(pl- 4)GlcNAc(p1-4)GlcNAc,, (oligosaccharide 2) were obtained from human placental fibronectin [23]. Gal(/~ 1-4)GlcNAc(pl-2)-

Transcription

1 Eur. J. Biochem. 36, (996) 0 FEBS 996 Comparative study of the sugar chains of alkaline phosphatases purified from rat liver and rat AH-30 hepatoma cells Occurrence of fucosylated high-mannose-type and hybrid-type sugar chains Tamao ENDO, Toshiyuki FUJIWARA, Yukio IKEHARAZ and Akira KOBATA Department of Biochemistry, Institute of Medical Science, University of Tokyo, Japan Department of Biochemistry, Fukuoka University School of Medicine, Japan (Received 8 September/l3 November 995) - EJB 95 58/5 The N-linked sugar chains of alkaline phosphatases, purified from rat AH-30 hepatoma and from normal rat liver, were released quantitatively as oligosaccharides by hydrazinolysis and were labeled by reduction with NaB3H,. A comparative study of their structures revealed that the following structural differences are induced by hepatocyte carcinogenesis : complex-type tetraantennary sugar chains and hybrid-type sugar chains appear; outer-chain moieties of the sugar chains of the hepatoma enzyme contain exclusively the Gal@l-4)GlcNAc groups (type chains) but those of the normal enzyme contain other Gal(p-)GlcNAc groups and type chains ; and novel fucosylated high-mannose-type sugar chains are found in the oligosaccharides of the hepatoma enzyme. Keywords: alkaline phosphatase ; rat liver; hepatoma; N-linked sugar chains ; fucosylated high mannose. Alkaline phosphatase in mammalian cells is a membranebound glycoprotein and is used as a marker enzyme for the plasma membrane [. Although alkaline phosphatase is present in large amounts in various tissues, its physiological function is not known. Because high levels of alkaline phosphatase were detected in the body fluids and tumor tissues of certain cancer patients, the enzyme has been used as a marker for the diagnosis of cancer -4. However, the diagnostic value of alkaline phosphatase for cancer has decreased with the finding of a relatively high incidence of alkaline-phosphatase-positive sera from patients with non-cancerous diseases [I]. Comparative studies of the sugar chains of several tumor glycoproteins and their normal counterparts revealed that the alterations of the sugar-chain structures of glycoproteins that are induced by malignant transformation are quite diverse [5-03. It was also observed, however, that the changes induced in a particular glycoprotein in a particular tumor are rather constant. Methods have been developed that discriminate between glycoproteins of malignant cells and their normal counterparts by means of analysis of their sugar chains [,. The accumulation of data on the sugar chains of alkaline phosphatases purified from normal tissues and various tumors is expected to lead to the development of a new method to detect specifically alkaline phosphatase from tumor cells. We reported previously the structures of sugar chains of alkaline phosphatase samples purified from human placenta [I 3 and from FL cells (Kasahara isozyme) 4. Here, we present Correspondence to T. Endo, Department of Glycobiology, Tokyo Metropolitan Institute of Gerontology, 35- Sakae-cho, Itabashi-ku, Tokyo, Japan 73 Abbreviufions, XylNAc, N-acetylxylosarnine; or, NaBiH,-reduced oligosaccharides. Enzyme. Alkaline phosphatase, orthophosphoric-monoester phosphohydrolase (EC 3..3.). Note. All sugars mentioned in this paper have the D configuration except for fucose which has the L configuration. a comparative study of the N-linked sugar chains of alkaline phosphatases purified from normal rat liver and from rat AH- 30 hepatoma cells. MATERIALS AND METHODS Enzymes and lectins. Diplococcal p-galactosidase and p- N-acetylhexosaminidase were purified from the culture fluid of Diplococcus pneunzoniae according to the method of Glasgow et al. [5]. P-N-Acetylhexosaminidase and a-mannosidase were purified from jack bean meal as described previously [6]. a- Mannosidases I and I were purified from Aspergillus saitoi according to the method reported previously [ 7. Bovine epididyma a-fucosidase and Arthrobacter ureafaciens sialidase were purchased from Sigma Chemicals Co. and Nacalai Tesque, respectively. Streptococcus 6646 K p-galactosidase [ 8 and endo- P-N-acetylglucosaminidase D were purchased from Seikagaku Corporation. Recombinant Aleuria-aurantia-lectin-Sepharose [I9 and Datura-stramonium-agglutinin- Sepharose 0 were prepared according to the cited references. Concanavalin-A - Sepharose and phytohemagglutinin-e,- Sepharose were purchased from Pharmacia Biotech and Seikagaku Corporation, respectively. Purification of alkaline phosphatases from AH-30 hepatoma cells and rat liver. Rat liver alkaline phosphatase and rat ascites hepatoma AH-30 alkaline phosphatase were purified as described previously [,. Both alkaline phosphatase samples gave a single protein band of 7 kda when analyzed by SDSPAGE. Oligosaccharides. Gal(~-4)GlcNAc(~l-6)[Gal(~-4)Glc- NAc(Pl-)ManI (al-6) { Gal(pl-4)GlcNAc(p-4) [Gal(p-4)Glc- NAc(P-)]Man(a-3)}Man(p-4)GcNAc@-4)GcNAc0, (oligosaccharide ) and Gal(p-4)GlcNAc(pl-6)[Gal(~l-4)GlcNAc- (/3-)] Man(a-6) [Gal(~l-4)GlcNAc(B-)Man(a-3)] (pl- 4)GlcNAc(p-4)GlcNAc,, (oligosaccharide ) were obtained from human placental fibronectin [3]. Gal(/~ -4)GlcNAc(pl-)-

2 580 Endo et al. (Eur: J. Biochem. 36) Man(a-6) { Gal(pl-4)GlcNAc(pl-4) [Gal(pl-4)GlcNAc(pl-)]- Man(a-3)) Man(~-4)GlcNAc(~-4)GlcNAc,,. (oligosaccharide 3), Gal(,& -4)GlcNAc(~l-)Man(a-6)[Gal(p-4)GlcNAc(~-) Man (al-3)] Ma n(p-4) GlcNAc (pl-4) [Fuc (al-6)] GlcNAc,,, (oligosaccharide 4), and Gal(~-4)GlcNAc(~-)Man(al-6) [Gal (p-4) GlcNAc (pl-) Man (al-3)] Man (pl-4) GlcNAc (pl-4) GlcNAc,, (oligosaccharide 5) were prepared from human chorionic gonadotropin purified from the urine of a patient with invasive mole [7]. Gal(p-4) GlcNAc (pl-) Man (al-6) [Glc- NAc u-4)] [Gal (Dl -4) GlcNAc(pl-)Man(al-3)]Man(pl- 4)GlcNAc(P-4)GlcNAc0, (oligosaccharide 6), Man(al-6)- [Man(al-3)]Man(crl-6)[Man(al-3)] Man (pl-4)glcnac (pl-4)- GlcNAc,,(oligosaccharide 7), and [Man (al-),-,-labelled oligosaccharide 7 (oligosaccharides 8A- SD) were obtained from human IgM myeloma protein as described previously 4. Man(al-6)[Man(al-3)]Man(pl-4)GlcNAc(p-4)[Fuc(al-6)]Glc- NAc,, (oligosaccharide 9) and Man(al-6)[Man(al-3)]Man(pl- 4)GlcNAc(~l-4)GlcNAc,,. (oligosaccharide 0) were prepared from oligosaccharide 4 and oligosaccharide 5, respectively, by digestion with a mixture of diplococcal p-galactosidase and diplococcal p-n-acetylhexosaminidase. Man(pl-4)GlcNAc(P- 4)[Fuc(al-6)]GlcNAc,. (oligosaccharide l), and Man(p- 4)GlcNAc(~-4)GlcNAc,, (oligosaccharide ) were prepared from oligosaccharide 9 and oligosaccharide 0, respectively, by digestion with jack bean a-mannosidase. Man(al-6)[Gal(pl- 3)GlcNAc(pl-)Man(a-3)]Man(pl -4)GlcNAc~l-4)GlcNAc0, (oligosaccharide 3) was obtained from bovine prothrombin as described previously [5]. The structures of each of these standards was verified by sequential exoglycosidase digestion and methylation analysis. The details were described in each cited reference [7, 3-5. Periodate oxidation. Radioactive oligosaccharide (X 0 cpm) was dissolved in 00 pl 0.05 M sodium acetate, ph 4.5, 0.04 M sodium periodate and the mixture was kept at 5 C for 6 h in the dark. Ethylene glycol (0 pl) was added, and the mixture was kept at 5 C in the dark for h. 0 mg NaBH, in 0. M sodium borate, ph 9.5, was added and the mixture was kept at 30 C for 3 h. The reaction was stopped by adding 00 pl acetic acid, and the mixture was passed through a mixed-bed column (0.5 cmx3 cm) of Bio-Rad AG-50 (H form) and AG- 3 OH^- form). The column was washed with three bed vol. distilled water. The eluate was evaporated repeatedly with methanol to remove boric acid. The residue was dissolved in 0.3 ml 0.05 M H,SO, and heated at 76 C for h. The reaction mixture was passed through a column of AG-3 (OH- form), and the radioactive products in the effluent were analyzed by Bio-Gel P-4 column chromatography. Release of N-linked sugar chains of hepatoma and normal rat liver enzymes as oligosaccharides. Purified rat liver alkaline phosphatase (00 pg) and AH- 30 hepatoma alkaline phosphatase ( mg) were thoroughly dried over P,O, in vucuo and subjected to hydrazinolysis [6] at 00 C for 9 h. Liberated oligosaccharides were N-acetylated and purified as described previously [6]. This procedure releases N-linked sugar chains of glycoproteins quantitatively as oligosaccharides. The oligosaccharide fractions thus iobtained were reduced with NaBIH, (600 mci/mmol; New England Nuclear) after addition of 0 nmol xylose as an internal standard. The radioactive oligosaccharide fraction, and xylose were separated by paper chromatography with -butanol/ethanol/water (4: :, by vol.) as a solvent. Based on the radioactivities incorporated into the two sugar fractions and the molecular mass of alkaline phosphatase, the amounts of the total sugar chains released from mol of both alkaline phosphatase samples were calculated to be approximately 4 mol. AH-30 hepatoma alkaline phosphatase (.5 mg) was subjected to hydrazinolysis as described above and reduced FRACTION NUMBER Fig.. Anion-exchange column chromatography of the radioactive oligosaccharides liberated from the alkaline phosphatases of rat AH- 30 hepatoma cells (A) and rat normal liver (B). H-labeled oligosaccharides were subjected to FPLC on a Mono Q HR5/5 column, equilibrated with 5 mm sodium acetate, ph 4.0, and were eluted with a inear gradient from 5 mm to 300 mm sodium acetate, ph 4.0, at a flow rate of ml/min. Arrows,, 3, 4, and 5 indicate the elution positions of N-linked oligosaccharides which contained 0-4 sialic acids, respeitively, and which were obtained from fetuin [SS]. with NaB H, to obtain deuterium-labeled oligosaccharide mixtures for methylation analysis. To facilitate the detection of the deuterium-labeled oligosaccharides, 0% of the tritium-laheled oligosaccharides were added. All other experimental procedures were as described previously 7, 3, 4. RESULTS Fractionation of oligosaccharides from AH-30 and rat liver alkaline phosphatases by anion-exchange column chromatography. H-labeled oligosaccharide fractions were subjected to anion-exchange column chromatography on a Mono Q HR5/ 5 column. The sample from the AH-30 alkaline phosphatase gave a different fractionation profile to that from normal rat liver alkaline phosphatase (Fig. ). The oligosaccharide fraction from AH- 30 alkaline phosphatase was separated into one neutral (N) and four acidic (A-A4) fractions (Fig. A), while that from rat liver alkaline phosphatase was separated into one neutral (N ) and three acidic (Al -A3 ) fractions (Fig. B). The molar percentages of N, Al, A, A3 and A4, on the basis of their radioactivities, were 3,, 6, 4 and 6, respectively, and those of N, Al, A and A3 were 4, 9, 5 and 4, respectivel:: The pooled acidic oligosaccharides from both alkaline phosphitases were completely converted to neutral oligosaccharides (named AN for those from the AH-30 alkaline phosphatase and AN for those from the normal rat liver alkaline phosphatase). upon exhaustive digestion with A. ureafaciens sialidase indicating that the acidic nature of these oligosaccharides is due to sializ acid residues, A and Al contain one, A and A contain two, A3 and A3 contain three and A4 contains four sialic acid residues, as confirmed by partial desialylation as described pre\ iously ~3. Methylation analysis of deuterium-labeled-oligosaccharide mixtures. Because of the limited amount of each sample. methylation analysis of each component could not be performed. However, information of the glycosidic linkages is indispensable for the interpretation of the results obtained by lectin column chromatography and sequential exoglycosidase digestion. Therefore fractions N and AN were subjected to methylation analysis

3 Endo et al. (Eur: J. Biochem. 36) 58 Table. Methylation analysis of oligosaccharide fractions from rat AH-30 hepatoma alkaline phosphatase. Molar ratios were calculateds by taking the value of (,3,5-tri-O-methyl--N-methylacetamido--deoxyglucitol plus,3,5,6-tetra-o-methyl--n-methylacetamido--deoxyglucitol) as.o., not detected. Sugar Methylation and acetylation Molar ratio of sugars in fraction AN fraction AN Fucitol Galactitol Mannitol -N-methylacetamido -deoxyglucitol,3,4-tri-o-methyl-,5-di-o-acetyl,3,4,6-tetra-o-methyl-,5-di-o-acetyl,3,4,6-tetra-o-methyl-,5-di-o-acetyl 3,4,6-tri-O-methyl-,,5-tri-O-acetyl,4,6-tri-O-methyl-l,3,5-tri-O-acetyl,3,4-tri-O-methyl-,5,6-tri-O-acetyl 3,6-di-O-methyl-,,4,5-tetra-O-acetyl 3,4-di-O-methy-,,5,6-tetra-O-acetyl,4-di-O-methyl-l,3,5,6-tetra-O-acetyI -mono-o-methyl-l,3,4,5,6-penta-o-acetyl 3,4,6-tri-O-methyl-,5-di-O-acetyl 3,6-di-O-methyl-l,4,5-tri-O-acetyl,3,5-tri-O-methyl-4,6-di-O-acetyl,3,5,6-tetra-O-methyl-4-mono-O-acetyl 0. < I without further fractionation. All galactose residues of the oligosaccharides occur as non-reducing termini (Table ). This result indicated that -)repeating structures are not present in the outer-chain moieties. Detection of five O-methylated mannitols indicated that the mannose residues of the oligosaccharides in fraction N occur in the five forms, i.e., Man(-, -)Man(l-, -3)Man(l-, -6)Man(l- and ::jman(l-. Detection of seven 0-methylated mannitols in fraction AN indicated that the mannose residues in this fraction occur in the seven forms, i.e., Man(l-, -)Man(l-, -3)Man(l-, ::jman(l-, ::iman(l-, :gjman(land!lman(l-. Detection of, 3, 5, 6-tetra-0-methy--N-methylacetamido--deoxyglucitol and, 3, 5-tri-0-methyl--N-methylacetamido--deoxyglucitol indicated that the reducing termini of the oligosaccharides in both fractions occur as -4)GlcNAc and ::;GlcNAc. This result was in agreement with the data obtained by sequential exoglycosidase digestion and indicated that both fucosylated and non-fucosylated trimannosyl cores are included among the oligosaccharides. Detection of 3, 4, 6-tri-O-methyl-- N-methylacetamido--deoxyglucitol and 3, 6-di-O-methyl--Nmethylacetamido--deoxyglucitol indicated that the N-acetylglucosamine residues of the oligosaccharides in both fractions occur as non-reducing termini and as -4)GlcNAc(l-. Fractionation of neutral oligosaccharides by A. auratia-lectin-sepharose column chromatography and Bio-Gel P-4 column chromatography. Since preliminary studies indicated that all four fractions were complicated mixtures of fucosylated and non-fucosylated oligosaccharides, we decided to fractionate them first by A. aurantia-lectin- Sepharose column chromatography. As reported previously [9, 7, oligosaccharides with fucosylated trimannosyl cores are retained by the column and eluted with mm fucose. When subjected to A. aurantia-lectin-sepharose column chromatography, all four neutral oligosaccharide fractions (N, AN, N and AN ) were separated into two groups. The fractions retained by the column and eluted with mm fucose were named N(+F), AN (+F), N (SF) and AN (+F), and those not retained as N (-F), AN (-F), N (-F) and AN (-F). The molar ratios (mol/l00 mol) of N (-F), N (+F), AN (-F) and AN (+F), on the basis of their radioactivities, were 3, 9, and 47, respectively and those of N (-F), N (+F), AN (-F) and AN (+ F) were 4,, 39 and 9, respectively. If we consider the binding specificity of the A. auruntia-lectin - Sepharose column, we can assume that all oligosaccharides in fractions N (+F), AN (+F), N (+F) and AN (+F) contain a fucose residue linked to the C6 position of the proximal N-acetylglucosamine residue of their trimannosyl core and that oligosaccharides in fractions N (-F), AN (-F), N (-F) and AN (-F) lack fucose residues. These assumptions were supported by the results of methylation analysis described above and by sequential exoglycosidase digestion. Structural studies of oligosaccharides in these fractions, except for fraction N (+F), were performed. Because of the limited amount of fraction N (+F), further study of this fraction could not be performed. By means of Bio-Gel P-4 column chromatography, the seven fractions were separated into multiple components (Fig. ). The structure of each neutral component in Fig. was elucidated by sequential exoglycosidase digestion, lectin column chromatography and periodate oxidation (Smith degradation). Structures of oligosaccharides in fraction N (-F). When fraction N (-F) was subjected to Bio-Gel P-4 column chromatography, it was separated into eight radioactive components (a-a8; Fig. A). By digestion with jack bean a-mannosidase, al-a8 were converted to a radioactive component with the same mobility as oligosaccharide and 8- mannose residues were released, respectively (Fig. 3 A). That the radioactive trisaccharides have the same structure as oligosaccharide was confirmed by sequential digestion and jack Accordingly, a8 -a should be a series of high-mannose-type sugars with the structures, [Man(al-)],~,Man(@l-4)GlcNAc~l-4)GlcNAc. That the oligosaccharides a -a5 have the same structures as oligosaccharides 8A- 8D was confirmed by the following analytical procedures. The elution positions of the five peaks were the same as those of oligosaccharides 8A-8D. On incubation with A. saitoi a- mannosidase I, which cleaves only the Man(a-)Man linkage [8], more than 95% of a mixture of radioactive al-a4 were converted to a radioactive oligosaccharide with the same mobility as oligosaccharide 7, while a5, which has the same mobility as oligosaccharide 7, was resistant to this enzymatic digestion (Fig. 3B). The digestion products from al-a4, and a5, were further converted to Man(@-4)GlcNAc(/3-4)GlcNAc,T, Glc- NAc(j l-4)glcnac0, and H-labeled N-acetylglucosaminitol,

4 5 8 Endo et al. (Eur J. Biochem. 36) A 'I'IV~'ITV~ 'I i 'I 'I i 'I Y I * 04 A B A - G sz ELUTION VOLUME (ml) Fig.. Bio-Gel P-4 column chromatography of the neutral oligosaccharides obtained after fractionation on an A. uleuriu-lectin-sepharose column. (A) Fraction N (-F); (B) fraction N (+F); (C) fraction AN (-F); (D) fraction AN (+F); (E) fraction N' (-F); (F) fraction AN' (-F); (G) fraction AN' (+F). White arrows indice the elution positions of oligosaccharides standards : I, oligosaccharide ;, oligosaccharide 5. Arrowheads indicate the elution positions of glucose oligomers, and the numbers indicate the number of glucose units. with release of four mannose, one mannose, and one N-acetylglucosamine residue, respectively, by sequential digestion with jack bean a-mannosidase, p-mannosidase, and jack bean p-nacetylhexosaminidase, respectively (data not shown). In contrast, a6, a7 and a8 were all resistant to A. saitoi a- mannosidase I digestion. On digestion with jack bean a-mannosidase, the three components were completely converted to Man(pl-4)GlcNAc(pl-4)GlcNAc with release of three, two and one mannose residues, respectively. Therefore, the structures of oligosaccharides a6, a7 and a8 can be written as [Man(a-)l3- Man(pl-4)GlcNAc(pl -4)GlcNAc,, [Man( a -)],Man(pl-4)Glc- NAc@-4)GlcNAc0, and Man(n -),,Man@-4)GlcNAc(~l- 4)GlcNAc,, respectively. Detection of 3, 4, 6-tri-0-methylmannitol,, 4, 6-tri-Omethylmannitol, and, 3, 4-tri-0-methylmannitols as tri-0- methyl derivatives indicated that the following three structures can be considered for oligosaccharides a8 : Man(al-6)Man(pl- 4)GlcNAc(p-4)GlcNAco, (structure I), Man(al-3)Man(pl- 4)GlcNAc(pl-4)GlcNAco, (structure ), and Man(al-)- Man(pl-4)GlcNAc(j?I -4)GlcNAc,, (structure ). Since a8 is resistant to A. saitoi a-mannosidase I digestion, structure I can be eliminated as a possibility. To determine which of the remaining two possible structures is correct, a8 was subjected to Smith degradation. GlcNAc(~l-4)XylNAc,, was detected as the sole radioactive product on Bio-Gel P-4 column chromatography (data not shown). GlcNAc(p-4)XylNAc,, should be produced from structure I, and Man(pl-4)GlcNAc(pl-4)XylNAc,, should be obtained from structure. Accordingly, structure I was assigned to a8. ELUTION VOLUME (mi) Fig. 3. Sequential exoglycosidase digestion of components al-a8 and bl-b6. Components were treated with the enzymes indicated and subjected to chromatography on a Bio-Gel P-4 column. White arrows indicate the elution positions of oligosaccharides standards: I, oligosaccharide 7;, oligosaccharide 4;, oligosaccharide ; IV, oligosaccbaride. The arrowheads are the same as in Fig.. (A) A mixture of radioactive a -a8 (Fig. A) after digestion with jack bean a-mannosidase; (B) a mixture of al-a5 (Fig. A) after digestion with A. saitoi a-mannosidase I; (C) a mixture of b3-b6 (Fig. B) after digestion with jack bean a-mannosidase ; (D) b after digestion with diplococcal P-N-acetylhexosaminidase; (E) b' from (D) (solid line) and bl (Fig. B) (aotted line), after digestion with diplococcal D-galactosidase; (F) b' (solid line) and bl (dotted line) after sequential digestion with diplococcal P-galactosidase and diplococcal D-N-acetylhexosaminidase. Since a7 is a-mannosylated a8, three possible structures could be considered: Man(al-6)[Man(al-3)]Man(pl-4 iglc- NAc(p-4)GlcNAco, (structure IV), (structure V), and Man(a- 3)Man(al-6)Man(~-4)GlcNAc@l-4)GlcNAco, (structure VI). To determine which of these three structures is correct. radioactive a7 was subjected to Smith degradation. Analysis of the reaction mixture by means of Bio-Gel P-4 column chromatography revealed that 3H-labeled Man(~l-4)GlcNAc(p-4)XylNAc,, was the only radioactive product obtained (data not shown). Man(pl-4)GlcNAc(~-4)XylNAc,, should be produced from structure IV, and GlcNAc(~-4)XylNAco, should be obtained from structures V and VI. Therefore, structure IV is assigned to oligosaccharide a7. This conclusion was also supported by the finding that oligosaccharide a7 totally bound to a concanavalin- A-Sepharose column (data not shown). It is known that the presence of at least two a-mannosyl residues, either unsubstituted or substituted only at the C position, is required for binding to a concanavalin-a-sepharose column [9]. Since a6 is a-mannosylated a7 and a5 from which one mannose residue has been removed, two possible structures could be considered: Man(al-6)Man(al-6)[Man(al-3)]Man(~l-4)Glc- NAc(~l-4)GlcNAco, (structure VII) and Man(al-3)Mam a -6) [Man(al-3)]Man(~l-4)GlcNAc(pl-4)GlcNAc,,, (structure VIII).

5 After Smith degradation of a6, Man(al-6)Man(pl-4)Glc- NAc(p-4)XylNAc,, was obtained (data not shown). Man(pl-4)- GlcNAc(j3-4)XylNAcOT should be produced from structure VII, and Man(al-6)Man(pl-4)GlcNAc(pl-4)XylNAc0, should be obtained from structure VIII. Therefore, structure VIII was assigned to oligosaccharide a6. Based the results described, structures are proposed for components al-a8 (Table ). Endo et al. (Eur: J. Biochem. 36) 583 Structures of oligosaccharides in fraction N (+F). When the fraction N (+F) was subjected to Bio-Gel P-4 column chromatography it was separated into six radioactive components (blb6; Fig. B). The elution positions of four components (b3-b6) indicated that these components were one glucose unit larger than a5-a8 (Fig. A). Because the A. aurantia-lectin column retains N-linked oligosaccharides with Fuc(a-6)GlcNAc groups at their reducing termini and the effective size of this a-fucosyl residue is approximately one glucose unit [30], the differences in their mobilities of b3-b6 and a5-a8 were to be due to the presence of the a-fucosyl residue. This estimation was confirmed by the following studies. b3-b6 were all resistant to digestion with A. saitoi a-mannosidase I, P-galactosidase or P-N-acetylhexosaminidase (data not shown). Upon jack bean a-mannosidase digestion, however, the four components were completely converted to a radioactive oligosaccharide with the same mobility as oligosaccharide with the release of four, three, two and one mannose residues, respectively (Fig. 3 C). That the radioactive peak in Fig. 3 C has the structure, Man(p-)GlcNAc(Pl-)[Fuc(al-)]GlcNAc,,, was confirmed by sequential digestion with P-mannosidase, jack bean P-N-acetylhexosaminidase and a-fucosidase (data not shown). Further structural studies of b3-b6 were performed in the same manner as described for a5-a8. The results indicated that proposed structures of b3 -b6 are the fucosylated high-mannose-type sugar chains shown in Table. When b was digested with diplococcal P-N-acetylhexosaminidase, part of the peak was converted to an oligosaccharide with the same mobility as oligosaccharide 9, with the release of two N-acetylglucosamine residues, while the remainder of the peak (b') was resistant to such digestion (Fig. 3D). The molar ratio of component b' and oligosaccharide 9 (Fig. 3D), calculated on the basis of their radioactivities, was 39 : 6. Since diplococcal,!-n-acetylhexosaminidase cleaves the GlcNAc(P- )Man linkage but not the ClcNAc(P-4)Man or the Glc- NAc(P-6)Man linkage [3], the structure of the oligosaccharide in b which was cleaved by this enzyme was assigned as shown in Table. Upon sequential digestion with diplococcal P-galactosidase and diplococcal P-N-acetylhexosaminidase, one galactose residue (Fig. 3 E) and one N-acetylglucosamine residue (Fig. 3F), respectively, were released from b'. Based on the specificities of diplococcal P-galactosidase, which cleaves the Gal(P-4)GlcNAc linkage but not the Gal(p-3)GlcNAc or the Gal(p-6)GlcNAc linkages [3], and diplococcal,!-n-acetylhexosaminidase, as described above 3, the galactose and the N- acetylglucosamine residues were concluded to occur in component b' as the Gal(pl-4)GlcNAc(pl-)Man group. That the radioactive oligosaccharide obtained from b' after treatment with diplococcal P-galactosidase and diplococcal P-N-acetylhexosaminidase (Fig. 3 F) has the same structure as b4 was confirmed by the sequential exoglycosidase digestion and periodate oxidation as described for b4 (data not shown). The location of the Gal@- 4)GlcNAc group of b' was assigned by the following study. While b' was resistant to digestion with endo-p-n-acetylglucosaminidase D, the radioactive oligosaccharide obtained from b' after treatment with diplococcal P-galactosidase and diplococcal P-N-acetylhexosaminidase (Fig. 3 F) was completely converted to Fuc(a-6)GlcNAc by this enzymatic digestion (data not D ELUTION VOLUME (ml) Fig. 4. Sequential exoglycosidase digestion of components c3, c4, and c5. Components were treated with the enzymes, where indicated, and subjected to chromatography on a Bio-Gel P-4 column. White arrows indicate the elution positions of oligosaccharide standards : I, oligosaccharide 6;, oligosaccharide 5 ;, oligosaccharide 7; IV, oligosaccharide 0. The arrowheads are the same as in Fig.. (A) c5 (solid line) and c4 (dotted line) after digestion with diplococcal p-galactosidase ; (B) diplococcal-p-galactosidase-treated c5 (solid line) and diplococcal-p-galactosidase-treated c4 (dottet line) after digestion with diplococcal p- N-acetylhexosaminidase ; (C) the fraction of c3 that passed through a phytohemagglutin-e4-sepharose column (c3-); (D), the fraction of c3 that was retarded by a phytohemagglutin-e,-sepharose column (c3') ; (E) c3- (solid line) and c3' (dotted line) after digestion with diplococcal,!-galactosidase; (F) diplococcal-p-galactosidase-treated c3- (solid line) and diplococcal-p-galactosidase-treated c3' (dotted line) after digestion with diplococcal p-n-acetylhexosaminidase. shown). Endo-8-N-acetylglucosaminidase D cleaves sugars with the general structure R,(-4Man(al -3)[R(-6)]-Man(p-4)- GlcNAc(p-4)[R,(-6)]GlcNAc (in which R,, R, and R, represent either hydrogen or sugars) 33, 34. These results indicated that the Gal(/?-4)GlcNAc group is exclusively linked to the a-mannosy residue located at the C3 position of a P-mannosyl residue. The results indicated that b' has a hybrid-type structure as shown in Table. The radioactive oligosaccharide bl was resistant to diplococcal 8-N-acetylhexosaminidase treatment (data not shown) but one galactose residue and one N-acetylglucosamine residue were released upon sequential digestion with diplococcal,&galactosidase (Fig. 3 E) and diplococcal P-N-acetylhexosaminidase (Fig. 3F). The final product eluted at the same position as b3 (Fig. B). That the oligosaccharide obtained from bl after treatment with these two enzymes (Fig. 3F) had the same structure as b3 was confirmed by a series of analyses described above (data not shown). Based on these results, the structure of oligosaccharide bl was concluded to be a hybrid type, as shown in Table. I

6 5 84 Endo et al. (Eul: J. Biachem. 36) Table. Proposed structures of sugar chains of rat AH-30 hepatoma alkaline phosphatase. R,, GlcNAc(~-4)GlcNAc; R,, GlcNAc(J- 4)[Fuc(al-6)]GlcNAc. Structure R,& Molar ratio Present in (Mannl-),, Manul gmann Manal / ManPl -4R Mannl f3 mo/00 mol 4 a -a4 Mannl 6Manal Manal T3 g ManP-4R Manal / 4 a5 b3 Manal-3Mannl ManPl-4R / Manul a6 b4 Manal Manpl -4R Manal l3 a7 b5 a8 b6 Mannl, \;Manu Manall.3 Manpl-4R 8 7 c4 bl, d4 Manul-3Manal Manpl -4R GalPl-4GlcNAcP-Manal/ 3 7 c5 b, d5 GlcNAcpl -Manul GlcNAc~l-Manal/ :Man/(l-4R b Gal~l-4GlcNAc/ -Manal Gal~l-4GlcNAc~l-Manal / : ManPl-4R 4 6 c3 ~ d3 ~ GlcNAcPl Gal~l-4GlcNAc~l-Manal : M&-4R Gal~l-4GlcNAc~l-Manul/ <0.5 6 c3 d3

7 Endo et al. ( Em J. Biochem. 36) 585 Table. (continued) Structure WR Molar ratio Present in mo/00 mol Gal/?l-4GlcNAc~I -Manul Galbl-4GlcNAc/jY Manjl l-4r C lmanal 0 d Galpl-4GlcNAcpl, Gal~l-4GlcNAc~l \;Manal 6 Galpl-4GlcNAc~l ManPl-4R Gal~l-4GlcNAcPl -Manal y. 6 c d 9 cl dl GalPl-4GlcNAcpl Structures of oligosaccharides in fraction AN (-F). After pas- deoxyglucitol and -mono-0-methylmannitol also supported the sage through a Bio-Gel P-4 column, the fraction AN (-F) was presence of the bisecting N-acetylglucosamine residue. separated into five radioactive components (Fig. C). Upon affinity chromatography on a D. stramonium-ag- Upon sequential digestion with diplococcal P-galactosidase glutin- Sepharose column c was separated into a fraction that and diplococcal P-N-acetylhexosaminidase, one galactose resi- was retarded by the column (c ) and a fraction that bound to due and, one N-acetylglucosamine residue, respectively, were re- the column and was eluted with buffer containing a mixture ( % leased from c4 and c5 (Fig. 4A, 4B). The radioactive products mass/vol.) of N-acetylglucosamine oligomers (c ). The fracfrom c4 and c5 (Fig. 4B) eluted at the same positions as a5 and tions obtained were subjected to Bio-Gel P-4 column chromaa6 (Fig. A), respectively. That these radioactive products have tography. The elution positions of c (Fig. 5A) and c the same structures as a5 and a6 was confirmed by sequential (Fig. 5B) were exactly the same as those of oligosaccharide 3 exoglycosidase digestion as described above (data not shown). and oligosaccharide, respectively. Since c was retarded by a Accordingly, c4 and c5 should have the hybrid-type structures D. stramonium-agglutinin- Sepharose column, the oligosacchashown in Table. ride should contain the Gal(~-4)GlcNAc(~-4)[Gal(~-4)Glc- By means of affinity chromatography on a phytohemaggluti- NAc(P-)IMan group in its outer-chain moiety, as reported by nin E,- column, c3 was separated into fractions that passed Yamashita et al. 0. Digestion of CT, after Bio-Gel P-4 chrothrough (c3 -) or were retarded by (c3 ) the column. When sub- matography, with diplococcal /I-galactosidase released three jected to Bio-Gel P-4 column chromatography, c3- (Fig. 4C) galactose residues (Fig. 5 C) and subsequent digestion with jack and c3 (Fig. 4D) eluted at exactly the same positions as oligo- bean b-n-acetylhexosaminidase released three N-acetylglucossaccharide 5 and oligosaccharide 6, respectively. Upon sequen- amine residues. The radioactive product at this stage eluted at tial digestion with diplococcal P-galactosidase and diplococcal the same position as oligosaccharide 0 (data not shown). These P-N-acetylhexosaminidase, two galactose residues (Fig. 4 E) and results indicated that c3 is a triantennary oligosaccharide. Ditwo N-acetylglucosamine residues were released from c3- gestion of the diplococcal P-galactosidase-treated c3 with diplo- (Fig. 4F), and eluted at the same position as oligosaccharide 0. coccal P-N-acetylhexosaminidase released two N-acetylglucos- In contrast, two galactose residues were released from c3 upon amine residues (Fig. 5 D). If we consider the substrate specificity digestion with diplococcal P-galactosidase (Fig. 4 E) but only of diplococcal P-N-acetylhexosaminidase [3], we can propose one N-acetylglucosamine residue was releaseds upon digestion the structure of c shown in Table. This conclusion is comwith diplococcal P-N-acetylhexosaminidase (Fig. 4F). Finally, patible with the result that after sequential digestion with diploafter digestion of c3 with jack bean P-N-acetylhexosaminidase coccal P-galactosidase and diplococcal P-N-acetylhexosaminidigestion, which released two N-acetylglucosamine residues, the dase, c3 was completely resistant to digestion by A. saitoi a- product eluted at the same position as oligosaccharide 0 (data mannosidase I (data not shown). On the basis of the specificity not shown). As reported previously [3], diplococcal /I-N-acetyl- of the enzyme, which hydrolyzes a mannose residue from hexosaminidase cleaves only the GlcNAc(P-)Man linkage on the GlcNAc(P -)Man(a-6)[Man(a-3)]Man(~-4)GlcNAc(~- the Man(a-3) arm of GlcNAc~l-)Man(a-6)[GlcNAc(P- group but not from the Man(a-6)[GlcNAc(~l-)Man(al-3)]- 4)] [ GlcNAc(~-)Man(al-3)]Man(~l-4)GlcNAc(~l-4)GlcNAc. Man(pl-4)GlcNAc(P -group [ 7, the remaining GlcNAc(P-4) Accordingly, c3-and c3 should have the structures as shown residue should be linked to the Man(a-3) arm of the trimannoin Table. These structures are in agreement with the binding syl core. specificity of phytohemagglutinin E, at room temperature [35, Digestion of c+, after Bio-Gel P-4 chromatography, with 36. Detection of 3, 4, 6-tri-0-methyl -N-methylacetamido-- diplococcal P-galactosidase digestion released three galactose

8 5 86 Endo et al. (Eur: J. Biochem. 36) r086l WTTTTTVVT T V V T T ELUTION VOLUME (mi) Fig. 5. Sequential exoglycosidase digestion of components cl and c. Components were treated with the enzymes, where indicated, and subjected to chromatography on a Bio-Gel P-4 column. The white arrow indicates the elution position of oligosaccharide 0. The arrowheads are the same as in Fig.. (A) the fraction of c that was retarded on a D. strumonium-agglutinin - Sepharose column (c') ; (B) the fraction of c that bound to a D. stramonium-agglutinin-sepharose column (c'); (C), c' after digestion with diplococcal P-galactosidase ; (D), diplococcal-p-galactosidase-treated c' after digestion with diplococcal P-N-acetylhexosaminidase: (E), c' after digestion with diplococcal P-galactosidase; (F), diplococcal-p-galactosidase-treated c' after digestion with diplococcal P-N-acetylhexosaminidase ; (G) cl after digestion with diplococcal P-galactosidase ; (H), diplococcal-p-galactosidase-treated cl after digestion with diplococcal P-N-acetylhexosaminidase (solid line) or with jack bean fl-n-acetylhexosaminidase (dotted line). residues (Fig. 5E). The product of this digestion was then converted to oligosaccharide 0, with the release of three N-acetyl- Structures of oligosaccharides in fraction AN (+F). After pasglucosamine residues, by treatment with jack bean P-N-acetyl- sage through a Bio-Gel P-4 column, fraction AN (+F) was sepahexosaminidase (data not shown). Since c' bound to the rated into five radioactive components (dl -d5; Fig. ~ DThat J D. strumonium-agglutininsepharose column, c+ should con- d4 and d5 have the same structures as bl and b', respec:i\ely, tain the Gal(~l-4)GlcNAc~l-6)[Gal(~l-4)GlcNAc(~-)]Man was confirmed by sequential exoglycosidase digestion iis degroup [0]. Digestion of diplococcal-p-galactosidase-treated c' scribed above (data not shown). d was separated into a fraction with diplococcal p-n-acetylhexosaminidase released one N-ace- that was retarded by (d') and a fraction that bound to (~') a tylglucosamine residue (Fig. 5 F). This result indicated the pres- D. stramonium-agglutinin - Sepharose column. Peak d3 WBS sepence of a, 6-branched outer-chain moiety because diplococcal arated into that passed through fraction (d3-) and a fracticm that p-n-acetylhexosaminidase can not cleave the GlcNAc(P-)Man was retarded by (d3') by a phytohemagglutin-e,-sepharo\e col- linkage in the GlcNAc(pl-6)[GlcNAc(~l-)]Man group [3]. C+, after digestion with diplococcal p-galactosidase and diplococcal P-N-acetylhexosaminidase, was incubated with A. suitoi a-mannosidase, and one mannose residue was released (data not shown). Therefore the, 6-branched outer chain of c' should be exclusively located on the Man(a-6) arm as shown in Table. The mobility of cl was the same as that of oligosaccharide (Fig. C). When digested with diplococcal P-galactosidase, cl was converted to a radioactive component with the same mobil- ELUTION VOLUME (ml) Fig. 6. Sequential exoglycosidase digestion of components fl and f. Components were treated with the enzymes, where indicated, and subjected to chromatography on a Bio-Gel P-4 column. The white arrows indicate the elution positions of authentic oligosaccharides: I, oligosaccharide 3:, oligosaccharide 0. The arrowheads are the same as in Fig.. (A), f after digestion with 6646 K /I-galactosidase: (B), 064K- P-galactosidase-treated f after digestion with diplococcal P-N-acetylhexosaminidase; (C) f after digestion with a mixture of diplococcal P- galactosidase and diplococcal P-N-acetylhexosaminidase; (D), the fraction of fl that passed through the D. strumonium-agglutinin-septrarose column (fl-); (E) fl- after digestion with a mixture of diplococcal P- galactosidase and jack bean P-N-acetylhexosaminidase. ity as that of diplococcal-p-galactosidase-treated oligosaccharide, with the release of four galactose residues (Fig. 5 G). On further digestion of the product with diplococcal p-n-acetylhexosaminidase one N-acetylglucosamine residue was released (Fig. 5H), whereas it was converted to oligosaccharide 0. with the release of four N-acetylglucosamine residues upon subsequent treatment with jack bean p-n-acetylhexosaminidase (Fig. 5H). These results indicated that cl was a typical tetraantennary complex-type oligosaccharide (Table ). umn (data not shown). Upon sequential exoglycosidase digestion, dl, d, d+, d3- and d3' gave the same series of results as obtained from cl, c, c+, c3- and c3: respectively, except that the effective size of each peak was one glucose unit larger than that of the corresponding c peak (data not shown). Accordingly, dl, d', d+, d3- and d3' were concluded to be fucosylated forms of cl, c, c+, c3- and c3: respectively (Table ). Structures of oligosaccharides in fraction N' (-F). After passage through a Bio-Gel P-4 column, fraction N' (-F) was sepa-

9 Endo et al. (Em J. Biochem. 36) 587 Table 3. Proposed structures of sugar chains of normal rat liver alkaline phosphatase. R,, GlcNAc(P-4)GlcNAc; R,, GlcNAc(P-4)[Fuc(al- 6)IGlcNAc. Structure RilR, Molar ratio Present in (Manal-),., Manal : Manal Manal/ :ManP-4R Manal moll00 mol 4 el -es GalPl-4GlcNAcPI -Manal! ManPl-4R GalPl-4GlcNAc~-Manal/ s f 8 6 f 4 8 Gal~-4GlcNAc~-Manal GalPl-4GlcNAcPl L4 6Man/?l-4R Manaf GalPl-4GlcNAcPl P3 4 3 fl gl Gal~l-4GlcNAc~-Manal GalPl -( 3)GlcNAcPl : ManPl-4R L4 Manal / Gal~l-4GlcNAc~l Gal/ll-4GlcNAcPl :Mano GalPl-4GlcNAc/3/ Manp-4R Gal~l-4GlcNAcPl-Manal3 C0.S fl+ gl rated into five radioactive components (Fig. E). These components (el -e5) were found to have the same structures as a - a5, respectively, by a series of analyses described above. The confirmed structures of el -e5 are listed in Table 3. Structures of oligosaccharides in fraction AN (-F). By means of Bio-Gel P-4 column chromatography, fraction AN (-F) was separated into two radioactive components (Fig. F). After sequential digestion with 6646 K p-galactosidase, which can cleave both the Gal@ -4)GlcNAc linkage and the Gal(P- 3)GlcNAc linkage [37], and diplococcal p-n-acetylhexosaminidase, two galactose residues (Fig. 6A) and two N-acetylglucosamine residues (Fig. 6B), respectively, were released from f, and the final product eluted at the same position as oligosaccharide 0. That the final product had the same structure as oligosaccharide 0 was confirmed by further sequential exoglycosidase digestion (data not shown). These results indicated that f is a non-fucosylated biantennary oligosaccharide. When f was digested with a mixture of diplococcal p-galactosidase and diplococcal p-n-acetylhexosaminidase, 77 % was converted to oligosaccharide 0, and the remainder was converted to a radioactive oligosaccharide with the same size as oligosaccharide 3 (Fig. 6 C). The results of sequential exoglycosidase treatments indicated that f is a mixture of two isomeric biantennary oligosaccharides. One has two Gal(P-4)GlcNAc(p-)Man (type chain) groups and the other has a type chain and possibly a Gal(P-3)GlcNAc(p-)Man (type chain) group in its outerchain moieties. However, the exact structure of the outer chain, that was resistant to the enzymatic digestion could not be confirmed by methylation studies because of the limited amount of the sample. The absence of the bisecting N-acetylglucosamine residue, which was detected in the biantennary sugar chains of the AH- 30 enzyme, was confirmed by phytohemagglutinin- E,-Sepharose column chromatography. All oligosaccharides in f passed through the column without any interaction. fl was separated into fractions that passed through (f-), were retarded by (fl ) or bound to (fl +) D. stramonium-agglutinin-sepharose column. fl and fl were found to have the same structures as c and c: respectively, by a series of analyses described above. Upon Bio-Gel P-4 column chromatography, fl eluted as a single radioactive peak (Fig. 6D). Sequential digestion of fl- with 6646 K p-galactosidase and jack bean p-nacetylhexosaminidase, released three galactose residues and three N-acetylglucosamine residues respectively, and the final

10 588 Endo et al. (Eul: J. Biochem. 36) Table 4. Characteristics of the sugar chains of normal rat liver alkaline phosphatase and rat AH-30 hepatoma alkaline phosphatase. The enzymes from both sources contain four sugar chains/enzyme., not detected. ~- - - Sugar structures Sugar type Amount present in alkaline phosphatase trom normal liver AH-30 cells Core structures High-mannose type sugars R(-)G~CNAC(P~-~)GICNAC 79 R(-)GlcNAc(~-4)[Fuc(ul-6)]GlcNAc non-fucosylated fucos ylated Hybrid type sugars s Complex-type sugars biantennary non-bisected biantennary bisected triantennary C-, C-,4 triantennary C-, C-,6 tetraantennary Residues included in outer-chain moieties Gal(/l-4)GlcNAc(type chain) Gal(~l-[3])GlcNAc % product eluted at the same position as oligosaccharide 0. The structure of the pentasaccharide was confirmed by further sequential exoglycosidase digestion (data not shown). These results indicated that fl- is a triantennary oligosaccharide. Since it did not show any interaction with the D. stramionium agglutinin column, it contains neither the Gal(~l-4)GlcNAc(~l-4)[Gal(PI- 4)GlcNAc@-)]Man group nor the Gal@ -4)ClcNAc(PI - 6)[Gal(pl-4)GlcNAc(P-)]Man group. Two N-acetylglucosamine residues were released from degalactosylated fl ~ upon digestion with diplococcal P-N-acetylhexosaminidase (data not shown). This result indicated that fl- is a, 4-branched triantennary oligosaccharide. fl was digested sequentially with diplococcal /j'-galactosidase and jack bean P-N-acetylhexosaminidase and two galactose residues and two N-acetylglucosamine residues were released (Fig. 6E). Since the final product was totally resistant to diplococcal jj-galactosidase treatment but released a galactose residue upon treatment with 6646 K P-galactosidase, the P-galactosyl residue possibly occurs as the Gal(P-3)GlcNAc group. Since the radioactive component in Fig. 6E did not show any interaction with a concanavalin-a - Sepharose column and was resistant to A. saitoi a-mannosidase I digestion (data not shown), the Gal(~I-[3])GlcNAc(~l- group in this oligosaccharide should be exclusively located at the C4 position of Man(a- 3) arm as shown in Table 3. Structures of oligosaccharides in fraction AN' (+F). By means of Bio-Gel P-4 column chromatography, fraction AN' (+F) was separated into two radioactive components (Fig. G). gl and g (Table ) were found to be fucosylated forms of fl and f, respectively, based on the finding that the results obtained by sequential exoglycosidase digestion were similar to those for fl and f except that each radioactive product was one glucose unit larger than that obtained from fl and f (data not shown). DISCUSSION As summarized in Table 4, several prominent structural differences are found between the sugar chains of alkaline phosphatases purified from normal rat liver and from rat AH-I30 hepatoma cells. One of the most prominent findings is the detection of fucosylated high-mannose-type sugar chains in the hepa- toma alkaline phosphatase. Since Schachter's group [38, 3'9 and others [40, 4 reported that GDP-fucose :p-n-acetyiglucoiaminide (Fuc to Asn-linked GlcNAc)(al-6)-fucosyltransferasr: can act on sugar chains only after addition of the fl-n-acetylgi,mx- amine residue at the C position of the Man(a-3) arm (.if the trimannosyl core, it has been believed that fucosylation of highmannose-type sugars can never occur. Fucosylated high-.mannose-type sugar chains have not been detected among the N- linked sugar chains of more than 00 glycoproteins analyzzd by use of an A. aleuria-lectin-sepharose column [9]. The only exception was the detection of components b6 and b5 in some lysosomal glycoproteins However, these sugar i hains were considered to be degradation products of fucosylated complex-type sugar chains in lysosomes. Therefore, detection of a series of fucosylated high-mannose-type sugar chains in the hepatoma alkaline phosphatase, which is of non-lysosomal origin, is very unusual. Possibly, the a-fucosyltransferase in All-30 hepatoma may have a wider substrate specificity than that in normal rat liver. Fucosylated high-mannose-type sugar chains with the Man(a-%)Man group were not detected in the hepatoma alkaline phosphatase. This finding suggests that the cr-fucosyltransferase from tumor cells may not act on the high-niannose-type sugar chains larger than oligosaccharide 7. Another possibility is that the a-fucosyltransferase from tumor cells requires the GlcNAc(P-) residue on the Man(a-3) arm in a similar manner to that of the normal enzyme. In the tumor cells, however, non-lysosomal P-N-acetylglucosaminidase may act on (a-6)-fucosylated sugar chains such that fucosylated higt-i-mannose-type sugar chains are produced as observed in this study. Whether the appearance of fucosylated high-mannose-type sugar chains is a reflection of an abnormal expression of fucosyltransferase in malignant cells remains to be elucidated. In addition to fucosylated high-mannose-type sugar chains, tetraantennary sugar chains, hybrid-type sugar chains and bisected sugar chains are found in the hepatoma enzyme. Bisected sugar chains, which are not found in the normal liver enzyme. were found in y-glutamyltranspeptidase from rat hepatoma [5]. Accordingly, this phenomenon is considered to occur widely in the glycoproteins produced by rat hepatoma. Together with the novel appearance of tetraantennary sugar chains, enrichment of, 6-branched triantennary sugar chains in AH-I30 alkaline phosphatase (7 %) compared with normal liver

11 Endo et al. (Eur: J. Biochem. 36) 589 alkaline phosphatase ( %) was detected. These data indicated that the increase in highly branched sugar chains in the hepatoma enzyme can be ascribed to the enhanced expression of p- N-acetylglucosaminyltransferase V, which is widely observed in malignant cells [ Another alteration detected in the sugar chains of hepatoma alkaline phosphatase is the disappearance of the type chain, which occurs in the outer-chain moieties of complex-type sugar chains of normal liver alkaline phosphatase. A similar phenomenon was found in the sugar chains of carcinoembryonic antigen [37, 54. Less than 5% of the outer-chain moieties of the sugar chains of this antigen occur as the Gal(p-3)GlcNAc group compared with 33 % of those in non-specific cross-reacting antigen, a normal counterpart of carcinoembryonic antigen. Enhancement of type chains with concomitant decrease of type chains has also been observed in the glycosphingolipids of various tumor cells [55, 56. The various structural alterations of N-linked sugar chains in the hepatoma alkaline phosphatase as revealed by this study may be effectively used for the diagnosis and prognosis of liver cancer. However, in order to apply this finding to clinical use, it will be necessary to elucidate whether similar structural changes occur in the human liver alkaline phosphatase, during carcinogenesis, since glycosylation is known to be species specific [57]. Accumulation of data concerning the structures of the sugar chains of alkaline phosphatases from various tissues and tumors will become the basis on which to develop a new method to detect specifically the tumor alkaline phosphatase. This work was supported by the Grant-in-Aid for Scientific Research on Priority Areas (068, ) from the Ministry of Education, Science and Culture of Japan. We would like to express our gratitude to Ms Yukari Hayashi for assistance in purification of alkaline phosphalases. REFERENCES. Fishman, W. H. (974) Perspectives on alkaline phosphatase isozymes, Am. J. Med. 56, Fishman, W. H., Inglis, N. I., Stolbach, L. L. & Krant, M. J. (968) A serum alkaline phosphatase isozyme of human neoplastic cell origin, Cancer Res. 8, Nathanson, L. & Fishman, W. H. (97) New observation on the Regan isozyme of alkaline phosphatase in cancer patients, Cancer (Phila.) 7, Higashino, K., Hashinotume, M., Kang, K., Takahashi, Y. & Yamamura, Y. (97) Studies on a variant alkaline phosphatase in sera of patients with hepatocellular carcinoma, Clin. Chim. Actu 4, Yamashita, K., Hitoi, A,, Taniguchi, N., Yokosawa, N., Tsukada, Y. & Kobata, A. (983) Comparative study of the sugar chains of ;I-glutamyltranspeptidases purified from rat liver and rat AH-66 hepatoma cells, Cancer Res. 43, Mizuochi, T., Nishimura, R., Derappe, C., Taniguchi, T., Hamamoto, T., Mochizuki, M. & Kobata, A. (983) Structures of the asparagine-linked sugar chains of human chorionic gonadotropin produced in choriocarcinoma: appearance of triantennary sugar chains and unique biantennary sugar chains, J. Biol. Chem. 58, Endo, T., Nishimura, R., Kawano, T., Mochizuki, M. & Kobata, A. (987) Structural differences found in the asparagine-linked sugar chains of human chorionic gonadotropins purified from the urine of patients with invasive mole and with choriocarcinoma, Cuncer Res. 47, Yamashita, K., Koide, N., Endo, T., Iwaki, Y. & Kobata, A. (989) Altered glycosylation of serum transferrin of patients with hepatocellular carcinoma,. Biol. Chem. 64, Yamashita. K., Totani, K., Iwaki, Y., Takamisawa, I., Tateishi, N., Higashi, T., Sakamoto, Y. & Kobata, A. (989) Comparative study of the sugar chains of y-glutamyltranspeptidases purified from human hepatocellular carcinoma and from human liver, J. Biochem. (Tokyo) 05, Endo, T., Nishimura, R., Teshima, S., Ohkura, H., Baba, S. & Kobata, A. (99) Structures of the asparagine-linked sugar chains of human chorionic gonadotropin from a patient with extragonadal germ cell tumour, Eur: J. Cancer 7, Hitoi, A., Yamashita, K., Ohkawa, J. & Kobata, A. (984) Application of a Phaseolus vulgaris erythroagglutinating lectin agarose column for the specific detection of human hepatoma y-glutamyl transpeptidase in serum, Jpn. J. Cuncer Res. 75, Endo, T., Iino, K., Nozawa, S., Iizuka, R. & Kobata, A. (988) Immobilized Daturu strumonium agglutinin column chromatography, a novel method to discriminate the urinary hcgs of patients with invasive mole and choriocarcinoma from those of normal pregnant women and patients with hydatidiform mole, Jpn. J. Cancer Res. 79, Endo, T., Ohbayashi, H., Hayashi, Y., Ikehara, Y., Kochibe, N. & Kobata, A. (988) Structural study on the carbohydrate moiety of human placental alkaline phosphatase, J. Biochem. (Tokyo) 03, Endo, T., Higashino, K., Hada, T., Imanishi, H., Muratani, K., Kochibe, N. & Kobata, A. (990) Structures of the asparagine-linked oligosaccharides of an alkaline phosphatase, Kasahara isozyme, purified from FL amnion cells, Cancer Res. 50, Glasgow, L. R., Paulson, J. C. & Hill, R. L. (977) Systematic purification of five glycosidases from Streptococcus (Diplococcus) pneumoniae, J. Biol. Chem. 5, Li, Y.-T. & Li, S.-C. (97) a-mannosidase, p-n-acetylhexosaminidase, and P-galactosidase from jack bean meal, Methods Enzymol. 8, Amano, J. & Kobata, A. (986) Purification and characterization of a novel a-mannosidase from Aspergillus saitoi, J. Biochem. (To- kyo) 99, , 8. Kiyohara, T., Terao, T., Shiiri-Nakano, K. & Osawa, T. (976) Purification and characterization of P-N-acetyl hexosaminidase and p- galactosidase from Streptococcus 6646 K, J. Biochem. (Tokyo) 80, Fukumori, F., Takeuchi, N., Hagiwara, T., Ohbayashi, H., Endo, T., Kochibe, N., Nagata, Y. & Kobata, A. (990) Primary structure of a fucose-specific lectin obtained from a mushroom, Aleuria auruntiu, J. Biochem. (Tokyo) 07, Yamashita, K., Totani, K., Ohkura, T., Takasaki, S., Goldstein, I. J. & Kobata, A. (987) Carbohydrate binding properties of complex-type oligosaccharides on immobilized Datura stramonium lectin, J. Biol. Chem. 6, Kawahara, S., Ogata, S. & Ikehara, Y. (98) Chemical and immunological characterization of rat ascites hepatoma alkaline phosphatase: a comparison with the liver enzyme, J. Biochem. (Tokyo) 9, Miki, A,, Tanaka, Y., Ogata, S. & Ikehara, Y. (98) Selective preparation and characterization of membranous and soluble forms of alkaline phosphatase from rat tissues. A comparison with the serum enzyme, Eur: J. Biochem. 60, Takamoto, M., Endo, T., Isemura, M., Kochibe, N. & Kobata, A. (989) Structures of asparagine-linked oligosaccharides of human placental fibronectin, J. Biochem. (Tokyo) 05, Ohbayashi, H., Endo, T., Mihaesco, E., Gonzales, M. G., Kochibe, N. & Kobata, A. (989) Structural studies of the asparaginelinked sugar chains of two immunoglobulin M s purified from a patient with Waldenstrom s macroglobulinemia, Arch. Biochem. Biophys. 69, Mizuochi, T., Yamashita, K., Fujikawa, K., Kisiel, W. & Kobata, A. (979) The structures of the carbohydrate moieties of bovine blood coaggulation factor X, J. Biol. Chem. 54, Takasaki, S., Mizuochi, T. & Kobata, A. (98) Hydrazinolysis of asparagine-linked sugar chains to produce free oligosaccharides, Methods Enzymol. 83, Yamashita, K., Kochibe, N., Ohkura, T., Ueda, I. & Kobata. A. (985) Fractionation of L-fucose-containing oligosaccharides on immobilized Aleuria aurantia lectin, J. Bid. Chem. 60,

12 590 Endo et al. (Eul: J. Biochem. 36) 8. Yamashita, K., Ichishima, E., Arai, M. & Kobata, A. (980) An a- mannosidase purified from Asparagillus saitoi is specific for a, linkage, Biochem. Biophys. Res. Commun. 96, Ogata, S., Muramatsu, T. & Kobata, A. (975) Fractionation of glycopeptides by affinity column chromatography on concanavalin A-Sepharose, J. Biochem. (Tokyo) 78, Yamashita, K., Mizuochi, T. & Kobata, A. (98) Analysis of oligosaccharides by gel filtration, Methods Enzymol. 83, Yamashita, K., Ohkura, T., Yoshima, H. & Kobata, A. (98) Substrate specificity of Diplococcal p-n-acetylhexosaminidase, a useful enzyme for the structural studies of the complex-type asparagine-linked sugar chains, Biochem. Biophys. Rex Commun. 00, Paulson, J. C., Prieels, J. P., Glasgow, L. R. & Hill, R. L. (978) Sialyl- and fucosyltransferases in the biosynthesis of asparaginelinked oligosaccharides in glycoproteins, J. Bid. Chem. 53, Tai, T., Yamashita, K., Ogata-Arakawa, M., Koide, N., Muramatsu, T., Iwashita, S., Inoue, Y. & Kobata, A. (975) Structural studies of two ovalbumin glycopeptides in relation to the endo-p-n-acetylglucosaminidase specificity, J. Bid. Chem. 50, Mizuochi, T., Amano, J. & Kobata, A. (984) New evidence of the substrate specificity of endo-p-n-acetylglucosaminidase D, J. Biochem. (Tokyo) 95, Yamashita, K., Hitoi, A. & Kobata, A. (983) Structural determinants of Phaseolus vulgaris erythroagglutinating lectin for oligosaccharides, J. Bid. Chem. 58, Kobata, A. & Yamashita, K. (989) Affinity chromatography of oligosaccharides on Ed-phytohemagglutinin-agarose column, Methods Enzymol. 79, Yamashita, K., Totani, K., Iwaki, Y., Kuroki, M., Matsuoka, Y., Endo, T. & Kobata, A. (989) Carbohydrate structures of nonspecific cross-reacting antigen-, a glycoprotein purified from meconium as an antigen cross-reacting with anticarcinoembryonic antigen antibody: Occurrence of complex-type sugar chains with the Gal~-+3GlcNAc~-+3Gal~~4GlcNAc~l+ outer chains, J. Bid. Chem. 64, Longmore, G. D. & Schachter, H. (98) Product-identification and substrate-specificity studies of the GDP-L-fucose : -acetamido-- deoxy-p-d-glucoside (Fuc-Asn-linked GlcNAc) 6-a-~-fucosyltransferase in a Golgi-rich fraction from porcine liver, Carhohydr. Res. 00, Schachter, H., Narasimhan, S., Gleeson, P. & Vella, G. (983) Control of branching during the biosynthesis of asparagine-linked oligosaccharides, Can. J. Biochem. Cell Biol. 6, Voynow, J. A., Kaiser, R. S., Scanlin, T. F. & Glick. M. C. (99) Purification and characterization of GDP-L-fucose-N-acetyl p-dglucosaminide x-6fucosyltransferase from cultured human skin fibroblast: requirement of a specific biantennary oligosaccharide as substrate, J. Biol. Chem. 66, Shao, M.-C., Sokolik, C. W. & Wold, F. (994) Specificity studies of the GDP-[LI-fucose: -acetamido--deoxy-~-[~]-glucoside (Fuc Asn-linked GlcNAc) 6-n-[~]-fucosyltransferase from ratliver Golgi membranes, Carhohydr. Res. 5, Takahashi, T., Schimidt, P. G. & Tang, J. (984) Novel carbohydrate structures of cathepsin B from porcine spleen, J. Biol. Chem. 59, Taniguchi, T., Mizuochi, T., Towatari, T., Katsunuma, N. & Kobata, A. (985) Structural studies on the carbohydrate moieties of rat liver cathepsins B and H, J. Biochem. (Tokyo) 97, Howard, D. R., Natowicz, M. & Baenziger, J. U. (98) Structural studies of the endoglycosidase H-resistant oligosaccharides present on human p-glucuronidase, J. Bid. Chem. 57, Kozutsumi, Y., Nakao, Y., Teramura, T., Kawasaki, T., Yamashina, I., Mutsaers, J. H. G. M., van Halbeek, H. & Vliegenthart, J. F. G. (986) Structures of oligosaccharide chains of a-mannosidase from porcine kidney, J. Biochem. (Tobo) 99, Yamashita, K., Inui, K., Totani, K., Kochibe, N., Furukawa, M. & Okada, S. (990) Characteristics of asparagine-linked iugar chains of sphingolipid activator protein purified from nmnal human liver and GM, gangliosidosis (type ) liver, Biochemistry 9, Yamashita, K., Tachibana, Y., Ohkura, T. & Kobata, A. (985) Enzymatic basis for the structural changes of asparagine-linked 'sugar chains of membrane glycoproteins of baby hamster kidney cells induced by polyoma transformation, J. Biol. Chem. 60, Pierce, M. & Ardngo, J. (986) Rous sarcoma virus-transfcrmed baby hamster kidney cells express higher levels of asparaginelinked tri- and tetraantennary glycopeptides containing [GlcNAcp(,6)Man-a(,6)Man] and poly-n-acetyllactosamine sequences than baby hamster kidney cells, J. Biol. Chem. 6, Hiraizumi, S., Takasaki, S., Shiroki, K., Kochibe, N. & Kobata, A. (990) Transfection with fragments of the adenovirus gene induces tumorigenicity-associated alteration of N-linked sugar chains in rat cells, Arch. Biochem. Biophys. 80, Dennis, J. W. & Laferte, S. (989) Oncodevelopmental expression of -GlcNAc~l+6Manal-+6Man~l-branched asparagine-linked oligosaccharides in murine tissues and human breast carcinomas, Cancer Res. 49, Yagel, S., Feinmesser, R., Waghorne, C., Lala, P. K., Breitmm. M. L. & Dennid, J. W. (989) Evidence that p-6 branched Asnlinked oligosaccharides on metastatic tumor cells facilitate invasion of basement membranes, lnt. J. Cancer 44, Easton, E. W., Bolscher, J. G. M. & van den Eijnden, D. H. 99) Enzymatic amplification involving glycosyltransferases forms the basis for the increased size of asparagine-linked glycans at the surface of NIH 3T3 cells expressing the N-ras proto-oncogene, J. Biol. Chem. 66, Easton, E. W., Blokland, I., Geldof, A. A., Rao, B. R. & \.,in den Eijnden, D. H. (99) The metastatic potential of rat prosiate tumor variant R337-MatLyLu is correlated with an increa5ed activity of N-acetylglucosaminyl transferase and V, FEBS Lett. 308, Yamashita, K., Totani, K., Kuroki, M., Matsuoka, Y., Ueda, I. & Kobata, A. (987) Structural studies of the carbohydrate moieties of carcinoembryonic antigens, Cancer Res. 47, Holmes, E. H., Hakomori, S. & Ostrander, G. K. (987) Synthesis of type and type lacto series glycolipid antigens in human colonic adenocarcinoma and derived cell lines is due to activation of a normally unexpressed pl-3n-acetylglucosaminyltransferase, J. Bid. Chem. 6, Miyake, M., Kohno, N., Nudelman, E. D. & Hakomori, S. (989) Human IgG, monoclonal antibody directed to an unbranched repeating type chain (Gal~-.4GlcNAc~l~3Gal~+4Gl~:NAc~- +3Gal/?l-R) which is highly expressed in colonic and hepatocellular carcinoma, Cancer Rex 49, Kobata, A. & Yarnashita, K. (984) The sugar chains of yglutamyl transpeptidase, Pure & Appl. Chem. 56, Takasaki, S. & Kobata, A. (986) Asparagine-linked sugai chains of fetuin : occurrence of tetrasialyl triantennary sugar chajns containing the Gal(jjl-3)GlcNAc sequence, Biochemi., fry 5,