[CANCER RESEARCH 54, 55-61, January 1, 19941 Increase ......[CANCER RESEARCH 54, 55-61, January 1, 19941 Increase of Fucosylated Serum Cholinesterase in Relation to High Risk Groups

[CANCER RESEARCH 54, 55-61, January 1, 19941

Increase of Fucosylated Serum Cholinesterase in Relation to High Risk Groups for

Hepatocellular Carcinomas I

T a k a s h i O h k u r a , T o s h i k a z u H a d a , K a z u y a H i g a s h i n o , T o r u O h u e , N a o h i s a K o c h i b e , N o r i o K o i d e , a n d K a t s u k o Y a m a s h i t a 2

Department of Biochemistry, Sasaki Institute, Tokyo 101 [T. 0., K. Y]; Department of Biology, Faculty of Education, Gunma University, Gunma 371 [N. K.]; Department of Internal Medicine, Hyogo College of Medicine, Hyogo 663 [T. H., K. H., T. 0.]; and Department of Internal Medicine, Okayama University School of Medicine, Okayama 700 [N. K.], Japan

ABSTRACT

Serum cholinesterase (ChE) (E.C. 3.1.1.8) is a glycoprotein which has 36 potential sites of asparagine-N-linked sugar chains. The structures of oligosaccharides released from ChE on hydrazinolysis were studied by serial iectin affinity column chromatography, exoglycosidase digestion, and methylation analysis. Seventy-three % of the sugar chains occurred as biantennary oligosaccharides and the remainder as C-2 and C-2,4/C-2,6 branched tri- and tetraantennary oligosaccharides. Several percentages of the Lewis X antigenic determinant and fucosylated mannose core were linked to them, and their sialic acid residues were linked to nonreclucing terminal galactose residues at the C-3 and C-6 positions.

Aleuria aurantia iectin-reactive ChE with the Lewis X antigenic determinant increased in hepatocellular carcinomas and liver cirrhosis compared with chronic hepatitis; on the other hand, Aleuria aurantia lectin- reactive ChE did not change significantly after transcatheter arterial embolization and was not related to the serum levels of a-fetoprotein and carcinoembryonic antigen in patients with hepatoceilular carcinomas. Ac- cordingly, the analysis ofAleuria aurantia lectin-reactive ChE is clinically useful for differentiating liver cirrhosis from chronic hepatitis and to identify high risk groups for hepatocellular carcinomas, i.e., cirrhotic patients in Child's A grade.

I N T R O D U C T I O N

Approximately 80% of patients with HCC 3 in Japan have associated, underlying LC (1), and recently two-thirds of cirrhotic patients were reported to die of an associated HCC (2). Kobayashi et al. (3) examined the following serum risk factors for the development of HCC in patients with LC, i.e., age, sex, Child's classification (4), hepatitis B virus markers, alcohol intake, history of blood transfusion, and family history of chronic liver diseases, and found that Child's A grade is the most significant factor. Thus, the early discrimination of LC from CH is very important. Once the diagnosis of LC is made, the patient is thought to be in a precancerous state and thus must be carefully followed to detect small-sized HCC as early as possible. But thus far, few biochemical markers are available for discriminating LC from CH.

ChE (E.C. 3.1.1.8) formed in the liver is widely measured as a liver function test that shows protein synthetic activity. Although the activity of serum ChE has been reported to decrease in LC or HCC (5), it is impossible to discriminate LC from CH on the basis of serum ChE activity. Since the electrophoretic patterns of serum ChE in patients with LC were different from those of serum ChE in patients with CH

Received 6/17/93; accepted 10/29/93. The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture, and Special Funds from the Science and Technology Agency of the Japanese Government.

2 To whom requests for reprints should be addressed. 3 The abbreviations used are: HCC, hepatocellular carcinoma; LC, liver cirrhosis; CH,

chronic hepatitis; ChE, cholinesterase; RCA-I, Ricinus communis agglutinin-l; LCA, lentil lectin; L4-PHA, phytohemagglutinin-L4; E4-PHA, phytohemagglutinin-E,~; MAL, Macckia amurensis lectin; ConA, concanavalin A; AAL, Aleuria aurantia lectin; DSA, Datura stramonium agglutinin; TJA-I, Trichosanthes japonica agglutinin-I; i.d., inside diameter; GIcNAc, N-acetylglucosamine; Man, mannose; Neu, neuraminic acid.

55

(6, 7), but the difference disappeared with sialidase digestion (7), it is suggested that the sugar chains of ChE might be altered in patients with LC.

If the structural change of serum ChE in patients with LC can be easily and constantly detected, it may be useful for discriminating LC from CH and for identifying high risk groups for HCC. Because serum ChE consists of four subunits linked through sulfide bonds and each subunit has nine potential asparagine-N-linked sugar chain sites (8), alteration of the sugar chains should be magnified. In the present study, the sugar chain structures of serum ChE and the binding of serum ChE in patients with LC and HCC to an Aleur ia aurantia lectin column will be described.

MATERIALS AND M E T H O D S

Lectins, Chemicals, and Enzymes. RCA-I-agarose (4 mg/ml gel), LCA- agarose (4 mg/ml gel), La-PHA, E4-PHA, and MAL were purchased from Hohnen Oil Corp. (Tokyo, Japan). ConA-Sepharose was from Pharmacia Bio- technology, Inc. (Uppsala, Sweden). AAL-Sepharose (7 mg/ml gel), DSA- Sepharose (3 mg/ml gel), TJA-I-Sepharose (3 mg/ml gel), L4-PHA-Sepharose (9.8 mg/ml gel), E4-PHA-Sepharose (4.8 mg/ml gel), and MAL-Sepharose (10.7 mg/ml gel) were prepared according to the CNBr-method (9).

NaB3H4 (490 mCi/mmol) was purchased from New England Nuclear (Boston, MA). Methyl-a-D-glucopyranoside, methyl-a-o-mannopyranoside, L-fucose, chitin, lactose, Arthrobacter ureafaciens sialidase, and snail/3-mannosidase were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Salmonella typhimurium LT2 sialidase was purchased from Takara Shuzo Co., Ltd. (Kyoto, Japan). Jack bean /3-N-acetylhexosaminidase and ot-mannosidase were prepared by the methods of Li and Li (10). Diplococcal /3-galactosidase and /3-N-acetylhexosaminidase were purified from the culture fluid of Diplococcus pneumoniae according to the method of Glasgow et al. (11). Almond emulsin a-L-fucosidase I was purified as reported previously (12). Bio-Gel P-4 (<45 /xm) was obtained from Bio-Rad Laboratories (Richmond, CA).

Oligosaccharides. (Neu5Acot2--~6)1_z[Gal131-*4GlcNAc131-->2 Man- o~ 1--> 6(Gal/31 ---> 4GIcNAc/31--> 2Manor 1--> 3)Manl31 --.- 4GlcNAcl31--> 4 GIcNA- COT] (abbreviated as Neu5AcI_2.Gal2"GIcNAc2"Man3-GIcNAc'GlcNAcoT) and [Neu5Aca2--->6(3)]3{Gall31-->4GlcNAcl31--->2Manotl--->6 [Gall31--~4 GlcNAc/31--~4(Gall31---~4GlcNAcl31-->2) ManoH--~3]Man/31--~4 Glc- NAc/31---> 4GIcNAcoT} (abbreviated as Neu5mc3" Gal3" GIcNAc3. Man3 -GIcNAc-GlcNACoT) were obtained from ceruloplasmin by hydrazinolysis followed by reduction with NaB3H4 (13). Manal---~6(Manc~l---~3)Manl31--~4 GIcNAcI31--+4GIcNACoT(Man3"GlcNAc'GlcNACoT) and Manotl--> 6 (Manofl--->3)Man/31--,4GlcNAc/31 --,4(Fuccd-->6)GlcNAco-r (Man3.GlcNAc- Fuc.GlcNACoT) were prepared from Neu5Acz-Gal2"GlcNAc2"Man3- �9 GlcNAc'GlcNACoT (13) and Gal2.GIcNAca'Man3"GlcNAc'Fuc'GlcNAco-r (14) by serial digestion with sialidase, 13-galactosidase, and /3-N- acetylhexosaminidase, respectively.

Purification of Serum/Plasma ChE. Ammonium sulfate fractionation of 500 ml of plasma was performed, and the fraction that precipitated between 50 and 65% saturation was resuspended in 100 ml of 0.1 M phosphate buffer (pH 6.7) and then dialyzed against the same buffer. One-fifth of the enzyme solution was applied to a Blue-Sepharose CL-6B column (3.5 i.d. x 15 cm long) equilibrated with 0.1 M phosphate buffer (pH 6.7). The resulting pass- through fraction containing ChE was then applied to a ConA-Sepharose column (1.5 i.d. x 30 cm long) equilibrated with 0.1 M phosphate buffer (pH 6.7) containing 0.15 M NaC1. The eluate with 0.4 r~ methyl-ot-D-mannopyranoside was concentrated and then dialyzed against 25 mM histidine-HC1 buffer (pH

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S U G A R C H A I N S OF S E R U M C H O L I N E S T E R A S E IN LIVER DISEASES

6.3). The dialyzed sample was applied to a chromatofocusing column of PBE 94 gel (1.6 i.d. x 50 cm long) equilibrated with the above buffer, and then the column was eluted isocratically with 8 times-diluted polybuffer 74 (pH 3.0). The ChE eluted at pH 3.8 was concentrated and applied to a TSK G4000 SW column (0.75 i.d. x 30 cm long) equilibrated with 0.1 M phosphate buffer (pH 6.7) containing 0.15 M NaCI. The enzyme fraction was pooled, concentrated, and then stored at -20~ The reduced purified ChE gave a single band at Mr 80,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results of purification are summarized in Table 1.

Release of Asparagine-N-linked Sugar Chains from Serum ChE. Serum ChE (2 mg) was subjected to hydrazinolysis at 100~ for 8 h as reported previously (15). After N-acetylation, one half of the released oligosaccharides was reduced with NaB3I-L and the remaining half was reduced with NaB2H4 for methylation analysis. The total yield of radioactive oligosaccharides was 1.1 • 106 cpm.

Affinity Chromatography of Radioactive Oligosaccharides or Serum ChE on Immobilized Lectin Columns. Columns containing 1 ml of RCA- I-agarose, TJA-I-Sepharose, ConA-Sepharose, E4-PHA-Sepharose, L4-PHA- Sepharose, DSA-Sepharose, AAL-Sepharose, MAL-Sepharose, and LCA-agarose were equilibrated with 10 mM Tris-HC1 (pH 7.4) containing 0.02% NaN3 and 0.15 M NaC1, respectively. Tritium-labeled oligosaccharides or serum ChE dissolved in 100/.d of Tris-HC1 were applied to a column, which was then kept at 4~ for 15 min. Elution was started with 4-10 ml of Tris-HC1 at 4~ followed by 5 ml of Tris-HC1 containing 10 mM lactose (RCA-I column), 0.1 M lactose (TJA-I column), 0.4 M lactose (MAL column), 5 mM methyl-ot-o- glucopyranoside (ConA column), 1% N-acetylglucosamine oligomers (DSA column), 5 a M L-fucose (AAL column), and 0.2 M methyl-c~-t~-mannopyranoside (LCA column), respectively, at 20~

Glycosidase Digestion. Radioactive oligosaccharides were digested in one of the following ways at 37~ for 17 h except for Salmonella sialidase: Salmonella sialidase digestion, 10 milliunits of enzyme in 0.2 M citrate-phosphate buffer, pH 6.0 (20 p.1) for 2 h; Arthrobacter sialidase, 100 milliunits of enzyme in sodium acetate buffer, pH 5.0 (40/xl); digestion with a mixture of diplococcal/3-galactosidase and jack bean 13-N-acetylhexosaminidase, 2 milliunits of/3-galactosidase and 1 unit of 13-N-acetylhexosaminidase in 0.2 M citrate-phosphate buffer, pH 5.5 (20 txl); and digestion with almond Ot-L- fucosidase, 40 microunits of a-L-fucosidase in 0.2 M citrate-phosphate buffer, pH 6.0 (10/xl). The other enzyme digestions were performed according to the procedures described previously (14).

Analytical Methods. Methylation analysis of oligosaccharides was performed as reported previously (16). Analysis of partially O-methylated hexitols and N-acetylglucosaminitols was performed with a gas chromatograph-mass spectrometer (Model GC-MS JMS-SX 102; Japan Electron Optics Laboratory, Tokyo) equipped with a fused silica capillary column coated with cross-linked SPB-35 (0.25 mm i.d. x 30 m long) (17).

High-voltage paper electrophoresis was performed with pyridine-acetate buffer, pH 5.4 (pyridine:acetic acid:water, 3:1:387) at a potential of 73 V/cm for 90 min. Radiochromatoscanning was performed with a Raytest radiochro- matogram scanner (Model RITA-90).

Bio-Gel P-4 (<45 /~m) column chromatography (2 cm i.d. x 1.25 m long) was performed as reported previously (18). Radioactivity was determined with a Beckman liquid scintillation spectrometer (Model LS-6000 LL).

Serum Samples. Serum samples were obtained from 50 patients with HCC with LC or CH, 47 patients with LC, 40 patients with CH, and 20 normal controls. The serum samples were stored at -30~ until used. The diagnoses of liver diseases were made on the bases of the results of liver function tests,

Table 1 Purification of cholinesterase from human plasma

Total Total Specific protein units activity = Purification

Step (mg) ( IU) (IU/mg) (fold)

Plasma 40000 791.1 0.02 1 (NH4)2SO4 (50-65%) 11352 1054.8 0.09 4.5 Blue-Sepharose 7279 1107.5 0.15 7.5 Con A-Sepharose 1740 580.1 0.33 16.5 Chromatofocusing 42.5 332.3 7.82 391.0 G 4000 SW 4.38 152.9 34.91 1745.5

= Based on the hydrolysis of benzoylcholine chloride (Cholinesterase B-test Wako). One IU was taken as the amount of enzyme which cleaved 1 mol of substrate in 1 min at 37~

.'2_ >

, m

o m 0

= m

" 0

t~

A 1 2 3

B

(- ) 0 10 2 ~ 30 (§ D i s t a n c e f r o m o r i g i n ( c m )

Fig. 1. Paper electrophoresis of oligosaccharides released from serum ChE on hydrazinolysis followed by reduction with NaB3H4. Arrows, the migration positions of authentic oligosaccharides: 1, Neu5Ac.GaI2.GIcNAc2-Mana-GIcNAc'GIcNAcoT; 2, Neu5Acz. GaI2.GIcNAc2.Man3.GIcNAc'GIcNAcoT; 3, Neu5Aca.Gal3-GlcNAc3.Man3.GlcNAc" GlcNACoT. A, oligosaccharides obtained from serum ChE; B, radioactive fractions A1-A5 digested with Salmonella sialidase; C, radioactive acidic fractions in B digested with Arthrobacter sialidase.

ultrasonography, computed tomography, angiography, and histological exami- nation of liver specimens obtained at biopsy or operation. Serum ChE activity was measured with ChE B-test Wako (Wako Pure Chemical Co., Osaka, Japan). One IU was taken as the amount of enzyme which hydrolyzed i/zmol of benzoylcholine chloride in 1 min at 37~ The concentrations of a-fetoprotein and carcinoembryonic antigen in the samples were determined by an ELISA.

RESULTS

Structural Studies of Oligosaccharides Released from Serum ChE

Paper Electrophoresis of Tritium-labeled Oligosaccharides Re- leased from Serum ChE. When the tritium-labeled oligosaccharides released from serum ChE were subjected to paper electrophoresis,

they were separated into five acidic fractions (A1, A2, A3, A4, and A5) as shown in Fig. 1A. The percentage molar ratio of A1, A2, A3, A4, and A5 was calculated to be 39:14:24:11:12 on the basis of

their radioactivities. A part of these acidic oligosaccharides was converted to neutral oligosaccharides by Salmonella sialidase digestion, which specifically hydrolyzes Neu5Aca2--->3Gal{31--> groups (Ref. 19; Fig. 1B). The remaining acidic oligosaccharides were adsorbed to a TJA-I column, which specifically interacts with Neu5Aco~2~6- Gall31--->4GlcNAc group (20), and then converted to neutral oligosaccharides by exhaustive Arthrobacter sialidase digestion (Fig. 1C). The results indicated that the sialic acid residues are linked to {3-galactosyl residues at the C-3 or C-6 position. Furthermore, the oligosaccharides in fraction A1 were confirmed to be monosialyl

derivatives, those in fractions A2 and A3 to be disialyl derivatives, and those in A4 and A5 to be trisialyl derivatives by analyzing the number of acidic fractions produced from each fraction on partial

desialylation (data not shown). On digestion with a mixture of diplococcal /3-galactosidase, almond C~-L-fucosidase and jack bean 13-N-acetylhexosaminidase, which specifically hydrolyzes Gal/31 -->4GIcNAc and Gal/31-->4(Fucal-->3)GlcNAc groups, the radioac-

tive neutral fractions were converted to two radioactive oligosaccha-

56



SUGAR CHAINS OF SERUM CHOL1NESTERASE IN LIVER DISEASES

Table 2 Methylation analysis of desialylated oligosaccharides (AN) obtained from serum ChE

Partially methylated sugars AN

Fucitol 2,3,4-Tri-O-methyl- 1,5-di-O-acetyl

Galactitol 2,3,4,6-Tetra-O-methyl- 1,5-di-O-acetyl

Mannitol 3,4,6-Tri-O-methyl-l,2,5-tri-O-acetyl 3,6-Di-O-methyl-l,2,4,5-tetra-O-acetyl 3,4-Di-O-methyl-l,2,3,5-tetra-O-acetyl 2,4-Di-O-methyl-l,3,5,6-tetra-O-acetyl

N-Methylacetamido-2-deoxyglucitol 1,3,5,6-Tetra-O-methyl-4-mono-O-acetyl 1,3,5-Tri-O-methyl-4,6-di-O-acetyl 3,6-Di-O-methyl-l,4,5-tri-O-acetyl 6-Mono-O-methyl-l,3,4,5-tetra-O-acetyl

Molar ratio a trace b

2.3

1.7 0.3 0.1 1.0

0.6 trace

3.5 trace

= The values in the table were calculated by taking the value for 2,4-di-O-methyl mannitol as 1.0.

b Trace; less than 0.05.

rides with the same mobilities as authentic Man3.GlcNAc. Fuc-GlcNAcoT and Man3.GlcNAc-GlcNACoT, as judged on analysis of a Bio-Gel P-4 column. The structures of the hexaitol and pentaitol were further confirmed to be Man~l-->6(Manal-->3)Man/31--> 4GlcNActB1---->4(Fucal--~6)GlcNAcor and Manotl--->6(Manotl--->3) Man/31--> 4GlcNAc/31--->4GlcNACoT by sequential exoglycosidase digestion as described previously (14). These results indicated that all the oligosaccharides comprise complex type sugar chains with a fucosylated or nonfucosylated mannose core and contain different numbers of Gal/31-->4GIcNAc or Gall31--~4(Fuco~l--->3)GlcNAc units in their outer chain moieties. Methylation analysis of the desialylated oligosaccharides also indicated that two o~-mannosyl residues are substituted at the C-2, C-2 and C-4, and C-2 and C-6 positions, respectively, and all galactose residues occur as nonreducing termini, indicating that the Gal/31---~4GlcNAc/31-->3 repeating structures are not included in their outer chain moieties (Table 2).

Fractionation of the Oligosaccharides from Serum ChE by Se- rial Lectin Affinity Chromatography and Bio-Gel P-4 Column Chromatography. All of the neutral oligosaccharides bound on RCA-I-agarose column chromatography (Fig. 2..4). On ConA-Sepha-

rose column chromatography, these neutral oligosaccharides were separated into a pass-through fraction (ConA-), and a fraction that

bound to the column and was eluted with 5 mM methyl-a-glucopyranoside (ConA +) (Fig. 2B). The ConA + fraction passed through a DSA-Sepharose column (Fig. 2C) and was retarded on a Ea-PHA- Sepharose column (Fig. 2D). On an AAL-Sepharose column, the ConA + fraction was separated into a pass-through fraction (AAL-), and a fraction that bound to the column and was eluted with 5 mM L-fucose (AAL § (Fig. 2E). These fractions were named fractions I and II, respectively (Fig. 2E). The CortA- fraction was separated into three fractions on a DSA-Sepharose column chromatography, which were named the DSA-, DSA r and DSA § fractions, respectively (Fig. 2F). The DSA § fraction was retarded on a L4-PHA-Sepharose column (Fig. 2G) and passed through an AAL-Sepharose column (Fig. 2H). The DSA r fraction was only retarded on an E4-PHA-Sepharose column (Fig. 2/) passing through an AAL-Sepharose column (Fig. 2/) . These DSA § and DSA r fractions were named fractions III and IV, respectively. The DSA- fraction in Fig. 2F was retarded on an AAL- Sepharose column (Fig. 2K) and passed through an L4-PHA-Sepha- rose column (Fig. 2L). On an Ea-PHA-Sepharose column, the L4- PHA- fraction in Fig. 2L was separated into a pass-through fraction and a retarded fraction, which were named fractions V and VI, respectively (Fig. 2M). Fractions V and VI were digested with almond ot-L-fucosidase I, which specifically hydrolyzes Fuco~l--->3GlcNAc or Fucal--->4GlcNAc (21), and then were analyzed on a DSA-Sepharose column. Fraction V bound to the column and was eluted with 1% N-acetylglucosamine oligomers and was named fraction V' (Fig. 2N). Fraction VI was retarded on the column (Fig. 2 0 ) and was named fraction VI' .

When the six fractions (I-VI; Fig. 2) were analyzed by Bio-Gel P-4 column chromatography (18), fractions I, II, IV, V, and VI gave symmetrical single peaks, and fraction III was separated into two components, IIIa and IIIb, as summarized in Table 3.

Structures of the Neutral Oligosaccharides. Structural analysis revealed that fractions I -VI contained one oligosaccharide, respectively. Therefore, they will be called oligosaccharides I, II, IIIa, IIIb,

Fig. 2. Serial immobilized lectin column chromatography of the radioactive desialylated oligosaccharides derived from serum ChE. The tritium- labeled oligosaccharide fraction was chroma- tographed sequentially on various immobilized lec- ~, tin columns as described in "Materials and Meth- ,~_ ods." The resultant fractions were then subjected to .~ the next lectin column chromatography (arrows). *~ Small arrows, positions where buffers were switched to those containing the respective hap- .-0 tenic sugars as described in "Materials and "O

t~ Methods." IX:

aH-Desialylated oligosaccharides

' RCA ~

AAL B '~ "' '

0-- ~ -10---15 Con

o~ -~'-[-, -10 [L 2~'C I

_ d ~ _ �9 I . . . . a . . . . .

E4-PHA E4-PHA Almond a-L- Fucosidase

DSA ! . , ~ DSA AAL , O- ..~ 10

. . . . | . . . . . . . . . . .

0- - - -5 - -~10 -15 0 - - 5 10-- 15 0 5 10 0 5 10 Elut ion vo lume (ml)

57



SUGAR CHAINS OF SERUM CHOLINESTERASE IN LIVER DISEASES

Table 3 Behavior on several lectin columns, glucose units on a Bio-Gel P-4 column, numbers of released N-acetyllactosamine residues, and proposed structures of oligosaccharides I-VI

Behavior on lectin columns a Numbers of Oligosaccharides Glucose released (% molar ratio) ConA DSA E4-PHA L4-PHA AAL units t' N-acetyllactosamine c Proposed structures

I (67.5) + - r - - 13.5 2

II (5.5) + - r - + 14.5 2

I I I a ( 3 . 8 ) - + - r - 1 8 . 5 4

IIIb(V') (2.1) - + - r - 16.0 3

IV (VI') (14.9) - r r - - 16.5 3

V (2 .3) . . . . r 16.5 3 f

VI (3.9) - - r - r 17.0 3 f

Gall31~ 4GIcNAc/31---~ 2Manotl

"~ 6Man/31___>Rld 3

Gal/31~4GlcNAc/31~ 2Manal S

Gal/31--,4GlcNAc/31--> 2Manal ~ 6Man/31--> R2 e

Gal/31---, 4GIcNAc/31---> 2Manor 1 "~ 3

Gal/31-->4GIcNAc/31 "~ 6

2Manal -,~

Gal/31--,4GIcNAc/31/" 6Man/31~R 1 Gal/3--~ 4GalcNAc/31 "~4 3

/* 2Mancd

Gal/31 --> 4GlcNAc/31 ,z

Gal/31--,4GIcNAc/31 ",.a 6

2Maned

Gal/31--> 4GlcNAc/31/* "a 6Man/31-~.R1 3

Gal/31---> 4GlcNAc/31---> 2Manod Z

Gal/31--> 4GlcNAc/31--> 2Mana 1 "~6

Gal/31-->4GIcNAc/31 Man/31---> R l "a 4 / 3

2Manal

Gal/31~4GIcNAc/31 '

Fuctxl "~ 2

Gal/31~ 4Glciq3Ac/31 (6 )

2 Mantel,, S 6

Gal/31-->4GIcNAc/31 ( 6 ) 3 Man/31---> R1 S

Gal/31---> 4GlcNAc/31--> 2 Manal

Gal/31-~4GlcNAc/31~2Manal Fucotl

"-a 2 "a6 Man/31~R l Gal/31~4GIc/(I3Ac/31 ( ) 3

4 Manod/'

Gal/31--->4GlcNAc/31/~ 2 (4)

a +, -, r indicate bound, passed through, and retarded on the respective lectin columns, respectively. b Glucose units indicate the effective sizes of oligosaccharides on a Bio-Gel P-4 column. c Numbers of released N-acetyllactosamine residues on digestion with a mixture of diplococcal/3-galatosidase and jack bean/3-N-acetylhexosaminidase. d Rl indicates 4GIcNAc/31-~4GIcNAcoT. e R2 indicates 4GlcNAc/31-->4(Fucal~6)GlcNAcox. f Oligosaccharides V and VI digested with almond a-L-fucosidase were used.

IV, V, and VI, respectively. On digestion with a mixture of diplococ-

cal /3-galactosidase and jack bean/3-N-ace ty lhexosaminidase , differ-

ent numbers of N-acetyl lactosamine residues were released f rom oli-

gosaccharides I, 11, II1a, IIIb, and IV, and Ct-L-fucosidase digested

oligosaccharides V and VI (V' and VI ') . By compar ing the mobil i ty

o f each ol igosaccharide before and after these glycosidase diges-

tions, the numbers of N-acetyl lactosamine residues released f rom oli-

gosaccharides I, II, Ilia, Illb, IV, V' , and VI ' were calculated to be 2,

2, 4, 3, 3, 3, and 3, respectively (Table 2), and then these enzyme-

digested ol igosaccharides I, IIIa, IIib, IV, V' , and VI ' were con-

verted to t r i -mannosyl N,N'-diacetylchitobii tol ; and oligosaccharide

11 was converted to fucosyla ted t r i -mannosyl N,N'-diacetylchi tobi i tol

(data not shown). The glycosidic l inkages and locations of the outer

chains in these seven oligosaccharides were determined by methyl-

ation analysis and f rom the behavior of the ol igosaccharides on

immobi l ized ConA, DSA, E4-PHA, L4-PHA, and A A L columns

(Table 3). Oligosaccharides containing the Gal/31--->4GIcNAc/31--->6

(Gal/31---> 4 G l c N A c j31---> 2 ) M a n a 1---> 6Man/31--> 4GlcNAc/31---> 4GI-

c N A c o x group are retarded on an La-PHA column and adsorbed to a

D S A column and eluted with 1% N-acety lg lucosamine ol igomers (22).

58




M e a n + S D 60.1_+17.2 57.3_+17.5 21.1_+8.0 16.9_+5.4 1 0 0

8 0

LIJ

0 6 0 ,=

, , I

0

| t . . _

, - I < 1 : 4 0 <

2 0

n=50 n=47

: � 9 =�9

o � 9 � 9 1 4 9

�9 0 o O e o e

�9 "~ ":1 0~ 0 �9

OO

0 o � 9 o ~ e � 9

o o 0 �9

o � 9 � 9

0 �9 o � 9

o o o 0 o O o

n=40

. . . . . - e . . . .

oo

�9

"|:

n--20

o � 9

O : o � 9

e � 9 o o o � 9 o � 9

HCC LC CH NC Fig. 3. Mean + SD of serum ChE reactive to AAL-Sepharose in patients with HCC,

LC, CH, and in healthy individuals (NC). �9 and �9 in HCC, HCC developing after LC and HCC developing after CH, respectively.

Oligosaccharides containing the Gal/31---~ 4 GlcNAc/31----~ 2Manod--*6Man/31--* 4GlcNAc/31---~4GIcNAco.r group are retarded on an Ea-PHA column at 4~ (23); ones containing the Gal/31--*4GIcNAc/31--*4(GalIS1---~4GIcNAc/31 --~2)Man group are retarded on a DSA column (24); oligosaccharides with two C-2 substituted a-mannosyl residues bind to a ConA-Sepharose column and can be eluted with 5 mM methyl-a-glucopyranoside (22); oligosaccharides with a Lewis X antigenic determinant are retarded on an AAL column (25); and ol igosacchar ides with fucosylated proximal N-acetylglucosaminitol at the C-6 position bind to an AAL column (25). Accordingly, ConA+E4-PHA r oligosaccharide I should be a biantennary component with a nonfucosylated mannose core; C o n m + E 4 -

P H A r A A L + oligosaccharide II a biantennary component with a fucosylated mannose core; DSA+La-PHA r oligosaccharide Ilia a C-2,6 and C-2,4 branched tetraantennary component; DSA+La-PHA r oligosaccharide IIIb a C-2,6 and C-2 branched triantennary component; and DSArE4-PHA r oligosaccharide IV a C-2,4 and C-2 branched triantennary component. AAU oligosaccharide V was converted to a DSA + component (fraction V'; Fig. 2N) on almond Ot-L-fucosidase I digestion, indicating that it is a C-2,6 and C-2 branched triantennary component with a Lewis X antigenic determinant. A A L r E a - P H A r oligo-

saccharide VI was converted to a DSA' component (fraction VI'; Fig. 20) on almond C=-L-fucosidase I digestion, indicating that it is a C-2,4 and C-2 branched triantennary component with a Lewis X antigenic determinant. From the results so far described, the structures of oligosaccharides I-VI are proposed to be as shown in Table 3.

Sugar Chain Structural Changes of Serum Cholinesterase in Patients with Hepatocellular Carcinomas and Benign Liver Diseases

The activity of serum ChE has been reported to decrease in LC; however, it is impossible to discriminate LC from CH on the basis of serum ChE activity. We preliminarily investigated, by means of several lectin column chromatographies, whether the sugar chain structures of serum ChE are altered in HCC and benign liver diseases. All the serum ChE in patients with HCC, LC, CH, and in the healthy individuals bound to RCA, MAL, ConA, TJA-I, and DSA columns (data not shown). Interestingly, the rate of binding of serum ChE to an AAL-Sepharose column greatly increased in HCC and LC compared to that of CH and healthy individuals. Because fucosylation of the sugar chains is the molecular basis for AAL-reactive variation of serum ChE, in an attempt to increase the efficiency of discriminating CH from LC, ChE was studied by means of AAL-Sepharose column chromatography.

AAL-binding Serum ChE in Patients with HCC and Benign Liver Diseases. The percentages of AAL-binding serum ChE in patients with HCC and benign liver diseases are shown in Fig. 3. The sera studied were from 50 patients with HCC, 47 with LC, and 40 with CH. The mean percentages of AAL-binding serum ChE were 60.1 _ 17.2% (SD) in HCC, 57.3 _ 17.5% in LC, 21.1 _ 8.0% in CH, and 16.9 ___ 5.4% in healthy individuals (Fig. 3), and the percentage of fucosylated ChE was significantly higher in LC and HCC than in CH and healthy individuals (P < 0.01). The percentage of AAL-binding ChE in HCC was the same as in LC. Because 80% of HCC patients (42 of 50) had cirrhotic lesions in their livers, AAL-binding ChE may

H C C L C C H N C

~ 118O/o 117%

Jk_ :,5_ A A L

| m ~ - 3 - T

0.4 60% 17%

o o 4 a 0;=:4 a 0 - = 4 a Elu t ion v o l u m e (ml )

Fig. 4. Serial A A L and LGA column chromatography of serum ChE in HCC, LC, CH, and a healthy individual (NC).

4 0 0

AAL § LCA- A A L + LCA +

3 0 0 , - :. �9 l = l " :

i O " O �9 O e

,,,200- ":" "~, - | .... : .: ~= o .. | : 0 �9 00 �9 e � 9

o0�9 00 �9 oe�9149 �9 �9

1 0 0 - ":. t I " - :- . | . �9 �9 l" I �9 I �9 I �9 ~

,I , o,1 ~ 0 L "" . . . .

I

HCC LC CH NC HCC LC CH N C

Fig. 5. Serum concentrations (IU/liter) of AAL+LCA - ChE and AAL+LCA + ChE in HCC, LC, CH, and in healthy individuals (NC).

59



SUGAR CttAINS OF SERUM CHOLINESTERASE IN lAVER DISEASES

Table 4 Mean • SD serum activity (lU/liter) toward AAL +LCA +, and AAL +LCA-ChE in hepatic diseases

Mean _+ SD (IU/liter) Hepatic diseases Total ChE AAL + LCA-ChE a AAL + LCA + ChE h AAL-LCA-ChE c

HCC (n = 14) 683.6 + 235.6 251.2 +_ 57.0 145.0 +- 38.8 287.4 • 181.3 LC (n = 21) 647.4 _ 282.2 212.3 • 93.2 134.7 _+ 51.8 300.4 • 214.9 CH (n = 17) 1421.6 ___ 391.0 44.0 • 36.3 230.5 +-- 82.9 1147.1 --- 344.4 NC (n = 16) 1320.6 +-- 334.0 41.4 • 44.0 199.4 +_ 38.8 1320.6 +- 334.0

a AAL+LCA-ChE, ChE containing X-aatigenic determinant. b AAL+LCA+ChE, ChE containing fucosylated mannose core. c AAL-LCA-ChE, nonfucosylated ChE.

be produced in liver cirrhotic tissue and may be indicative of a predisposition to HCC. This hypothesis was supported by the following results; the percentage of AAL-binding ChE was not correlated with the level of serum ot-fetoprotein or carcinoembyonic antigen (data not shown). When the changes in the serum AAL-binding ChE percentage in 10 patients with HCC were examined before and after transcatheter arterial embolization treatment, the percentage of AAL- binding ChE did not change with the treatment except in one case (data not shown).

Serial Leetin Column Chromatography on AAL-Sepharose and LCA-Agarose Columns. AAL interacts with various fucosyl residues, including Fucal-->2Galf31-->4GlcNAc, Galf31-->4(Fuco11-->3) GIcNAc, Gal/31-->3(Fucotl-->4)GlcNAc, and Fucoll--->6GlcNAc residues (25). Because the Gall31-->4(Fuco~l-->3)GlcNAc group (Lewis X antigenic determinant) and the Fucctl-->6GlcNAc group are present in the sugar chains of serum ChE (see Table 3), the type of fucosyl residues in serum ChE which increases in high risk groups for HCC should be determined. LCA specifically interacts with --->2(-->6) Manct 1--> 6(--> 2Manol 1--> 3)Man/31----> 4GlcNAc/31--> 4(Fuco~ 1--> 6)Glc- NAc-->Asn (26). Accordingly, the problem should be resolved by AAL and LCA serial lectin column chromatographies. After the intact serum ChE in patients with HCC, LC, CH, and in a healthy individual had been separated on an AAL-Sepharose column, the respective AAL + ChE was sequentially applied to an LCA-agarose column. As shown in Fig. 4, most AAL + ChE in a patient with CH and in a healthy individual was bound to an LCA-agarose column and eluted with 0.2 M methyl-ot-mannopyranoside; on the other hand, the AAL + ChE in patients with HCC and LC was separated into LCA § and LCA- fractions, indicating that AAL+LCA + ChE should have oligosaccharides with a fucosylated mannose core (oligosaccharide II; Table 3), and AAL+LCA - ChE should have oligosaccharides containing a Lewis X antigenic determinant (oligosaccharides V and VI; Table 3). The percentage of AAL-reactive ChE in patients with HCC, LC, and CH did not alter by desialylation compared to that of intact ChE (data not shown). However, it could not be determined whether the Lewis X antigenic determinants were sialylated because we have not yet analyzed whether neutral oligosaccharides and sialylated oligosaccharides differ in their affinity to AAL column by using standards.

The serum concentrations of total ChE, AAL+LCA - ChE, AAL+LCA + ChE, and AAL-LCA- ChE in patients with HCC and LC (n = 14), LC (n = 21), CH (n - 17), and in healthy individuals (n = 16) were investigated. As shown in Fig. 5 and Table 4, AAL+LCA + ChE containing a fucosylated mannose core did not change or rather decreased in HCC and LC, and total serum ChE greatly decreased; on the other hand, AAL+LCA-ChE containing a

Lewis X antigenic determinant increased about 5 times in HCC and LC, in comparison with CH and healthy individuals. These results indicate that the analysis of AAL-reactive ChE is clinically useful for differentiating LC from CH and for identifying high risk goups for HCC.

60

DISCUSSION

This report deals with the structures of N-linked sugar chains of serum ChE and the increase of AAL-reactive serum ChE in relation to

high risk groups for HCC. The sugar chains of serum ChE were determined to be sialylated

bi-, tri-, and tetraantennary oligosaccharides containing a trace amount of a Lewis X antigenic determinant and a fucosylated mannose core, although it could not be determined in this study whether the Lewis X antigenic determinant is sialylated. These structures are generally observed in serum glycoproteins, including ceruloplasmin, al-anti- trypsin, orosomucoid, etc., which are produced in parenchymal cells of human liver and secreted (27). Furthermore, it was elucidated in this study that AAL-reactive serum ChE increases in LC and HCC with LC and that the measurement of AAL-binding serum ChE is useful not only for discriminating LC from CH but also for identifying high risk groups for HCC according to the results of Kobayashi et al.

(3). An increase of sugar chains containing Lewis X antigenic determi-

nants had already been found in serum transferrin from patients with HCC developing after LC (28). Because fucosylated transferrin is also produced in cirrhotic liver cells'* and Gal/31-->4(Fucal-->3)GlcNAc groups increased in sugar chains of al-acid glycoprotein purified from cirrhotic ascitic fluid (29), fucosylation seems to be a general phe- nomenon in glycoproteins produced in human cirrhotic liver cells. A similar increase of AAL-reactive ChE was observed in patients with primary biliary cirrhosis or lupoid cirrhosis (data not shown); on the other hand, sugar chains containing Lewis X antigenic determinant could not be detected in a-fetoprotein (30) or -y-glutamyltranspeptidase (31), which should be produced in hepatoma cells. These results also support the idea that the fucosylation may be related to cirrhotic liver cells irrespective of the primary pathological cause.

Patients infected with hepatitis virus B or C often develop chronic hepatitis, and the liver cells undergo repeated necrosis and regenera- tion. After some interval, the liver cells of the patients change irre- versibly, accompanied by fibrosis and nodules. The few liver cells remaining in the cirrhotic liver surrounded by nodules regenerate. Perhaps glycoproteins produced in such cirrhotic liver cells may syn- thesize sugar chains with a Lewis X antigenic determinant.

We previously reported that the expression of a fucosylated mannose core in ot-fetoprotein produced in hepatoma cells might be related to depolarization of hepatocytes by malignant transformation (30). On the contrary, the fucosylated mannose core of serum ChE did not increase in HCC patients, indicating that most serum ChE is not produced in malignant liver cells.

Although it has not yet been elucidated what biological function, if

any, serum glycoproteins with Lewis X antigenic determinants have, the functional role of the Lewis X antigenic determinant of glycopro-

4 Manuscript in preparation.




teins produced in cirrhotic tissues may be similar to that of the stage- specific embryonic antigen, which is related to cell compaction in the morula (32, 33).

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1994;54:55-61. Cancer Res Takashi Ohkura, Toshikazu Hada, Kazuya Higashino, et al. High Risk Groups for Hepatocellular CarcinomasIncrease of Fucosylated Serum Cholinesterase in Relation to

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