[CANCER RESEARCH 54, 55-61, January 1, 19941
Increase of Fucosylated Serum Cholinesterase in Relation to High Risk Groups for
Hepatocellular Carcinomas I
Takashi Ohkura, Toshikazu Hada, Kazuya Higashino, Toru Ohue, Naohisa Kochibe, Norio Koide, and
Katsuko Yamashita 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 lectinreactive 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. Accordingly, 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.
(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 A l e u r i a aurantia lectin
column will be described.
M A T E R I A L S AND M E T H O D S
Lectins, Chemicals, and Enzymes. RCA-I-agarose (4 mg/ml gel), LCAagarose (4 mg/ml gel), La-PHA, E4-PHA, and MAL were purchased from
Hohnen Oil Corp. (Tokyo, Japan). ConA-Sepharose was from Pharmacia Biotechnology, Inc. (Uppsala, Sweden). AAL-Sepharose (7 mg/ml gel), DSASepharose (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).
INTRODUCTION
NaB3H4 (490 mCi/mmol) was purchased from New England Nuclear
(Boston, MA). Methyl-a-D-glucopyranoside, methyl-a-o-mannopyranoside,
Approximately 80% of patients with HCC 3 in Japan have associL-fucose, chitin, lactose, Arthrobacter ureafaciens sialidase, and snail/3-manated, underlying LC (1), and recently two-thirds of cirrhotic patients
nosidase were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Salmonella
were reported to die of an associated HCC (2). Kobayashi et al. (3)
typhimurium LT2 sialidase was purchased from Takara Shuzo Co., Ltd. (Kyoto,
examined the following serum risk factors for the development of Japan). Jack bean /3-N-acetylhexosaminidase and ot-mannosidase were preHCC in patients with LC, i.e., age, sex, Child's classification (4),
pared by the methods of Li and Li (10). Diplococcal /3-galactosidase and
/3-N-acetylhexosaminidase were purified from the culture fluid of Diplococcus
hepatitis B virus markers, alcohol intake, history of blood transfusion,
and family history of chronic liver diseases, and found that Child's A 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
grade is the most significant factor. Thus, the early discrimination of
/xm) was obtained from Bio-Rad Laboratories (Richmond, CA).
LC from CH is very important. Once the diagnosis of LC is made, the
Oligosaccharides. (Neu5Acot2--~6)1_z[Gal131-*4GlcNAc131-->2 Manpatient is thought to be in a precancerous state and thus must be
o~1-->6(Gal/31--->4GIcNAc/31-->2Manor1-->3)Manl31--.-4GlcNAcl31-->4 GIcNAcarefully followed to detect small-sized HCC as early as possible. But
COT] (abbreviated as Neu5AcI_2.Gal2"GIcNAc2"Man3-GIcNAc'GlcNAcoT)
thus far, few biochemical markers are available for discriminating LC
and [Neu5Aca2--->6(3)]3{Gall31-->4GlcNAcl31--->2Manotl--->6 [Gall31--~4
from CH.
GlcNAc/31--~4(Gall31---~4GlcNAcl31-->2) ManoH--~3]Man/31--~4 GlcChE (E.C. 3.1.1.8) formed in the liver is widely measured as a liver
NAc/31--->4GIcNAcoT} (abbreviated as Neu5mc3" Gal3" GIcNAc3. Man3
function test that shows protein synthetic activity. Although the ac-GIcNAc-GlcNACoT)were obtained from ceruloplasmin by hydrazinolysis followed by reduction with NaB3H4 (13). Manal---~6(Manc~l---~3)Manl31--~4
tivity of serum ChE has been reported to decrease in LC or HCC (5),
GIcNAcI31--+4GIcNACoT(Man3"GlcNAc'GlcNACoT) and Manotl--> 6
it is impossible to discriminate LC from CH on the basis of serum ChE
(Manofl--->3)Man/31--,4GlcNAc/31--,4(Fuccd-->6)GlcNAco-r (Man3.GlcNAcactivity. Since the electrophoretic patterns of serum ChE in patients
Fuc.GlcNACoT) were prepared from Neu5Acz-Gal2"GlcNAc2"Man3with LC were different from those of serum ChE in patients with CH
9GlcNAc'GlcNACoT (13) and Gal2.GIcNAca'Man3"GlcNAc'Fuc'GlcNAco-r
(14) by serial digestion with sialidase, 13-galactosidase, and /3-NReceived6/17/93; accepted 10/29/93.
acetylhexosaminidase, respectively.
The costs of publicationof this article were defrayedin part by the paymentof page
Purification of Serum/Plasma ChE. Ammonium sulfate fractionation of
charges. This article must thereforebe hereby markedadvertisement in accordancewith
500 ml of plasma was performed, and the fraction that precipitated between 50
18 U.S.C. Section 1734 solely to indicatethis fact.
1This work was supportedby a Grant-in-Aidfor Cancer Researchfrom the Ministry and 65% saturation was resuspended in 100 ml of 0.1 Mphosphate buffer (pH
of Education,Scienceand Culture, and Special Funds from the Scienceand Technology 6.7) and then dialyzed against the same buffer. One-fifth of the enzyme
Agency of the Japanese Government.
solution was applied to a Blue-Sepharose CL-6B column (3.5 i.d. x 15 cm
2 To whom requests for reprints shouldbe addressed.
3 The abbreviationsused are: HCC, hepatocellularcarcinoma;LC, liver cirrhosis; CH, long) equilibrated with 0.1 M phosphate buffer (pH 6.7). The resulting passchronichepatitis;ChE,cholinesterase;RCA-I,Ricinus communis agglutinin-l;LCA,lentil through fraction containing ChE was then applied to a ConA-Sepharose collectin; L4-PHA, phytohemagglutinin-L4; E4-PHA, phytohemagglutinin-E,~; MAL, umn (1.5 i.d. x 30 cm long) equilibrated with 0.1 Mphosphate buffer (pH 6.7)
Macckia amurensis lectin; ConA, concanavalinA; AAL, Aleuria aurantia lectin; DSA,
Datura stramonium agglutinin;TJA-I, Trichosanthes japonica agglutinin-I;i.d., inside 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
diameter; GIcNAc,N-acetylglucosamine;Man, mannose;Neu, neuraminicacid.
55
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Research.
SUGAR CHAINS
OF SERUM
CHOLINESTERASE
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.
IN L I V E R
A
DISEASES
1
2
3
.'2_
>
,m
o
m
0
B
= m
"0
t~
Affinity Chromatography of Radioactive Oligosaccharides or Serum
ChE on Immobilized Lectin Columns. Columns containing 1 ml of RCAI-agarose, TJA-I-Sepharose, ConA-Sepharose, E4-PHA-Sepharose, L4-PHASepharose, 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 MNaC1, 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-oglucopyranoside (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-Lfucosidase, 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 radiochromatogram 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,
(-)
0
Distance
10
from
2 ~
origin(cm)
30 (§
Fig. 1. Paper electrophoresisof oligosaccharidesreleased from serum ChE on hydrazinolysis followedby reductionwith NaB3H4.Arrows, the migrationpositions 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, oligosaccharidesobtainedfrom serumChE;B, radioactivefractionsA1-A5
digested with Salmonella sialidase; C, radioactive acidic fractions in B digested with
Arthrobacter sialidase.
ultrasonography, computed tomography, angiography, and histological examination 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 Released 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~2~6Gall31--->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 radioactive neutral fractions were converted to two radioactive oligosaccha-
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 benzoylcholinechloride (CholinesteraseB-test Wako).
One IU was taken as the amount of enzymewhich cleaved 1 mol of substrate in 1 min at
37~
56
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SUGAR CHAINS OF SERUM CHOL1NESTERASE IN LIVER DISEASES
Table 2 Methylation analysis of desialylated oligosaccharides (AN) obtained from
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-PHASepharose 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 AALSepharose column (Fig. 2K) and passed through an L4-PHA-Sepharose column (Fig. 2L). On an Ea-PHA-Sepharose column, the L4PHA- 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,
serum ChE
Partially methylated sugars
AN
Fucitol
Molar ratioa
traceb
2,3,4-Tri-O-methyl-1,5-di-O-acetyl
Galactitol
2,3,4,6-Tetra-O-methyl-1,5-di-O-acetyl
2.3
Mannitol
3,4,6-Tri-O-methyl-l,2,5-tri-O-acetyl
1.7
3,6-Di-O-methyl-l,2,4,5-tetra-O-acetyl
0.3
3,4-Di-O-methyl-l,2,3,5-tetra-O-acetyl
0.1
2,4-Di-O-methyl-l,3,5,6-tetra-O-acetyl
1.0
N-Methylacetamido-2-deoxyglucitol
1,3,5,6-Tetra-O-methyl-4-mono-O-acetyl
0.6
1,3,5-Tri-O-methyl-4,6-di-O-acetyl
trace
3,6-Di-O-methyl-l,4,5-tri-O-acetyl
3.5
6-Mono-O-methyl-l,3,4,5-tetra-O-acetyl
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 Serial 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-
aH-Desialylated
'
Fig. 2. Serial immobilized lectin column chromatography of the radioactive desialylated oligosaccharides derived from serum ChE. The tritiumlabeled oligosaccharide fraction was chromatographed sequentiallyon various immobilized lectin columns as described in "Materials and Methods." The resultant fractionswere then subjected to
the next lectin column chromatography (arrows).
Small arrows, positions where buffers were
switched to those containing the respective haptenic sugars as described in "Materials and
Methods."
~
RCA
AAL
B
0-- ~
-10---15
[L
2~'C
oligosaccharides
'~ "'
'
Con
I
o~ -~'-[-,
-10
~,
,~_
.~
*~
_ d
~
_
9
I
. . . .
a. . . . .
.-0
"O
t~
IX:
E4-PHA
Almond a-L- Fucosidase
DSA !. ,
~
DSA
. . . .
| .
.
.
0----5--~10
.
.
.
.
.
-15 0 - -
.
.
E4-PHA
AAL
,
O- ..~
10
0
10
.
5
10-- 15 0
5
10
Elution v o l u m e (ml)
57
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Research.
5
SUGAR CHAINS OF SERUM CHOLINESTERASEIN 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 columnsa
Oligosaccharides
(% molar ratio)
I
(67.5)
ConA
DSA
+
-
E4-PHA
r
L4-PHA
-
AAL
Glucose
unitst'
Numbers of
released
N-acetyllactosamine
c
-
13.5
2
Proposed structures
Gall31~ 4GIcNAc/31---~2Manotl
"~6Man/31___>Rld
3
Gal/31~4GlcNAc/31~ 2Manal S
II
(5.5)
+
-
r
-
+
14.5
2
Gal/31--,4GlcNAc/31-->2Manal ~ 6Man/31-->R2e
Gal/31---,4GIcNAc/31--->2Manor1 "~3
IIIa
(3.8)
-
+
-
r
-
18.5
4
Gal/31-->4GIcNAc/31"~6
2Manal -,~
Gal/31--,4GIcNAc/31/"
Gal/3--~4GalcNAc/31 "~4
/*
6Man/31~R 1
3
2Mancd
Gal/31-->4GlcNAc/31,z
IIIb(V')
(2.1)
-
+
-
r
-
16.0
3
Gal/31--,4GIcNAc/31
",.a6
2Maned
Gal/31-->4GlcNAc/31/*
"a6Man/31-~.R1
3
Gal/31--->4GlcNAc/31--->2Manod Z
IV (VI')
(14.9)
-
r
r
-
-
16.5
3
Gal/31-->4GlcNAc/31-->2Mana 1
"~6
Gal/31-->4GIcNAc/31
Man/31--->Rl
"a4
/3
2Manal
Gal/31~4GIcNAc/31 '
V
(2.3)
.
.
.
.
r
16.5
3f
Fuctxl
"~
2
Gal/31~ 4Glciq3Ac/31( 6 )
2 Mantel,,
6
S
Gal/31-->4GIcNAc/31( 6 )
3 Man/31--->R1
S
Gal/31--->4GlcNAc/31-->2 Manal
VI
(3.9)
-
-
r
-
r
17.0
3f
Gal/31-~4GlcNAc/31~2Manal
Fucotl
"-a
2
"a6
Gal/31~4GIc/(I3Ac/31( )
3Man/31~R l
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 o f diplococcal/3-galactosidase and j a c k b e a n / 3 - N - a c e t y l h e x o s a m i n i d a s e , different numbers o f N-acetyllactosamine residues w e r e released f r o m oligosaccharides I, 11, II1a, IIIb, and IV, and Ct-L-fucosidase digested
oligosaccharides V and VI (V' and VI'). By c o m p a r i n g the mobility
o f each oligosaccharide before and after these glycosidase diges-
verted to tri-mannosyl N,N'-diacetylchitobiitol; and oligosaccharide
11 was converted to fucosylated tri-mannosyl N,N'-diacetylchitobiitol
(data not shown). The glycosidic linkages and locations o f the outer
chains in these seven oligosaccharides were determined by methylation analysis and f r o m the behavior o f the oligosaccharides on
immobilized ConA, DSA, E4-PHA, L4-PHA, and A A L columns
tions, the n u m b e r s o f N-acetyllactosamine residues released f r o m oligosaccharides I, II, Ilia, Illb, IV, V', and VI' w e r e calculated to be 2,
2, 4, 3, 3, 3, and 3, respectively (Table 2), and then these e n z y m e digested oligosaccharides I, IIIa, IIib, IV, V', and VI' w e r e con-
(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---> 4GIc N A c o x group are retarded on an L a - P H A c o l u m n and adsorbed to a
D S A column and eluted with 1% N - a c e t y l g l u c o s a m i n e oligomers (22).
58
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Research.
S U G A R C H A I N S OF S E R U M
Mean+SD
100
80
60.1_+17.2
57.3_+17.5
21.1_+8.0
16.9_+5.4
n=50
n=47
n=40
n--20
:9
=9
o9
9149
9
LIJ
0
,=
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.
oOe
oe
9"~
0
C H O L I N E S T E R A S E IN L I V E R DISEASES
60
,,I
0~
":1
0
OO
9
0
o9
e9
0
9
o~
0
|
t.._
oo
,-I
o9
<1:40
<
o9
AAL-binding Serum ChE in Patients with HCC and Benign
Liver Diseases. The percentages of AAL-binding serum ChE in pa-
9
0
9
0
ooo
oOo
. . . .
tients 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
.-e . . . .
oo
9
20
o9
O:o 9
e9
"|:
o9
CH
NC
oo
o9
HCC
HCC
LC
Fig. 3. Mean + SD of serum ChE reactiveto AAL-Sepharosein patientswithHCC,
LC, CH, and in healthyindividuals(NC). 9 and 9 in HCC, HCC developingafterLC and
HCC developingafter CH, respectively.
LC
CH
~
|
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 o l i g o s a c c h a r i d e s with fucosylated proximal
N-acetylglucosaminitol at the C-6 position bind to an AAL column
(25). Accordingly, ConA+E4-PHAr 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-PHAr oligosaccharide IV a C-2,4 and C-2 branched triantennary component. A A U 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 oligosaccharide VI was converted to a DSA' component (fraction VI'; Fig.
2 0 ) 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.
m
~
NC
118O/o
117%
Jk_
:,5_
-3-
AAL
T
0.4 60%
17%
o
o
4
a 0;=:4
Elution
volume
a 0-=4
a
(ml)
Fig. 4. Serial A A L and LGA column chromatography of serum ChE in HCC, LC, CH,
and a healthyindividual(NC).
A A L + LCA +
AAL§ LCA-
400
300
,
l
O
i
,,,200- ":"
~=
o
0
=l"
"
9 O
"~,
-
9
":.
I
I
0 L
HCC
LC
t
9l"
9
,I
I "
II
9
9
, o,1~
""
....
CH
NC
| ....
00
o09 9
00
100-
:. 9
:
O
-
:-
e
:
|
.:
:
oe 9149
9
9
.|.
9
..
9e9
~
HCC
LC
CH
NC
Fig. 5. Serumconcentrations(IU/liter)of AAL+LCA- ChE and AAL+LCA+ ChE in
HCC, LC, CH, and in healthyindividuals(NC).
59
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1994 American Association for Cancer
Research.
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
Hepatic
diseases
HCC (n = 14)
LC (n = 21)
CH (n = 17)
NC (n = 16)
Mean _+ SD (IU/liter)
Total ChE
683.6 + 235.6
647.4 _ 282.2
1421.6 ___391.0
1320.6 +--334.0
AAL+LCA-ChEa
251.2 +_57.0
212.3 • 93.2
44.0 • 36.3
41.4 • 44.0
AAL+LCA+ChEh
145.0 +- 38.8
134.7 _+51.8
230.5 +--82.9
199.4 +_38.8
AAL-LCA-ChEc
287.4 • 181.3
300.4 • 214.9
1147.1 --- 344.4
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.
DISCUSSION
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 AALbinding ChE did not change with the treatment except in one case
(data not shown).
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-antitrypsin, 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.
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)GlcNAc-->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.
(3).
An increase of sugar chains containing Lewis X antigenic determinants 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 phenomenon 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 regeneration. After some interval, the liver cells of the patients change irreversibly, 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 synthesize 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.
60
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Research.
SUGAR CHAINS OF SERUM CHOLINESTERASE IN LIVER DISEASES
teins produced in cirrhotic tissues may be similar to that of the stagespecific embryonic antigen, which is related to cell compaction in the
morula (32, 33).
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Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1994 American Association for Cancer
Research.
Increase of Fucosylated Serum Cholinesterase in Relation to
High Risk Groups for Hepatocellular Carcinomas
Takashi Ohkura, Toshikazu Hada, Kazuya Higashino, et al.
Cancer Res 1994;54:55-61.
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