed to the nonreducing ends of high-mannose type oligosac­charides with the composition of Gal,Man 6 GlcNAc2. The galactofuranose was of the /3-anomeric form from its sensi­tivity to fi-galactofuranosidase from Penicillium charlesii/ S) but the position of its linkage to a mannose residue has not been identified. Moraes et al. 32) subsequently reported that galactofuranose-containing high-mannose type oligo­saccharides with compositions of Gal, _ 3Man9GlcNAc2 and Gal,Man 7 .8GlcNAc2 occurred in glycoproteins of the parasitic protozoan Leptomonas samudio In this case, the galactose residue was found to be attached to the C-2 posi­tion of a mannose at the nonreducing end. The anomeric configuration has not been identified. Groisman and de Lederkremer33 ) reported that galactofuranose-containing oligosaccharides were also present in the fungus Ascoholus furfuraceus. On methylation analysis and 13C-NMR, it was found that the galactofuranose residues in A. fiu'­furaceus were of the /3-anomeric form and linked to position 2 of the mannose residue in the oligosaccharides sensitive to endo-fi-N-acetylglucosaminidase H. Recently, galacto­furanose-containing high-mannose type oligosaccharides with similar structures to those of protozoan oligosac­charides were reported in an :x-galactosidase28 ) and an :x-glucosidase29 ) from the fungus Aspergillus niger. In A. niger, the galactofuranose residues were found to be linked to nonreducing mannose (Man-A) of Mans _ 8GlcNAc2 via an :x-( 1-+2)-linkage. Interestingly, the galactofuranose residues in Acremonium sp. AOD oligosaccharides were not linked to external mannose but internal mannose residue (Man-4'), in contrast to all other reported cases. Further­more, the anomeric configuration of the galactofuranose in Acremonium sp. AOD oligosaccharides differed from that of A. niger enzyme oligosaccharides.

It should be noted that the galactofuranose-containing oligosaccharides from Leptomonas samueli were sensitive to endo-fi-N-acetylglucosaminidase H,32) but those from Acremonium sp. AOD reported in this study were resistant to it. Maley et al. 34) reported that endo-fJ-N-acetylglucos­aminidase H required for hydrolysis the minimum struc­tural unit, Man:xI-+3Man:xI-+6Manfil-+4GlcNAcfil-+ 4GlcNAc. In addition, Yamashita 3S) reported that the pres­ence of an :x-( 1-+3)-linked mannose residue (Man-A) to an:x-( 1-+6)-linked mannose residue (Man-4') was essential for a substrate of the enzyme. Interestingly, although the structures of the galactofuranose-containing oligosaccha­rides from Acremonium AOD met these requirements, they were not hydrolyzed under these conditions (0.1 units of the enzyme, 3TC, 16 h). These results indicated that the at­tachment of Galfto the internal mannose residue (Man-4') via fi-( 1-+2)-linkage inhibited the action of endo-fJ-N-ace­tylglucosaminidase H.

The oligosaccharide structures of the fungus, Acremonium

sp., AOD differ markedly from those of plant AOD. 8) The

biosynthesis and biological functions of galactofuranose­containing high-mannose type oligosaccharides in Acremo­Ilium sp. remain unknown.

References I) M. H. Lee and C. R. Dav.'son. J. BioI. Chem .. 246. 6596 6602 (1973).

2) T. Nakamura. N. Makino. and Y. Ogura. J. Biocllem .. 64. lIN 195 ( 19(8).

3) G. A. White and R. M. Krupka. Ar('h. Bio('lIel1l. Biophys .• 110. 448 461 (1965).

4) J. Funaki. K. Abc. S. Honma. and K. Aida. /I/ippon Eil'i) Shokur,l'!J Gakkaishi. 40. 47 51 (1987).

5) W. A. Volk and J. L. Larsen. Biochim. Biophys. Acta. 67. 576 580 (1963).

6) S. Murao. H. Itoh. T. Yajima. Y. Ozaki. S. Fukuyasu. and T. Shin. Biosci. Biotech. Biochem .. 56. 847 852 (1992).

7) J. Ohkawa. N. Okada. A. Shinmyo. and M. Takano. Proc. /\latl. Acad. Sci. [/.SA .. 86. 1239 1243 (1989).

8) G. O·Andrea. J. B. Bouwstra. J. P. Kamerling. and J. F. G. Vliegenthart. Gll'cocoll/ugate 1.. 5. 151 157 ( 1988).

9) J. Hirose. T. Sakurai. K. Imamura. H. Watanabe. H. Iwamoto. K. Hiromi. H. Itoh. T. Shin. and S. Murao. 1. Biochel1l .. 115.811 813 ( 1994).

10) M. Esaka. T. Hattori. K. Fujisawa. S. Sakajo. and T. Asahi. EliI'. 1. Biochem .. 191. 537 541 (1990).

II) Y.-T. Li and S.-c. Li. Methods EI1::.ymol .. 28. 702 713 (1972). 12) J. Amano and A. Kobata. J. Bio('hel1l .. 99.1645 1654 (1986). 13) M. Z. Jones. E. J. S. Rathke. D. A. Gage. C. E. Costello. K.

Murakami. M. Ohta. and F. Matsuura. J. Inher. Mewh. Dis .. 15. 57 67 (1992).

14) M. Ohta. J. Hamako. S. Yamamoto. H. Hatta. M. Kim. T. Yamamoto. S. Oka. T. Mizuochi. and F. Matsuura. Gly('o('oll/llgate J .. 8. 400413 (1991).

15) F. Matsuura. M. Ohta. K. Murakami. K. Hirano. and C. C. Swcelcy. Biomed. Chromatogr .• 6.77-83 (1992).

16) F. Matsuura. H. A. Nunez. G. A. Grabowski. and C. C. Sweelev. Arch. Bioc/lem. Biophys .. 207. 337 352 (1981). -

17) F. Matsuura and A. Imaoka. GlYCOCOll/ligate J .. 5. 13 26 (1988). 18) M. Ohta. M. Kobatake. A. Matsumura. and F. Matsuura. Agric.

BioI. Chem .• 54. 1045 1047 (1990). 19) F. Matsuura. M. Ohta. K. Murakami. and Y. Matsuki.

GlYCOcoll/ligate J .. 10. 202·213 ( 1993).

20) K. Akasaki. Y. Yamaguchi. M. Ohta. F. Matsuura. K. Furuno. and H. Tsuji. Chem. Pharm. Bull .. 38. 2766 2770 (1990).

21) R. Takano. K. Kamei-Hayashi. S. Hara. and S. Hirase. Bio.lci. Biotech. Bioc/zem .. 57. 1195-1197 (1993).

22) I. Ciucanu and F. Kerek. Carho/zydr. Res .. 131. 209 217 (1984). 23) c.-J. Liang. K. Yamashita. C. G. Muellenberg. H. Shichi. and A.

Kobata. J. BioI. ('hem .. 254. 6414 6418 (1979).

24) T. Mizuochi. Y. Nishimura. K. Kato. and A. Kobata. Arch. Biochelll. Bioj1hy.I .. 209. 298 303 (1981).

25) O. H. Mendelzon and A. J. Parodi. J. BioI. Chem .. 261. 2129 2133 ( 1986).

26) J. F. G. Vliegenthart. L. Dorland. and H. Halbeek. Adl'. Carhohrdr. Chelll. Biochem .. 41. 209 374 (1983). .

27) D. R. Anderson and W. J. Grimes. Allal. Biochel1l .. 146.1322 (1985). 28) T. Takayanagi. K. Kushida. K. Idonuma. and K. Ajisaka.

Glycocolljugate J .. 9. 229 234 ( 1992). 29) T. Takayanagi. A. Kimura. S. Chiba. and K. Ajisaka. Carho/mlr.

Res .. 256. 149 158 (1994). -

30) E. D. Green. G. Adell. J. U. Baenziger. S. Wilson. and H. Van Halbeek. 1. BioI. Chem .. 263. 18253 18268 (1988).

31) O. H. Mendelzon. J. O. Prcviato. and A. J. Parodi. A/ol. Biochefll. Parasitol .. 18.355 367 (1986).

32) C. T. Moraes. M. Bosch. and A. J. Parodi. Biochcmistr)'. 27. 1543 1549 (1988). .

33) J. F. Groisman and R. M. de Lederkremer. EliI'. J. Biochem .. 165. 327 332 (1987).

34) F. Maley. R. B. Trimble. A. L. Tarentino. and T. H. Plummer Jr.. Allai. Biochem .. 180. 195 204(1989).

35) K. Yamashita. Proteill. /\'uc/eic Acicl alld EII::.yme. 30. 143 146 (1985).

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