[CANCER RESEARCH 44.1557-1567,
April 1984]
Structures of the Oligosaccharide Chains of Two Forms of ai-Acid
Glycoprotein Purified from Liver Métastasesof Lung, Colon, and
Breast Tumors1
E. V. Chandrasekaran,
Marco Davila, Daniel Nixon,2 and Joseph Mendicino
Department ot Biochemistry, University of Georgia, Athens, Georgia 30602
ABSTRACT
Two forms of «i-acidglycoprotein with common immunological
determinants and almost identical amino acid compositions but
different amounts of carbohydrate were isolated from liver mé
tastases of primary colon, lung, and breast tumors by extraction
with perchloric acid, gel filtration on Sepharose CL-6B and
Sephadex G-200, and affinity chromatography on concanavalin
A:agarose and Ricinus commuais agglutinin hagarose. Both
forms of the antigen yielded single bands which stained for
protein and carbohydrate when examined by disc gel electrophoresis and immunodiffusion. The molecular weights of the two
forms were 45,000 and 37,000 respectively. The larger form
contained about five to six Oligosaccharide chains, whereas the
smaller form had only three to four chains. The composition and
structures of the Oligosaccharide chains in the two forms of this
glycoprotein were very similar. Each contained di-, tri-, and
tetraantennary complex-type Oligosaccharide chains. The diantennary Oligosaccharide chains caused both forms of a^acid
glycoprotein to be retained by concanavalin A-agarose columns.
The lower-molecular-weight
form contained fewer chains and
correspondingly fewer terminal galactosyl residues. This resulted
in the separation of this species from the higher-molecular-weight
form on columns containing R. commuais agglutinin I.
Three types of reduced oligosaccharides were released from
the light and heavy forms of a^acid glycoprotein by treatment
with alkaline borohydride or by hydrazinolysis. These chains
were isolated by chromatography on concanavalin A:agarose
and Bio-Gel P-6 columns. The arrangement and linkage of sugars
in the purified oligosaccharides were determined by periodate
oxidation, sequential hydrolysis with glycosidases, and methylation analysis.
The major Oligosaccharide chain, comprising 50 to 55% of the
carbohydrate, had a triantennary structure as shown in the
structure:
GlcNAc -
NeuNAc
Man
1'6
Man
NeuNAc -
Gal -
-GlcNAc
- GlcNAc
.GlcNAcol
'"
tc.1,3
Fuc
in which NeuNAc is A/-acetylneuraminic acid, Gal is galactose,
GlcNAc is A/-acetylglucosamine, Man is mannose, GlcNAcol is
A/-acetylglucosaminitol, and Fuc is fucose. Tetraantennary chains
comprised about 25 to 30% of the carbohydrate, and the addi
tional outer chain was attached to the a1,6-mannosyl residue
through a 01,6-linked GlcNAc unit. The remaining 15 to 20% of
the Oligosaccharide chains had a diantennary structure. The
extent of sialylation of these chains varied in samples isolated
from tumors of the same histological type from different individ
uals. However, a relatively constant proportion of the three types
of chains was present in different forms of the glycoprotein
isolated from liver métastases.
INTRODUCTION
An increased level of «t-AGP3with abnormal carbohydrate
chains has been reported to be present in the serum of patients
with advanced neoplastic diseases (32). A very irregular pattern
in the hexose, glucosamine, and sialic acid components of this
glycoprotein compared to that found in normal o,-AGP sug
gested that multiple disturbances in the synthesis of the carbo
hydrate side chains may have taken place. It should be noted
that this glycoprotein (33) is normally formed in liver (3), a principle
site of metastasis of carcinoma derived from the digestive system
(10, 37). Many earlier investigators using perchloric acid or
trichloroacetic acid for extraction showed that the level of «1AGP in serum is increased in cancer patients (18, 23). The
function of «-AGPin pathophysiological states is not yet known.
Chu ef al. (7) have demonstrated that an increase in ai-AGP
levels in plasma is as sensitive an assay as are carcinoembryonic
antigen levels in the detection of colorectal cancer. Recently, an
unusual «i-AGP,which binds to pteridine, has also been isolated
from the serum of patients with neoplastic diseases (39). Earlier
workers clearly demonstrated that the level of «t-AGP is in
creased in many unrelated disease states. Nicollet ef al. (25)
reported an enhancement in the species of arAGP which binds
to Con A: Sepharose 4B columns in sera of patients with acute
inflammation.
Using high-resolution 'H-nuclear magnetic resonance spectroscopy, Fournet ef al. (9) have found that «i-AGPisolated from
normal human serum contains di-, tri-, and tetraantennary com
plex-type Oligosaccharide chains. A recent investigation of the
carbohydrate structure of «i-AGPby Yoshima ef al. (38) showed
that more than 90% of the Oligosaccharide chains in plasma a,AGP had tetraantennary structures. During the course of our
1This investigation was supported by USPHS Grants CA 23703 and CA 28815,
awarded by the National Cancer Institute, Department of Health. Education and
Welfare.
2 Present address: Department of Medicine, Emory University, School of Medi
cine, Atlanta, GA 30322.
Received September 22, 1983; accepted January 4, 1984.
APRIL
1984
3The abbreviations used are: a^AGP, ai-acid glycoprotein; ai-AGPL, liver
métastasesof primary tumor of lung; o,-AGPc. liver métastasesof colon tumor;
td-AGPn, liver métastases of breast tumor; NeuNAc, N-acetylneuraminic acid;
GlcNAcol, N-acetylglucosaminitol; Con A, concanavalin A; GlcNAc, N-acetylglucosamine; RCA,», Ricinus communis agglutinin I.
1557
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E V. Chandrasekaran et al.
investigations on the isolation and structures of glycoproteins in
metastatic liver (6), we have isolated 2 major species of «i-AGP
from liver métastasesof primary lung, breast, and colon cancers.
The carbohydrate structure of these 2 forms of c^-AGP is pre
sented in this paper.
MATERIALS AND METHODS
Isolation of <(,-AGP. Liver métastasesof primary colon, breast, and
lung tumors were obtained at autopsy and frozen at -20°. The liver
from a single individual was used for each preparation, and the majority
of livers selected for these studies had over 70% metastatic tumor at
autopsy. a,-AGP was purified from liver métastases by extraction with
perchloric acid, by gel filtration on Sepharose CL-6B and Sephacryl S200 columns, and by affinity chromatography on Con Aragarose and
RCA,.,„:agarosecolumns. Purified u,-AGP was obtained by these pro
cedures from more than 15 different preparations.
Crude extracts were prepared by homogenizing 2700 g of tissue in
2700 ml of 0.05 M Tris-HCI (pH 7.5) for 2 min in a Waring blender. Then
5400 ml of 2 M perchloric acid were added, and the suspension was
centrifugea at 27,000 x g for 15 min. The supernatant was adjusted to
pH 7.5 with NaOH, and sodium azide was added to a final concentration
of 0.04%. This solution was dialyzed against distilled water containing
0.04% sodium azide and concentrated by blowing air at room tempera
ture over the dialysis tubing. The solution was then dialyzed against 0.05
M sodium phosphate, pH 7.0:0.15 M NaCI:0.04% sodium azide and
applied in 50-ml aliquots to Sepharose CL-6B columns (5 x 75 cm) which
60
I20
I80
240
VOLUME (ml)
Chart 2. Affinity chromatography of ai-AGP on Con A: Sepharose 4B. The
column (2.2 x 15 cm) was equilibrated with 0.05 M sodium acetate, pH 6.5:1 M
NaCI: 1 mM CaCI-,: 1 mM MnClz:1 ni M MgCI2, and the sample was dialyzed against
this solution and applied to the column. Fractions of 2 ml were collected and, at
the volume indicated by the arrow, elution was initiated with 2 HIM EDTA:0.05 M
glycine-HCI, pH 2.0:0.1 M NaCI. Protein was measured by the Polin procedure,
carbohydrate was measured by the anthrone reaction, and immunological activity
was measured with ariti-,,,-AGP serum with the standard radioimmunoassay. 0.0.,
absórbanos.
0.6-
AGPL (•—•
<X,AGPC
<X,AGP.<*-.)
were equilibrated against the same buffer. The columns were eluted with
this buffer and more than 90% of the a,-AGP emerged from the column
in a single low-molecular-weight peak (Chart 1A, Peak III) between 1250
ml and 1500 ml. The fractions in this peak were collected, concentrated
as described previously, and applied to a Sephacryl S-200 column. <*iAGP was eluted in a single peak from this column (Chart IB). The
fractions in this peak were combined, concentrated by ultrafiltration, and
applied to a Sephadex G-50 column (2.4 x 40 cm). «i-AGP was eluted
from this column with 50 mw sodium phosphate, pH 7.5. A single included
peak containing nearly all of the ai-AGP emerged from the column after
48
I2
FRACTION
16
20
24
26
NUMBER
Charts. Affinity chromatography of «i-AGP on RCA,.,,, Sepharose 4B. Two
different preparations of «i-AGPtaken through affinity chromatography on the Con
A:Sepharose 4B step were fractionated on RCA120:Sepharose columns (0.9 x 7
cm). The columns were equilibrated with 0.05 M potassium phosphate, pH 8.0, and
25 mg of each sample of a,-AGP were dialyzed against this buffer and applied to
the column. Fractions of 5 ml were collected. The columns were washed with this
buffer; after Fraction 18, they were eluted with 0.05 M potassium phosphate, pH
8.0, containing 0.1 M lactose. The elution patterns were obtained by measuring
protein absorption at 280 nm. Curve •was obtained with «,-AGPL from liver
métastasesof lung tumor, Curve O was obtained with «,-AGPc from liver métas
tases of colon tumor, and Curve A was obtained with a,-AGPB from liver métastases
of breast tumor.
a smaller higher-molecular-weight
protein peak which eluted in the void
volume.
Fractions containing «,-AGP were combined,
800
IOOO
I200
I400
O.I2
O.I4
OJ6
VOLUME
(liters)
Chartl. A, gel filtration of a,-AGP on Sepharose CL-6B. The dialyzed and
concentrated perchloric acid extract, 50 ml, was applied to the column (5 x 75 cm)
which was previously equilibrated with 0.05 M sodium phosphate, pH 7.5:0.15 M
NaCI:0.04% sodium azide. Elution was carried out with the same buffer solution,
and fractions of 15 ml were collected at a flow rate of 50 ml/hr. Carbohydrate was
measured by the anthrone procedure with a 0.1-ml aliquot of each fraction.
Immunological activity with anti-,,,-AGP serum was measured with the standard
dilution radioimmunoassay Neatly all, 90%, of the ,,,-AGP was found in Peak III.
B. elution profile of ai-AGP on Sephacryl S-200. The column (1.8 x 90 cm) was
equilibrated with 50 my sodium phosphate, pH 7.5:0.15 M NaCI. The glycoprotein
fraction obtained by gel filtration on Sepharose CL-6B (A, Peak III) was concentrated
by ultrafiltration to 2 ml and applied to the column. The column was eluted with the
same buffer, and aliquots were removed from 2.5-ml fractions and assayed for
protein, carbohydrate, and reactivity with anti-ni-AGP antibody. The immunological
activity and protein peaks coincided with the single carbohydrate peak shown in
B.
1558
concentrated,
and di
alyzed against 0.05 M sodium acetate, pH 6.5:1.0 M NaCI:1 mM CaCI2:1
HIMMgCI2:1 mM MnCI;,. The solution was passed into a Con A: Sepharose
4B column (2.2 x 15 cm) which was equilibrated against the same buffer.
More than 90% of the carbohydrate bound to the column. at-AGP was
eluted from this column with 0.05 M glycine, pH 2.0:2 rnw EDTA:0.1 M
NaCI, as shown in Chart 2. Samples from the second bound peak
contained nearly equal amounts of protein and total hexose, and more
than 90% of the arAGP was recovered in this peak. The fractions were
combined, dialyzed against 0.05 M sodium phosphate (pH 8.0), and
concentrated by ultrafiltration to 10 ml. The sample, 20 i/mol of neutral
sugar, was applied to a RCA120:Sepharose 4B column. The column was
washed with 0.05 M potassium phosphate (pH 8.0), and the bound
glycoprotein was then eluted with this buffer containing 0.1 M lactose,
as shown in Chart 3. Both the bound and unbound fractions contained
«,-AGP.
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VOL. 44
-AGP from Liver Métastases
Immunoassay of ai-AGP. A radioimmunoassay utilizing 125l-a,-AGP
was used to measure the amount of a,-AGP by procedures described in
a previous report (6). Rabbit antiserum to purified samples of a,-AGP
isolated from individual liver métastases were prepared by 3 s.c. injec
tions of 100 M9 of the glycoprotein in complete Freund's adjuvant every
other week. Antisera collected after 4 weeks were used for immunoassay. The anti «i-AGP was partially purified by chromatography on DEAEcellulose and adsorption on Protein A:agarose columns (6). Immunodiffusion was carried out in buffered 1% agarose plates as described in a
previous report (6).
Amino Acid and Protein Analysis. Amino acid composition was
determined with a Beckman Model 120C analyzer, and corrections were
applied as described previously (6,14).
The concentration of protein was measured colorimetrically with crys
talline 0,-AGP, provided by Dr. K. Schmid, Boston University, as a
standard. Polyacrylamide gel electrophoresis at pH 8.5 was carried out
by the procedure of Segrest and Jackson (35), Holden er al. (12, 13),
and O'Farrell (25). Gels were stained for protein with Coomassie blue.
Carbohydrate was detected by the procedure of Segrest and Jackson
(35). Gel electrophoresis in the presence of sodium dodecyl sulfate was
earned out by procedures described in a previous report (6).
Release of Reduced Oligosaccharides from <n-AGP. When purified
samples of a,-AGP were treated with 0.1 N NaOH containing 1 M NaBH4
for 36 hr at 45° under nitrogen, no reduced Oligosaccharides were
released, indicating that the <v,-AGP samples do not contain 0-serine- or
O-threonine-linked carbohydrate chains. The W-asparagine-linked chains
were released by incubating 100 mg of «rAGP in 10 ml of 1 N NaOH
containing 4 M NaBH4 for 24 hr at 80° (40). The samples were then
cooled to 3°,adjusted to pH 5.0 by careful addition of acetic acid, and
desalted on Bio-Gel P-6 columns (2.2 x 200 cm). They were concen
trated, A/-acetylated with acetic anhydride, passed through Dowex 50H+ columns, and concentrated as described previously (6). Boric acid
was removed by evaporation with methanol. The yield of reduced Oligo
saccharides was about 90% based on the total GlcNAc and neutral
sugar content of the «i-AGP samples.
The oligosaccharide chains were also released by hydrazinolysis.
Purified «,-AGP, 50 mg, was lyophilized and further dried over HuSCu in
a vacuum desiccator for 24 hr. The sample was dissolved in 1 ml of
anhydrous hydrazine and heated in a sealed vial at 100° for 10 hr.
Afterwards, it was evaporated to dryness in a vacuum desiccator over
H2S04. Residual hydrazine was removed by flashing with toluene in a
rotary evaporator. The final residue was dissolved in 5 ml of 0.1 M
NaHCOa and N-acetylated by the addition of acetic anhydride. After 4
hr, the solution was passed through a Dowex SO-H* column (2.2 x 5
cm), and the column was washed with 5 volumes of water. The effluent
and wash were combined and concentrated to dryness. The sample was
dissolved in water and applied to Whatman No. 3MM paper. Following
development with 1-butanol:ethanol:water
(4:1:1) for 48 hr, the Oligo
saccharides were eluted from the paper (0 to 5 cm from origin) with
water. The sample was dried, dissolved in 0.6 ml of 0.05 N NaOH
containing 0.1 ml of sodium borotritide (25 mCi/ml), and incubated at
30°for 4 hr. NaBH4,10 mg, was added, and the sample was left at 5°
overnight. Excess borohydride was destroyed by the addition of acetic
acid, and the sample was evaporated to dryness. This material was
dissolved in 2 ml of water and desalted on a Bio-Gel P-6 column (2.2 x
200 cm). A broad radioactive peak, which contained all the carbohydrate,
was collected, concentrated to dryness, and dissolved in 1 ml of water.
The overall recovery of carbohydrate, estimated by neutral sugar deter
mination, was greater than 90% in different preparations.
Determination of the Number and Size of Oligosaccharide Chains
in a,-AGP. Individual oligosaccharide chains were isolated by gel filtration
on Bio-Gel P-6 columns, affinity chromatography on Con A: Sepharose
4B, and ion-exchange chromatography on DEAE-oellulose columns.
The molecular weights of the intact chains and samples treated with
glycosidases were estimated by gel filtration on calibrated Bio-Gel P-6
columns (2.2 x 200 cm) (6,19). Standard reduced diantennary Oligosac
charides were prepared from porcine IgG and human light chain SmX
APRIL 1984
(6,19). The standard reduced triantennary oligosaccharide chains were
prepared from fetuin by treatment with alkaline borohydride (6). [14C]Glycogen was used to indicate the void volume, and [14C]galactose was
used to measure the total volume of the column.
The number of oligosaccharide chains was assayed by determination
of GlcNAcol after acid hydrolysis. Samples were hydrolyzed in 4 N HCI
for 6 hr at 100°and evaporated to dryness under vacuum in the presence
of NaOH. They were then acetylated with 2 /imol of [14CH3]COOH
(specific activity, 3 cpm/pmol) in the presence of 10 ^mol of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline
as described in an earlier report
(30). The samples were desalted on Dowex 1-HCO3 (2.2 x 3 cm) and
Dowex 50-H* columns (2.2 x 3 cm) and concentrated, and radioactive
GlcNAc and GlcNAcol were isolated by paper chromatography
in bu-
tanohpyridine:water
(6:4:3). The amount of GlcNAcol is calculated by
dividing the total counts by the specific activity of the acetate.
Sequential Hydrolysis with Exoglycosidases. Treatment of Oligo
saccharides with glycosidases was carried out essentially as described
in a previous report (5). Neuraminidases obtained from Boehringer Mann
heim were purified on Sepharose 6B columns containing covalently
bound A/-(p-aminophenyl)oxamic
acid (Sigma). /3-Galactosidase, ß-Nacetylglucosaminidase, and «-mannosidase were purified from jack bean
and seminal fluid. /3-Mannosidase was isolated from Aspergillus niger.
Digestions were carried out in 0.01 M sodium acetate, pH 5.0, for 4 days
at 37°with 1 unit (1 ¿¡mol
of p-nitrophenylglycoside/min)
of each glycosidase. Additional enzyme, 1 unit, was added after 48 hr. Afterwards, the
solution was heated at 100°for 1 min, and it was applied to a Bio-Gel
P-6 column (2.2 x 200 cm), which was eluted with 0.1 M pyridinium
acetate, pH 5.5. The amount of sugar released was determined as
described previously (29, 30). The oligosaccharide isolated after treat
ment with each glycosidase was concentrated to dryness and dissolved
in buffer. An aliquot was removed for permethylation analysis, and the
sample was then treated with another glycosidase.
Methylation Analysis. Permethylation was performed by a modifica
tion of the procedures of Hakomori, and the methylated samples were
hydrolyzed, reduced, and acetylated as described in our previous studies
(6, 29). The partially methylated alditol and hexosaminitol acetates were
examined by gas chromatography column (0.3 x 200 cm) containing 3%
OV-225 on Supelcoport (80 to 100 mesh), or 3% SE-30 on 100/120
Gas-Chrom Q (0.3 x 400 cm). Retention times were determined relative
to 2,3,4,6-tetramethylglucitol
acetate. Peaks were identified by compar
ison with the retention times of authentic partially methylated alditol and
hexosaminitol acetates. The partially methylated alditol and hexosamini
tol acetate peaks separated by gas chromatography were identified by
mass spectrometric analysis. Mass spectrometry was performed on a
Finnigan Model 4000 quadripole automated gas chromatograph-mass
spectrometer. Identification of each peak was made by comparing its
relative retention time and mass spectra with known standards.
Other Procedures. The compositions of all Oligosaccharides were
determined by isotope dilution and specific colorimetrie and enzymatic
methods following acid hydrolysis as described in our previous reports
(29,30), except that glucosamine content was determined by amino acid
analysis after hydrolysis in 4 N HCI at 100°for 6 hr. Periodate oxidations
were performed as described previously (6). The anthrone and phenolsulfuric acid methods
determined with the
electrophoresis were
previous reports (20,
were used to detect carbohydrate. Sialic acid was
resorcinol reagent. Paper chromatography and
performed according to procedures described in
22). Oliosaccharides were detected with the perio-
date:alkaline silver nitrate reagent. All of the Oligosaccharides examined
in these studies were purified to homogeneity by repeated gel filtration
on Bio-Gel P-6 (400 mesh) columns (2.2 x 200 cm) with 0.1 M pyridinium
acetate, pH 5.5.
RESULTS
Separation of 2 Forms of «,-AGPby Affinity Chromatogra
phy on RCA120:Sepharose. When preparations of purified <n1559
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E. V. Chandrasekaran et al.
AGP were examined by gel electrophoresis, 2 bands which
stained for both carbohydrate and protein were observed (Fig.
1, Gels A, B, and C). These 2 components were not separated
by exclusion chromatography on Sephacryl S-200 (Chart 1S).
However, when the samples were passed into RCAi20:Sepharose columns, the larger component (Fig. 1, upper band, Gels
A, B, and C) bound to the column, and the smaller component
(lower band) passed through the column. The bound form was
eluted with 0.1 M lactose, and both samples were concentrated
and dialyzed against 0.01 M Tris-HCI, pH 7.O. The percentage of
purified c^-AGP which bound to the RCA120:Sepharose column
varied greatly with different liver métastases. As seen in Chart
3, about 15% of «i-AGPL, 35% of cn-AGPc, and 65% of «1AGPB bound to the column. The amount of a,-AGP bound was
related to the number of oligosaccharide chains present and the
extent of desialylation of these chains. Species of i^-AGP which
do not contain a sufficient number of terminal galactosyl residues
per polypeptide chain are not retained in this column. All of the
01-AGP present in the preparation which bound to Con
A: Sepharose 4B was recovered in the fractions which bound or
passed through RCA120:Sepharose. Nearly all of the protein
present in these fractions could be accounted for as «i-AGP.
These results clearly demonstrate that the purified glycoprotein
obtained by chromatography on Con A :Sepharose can be further
separated into 2 fractions.
Physical and Immunological Properties of the 2 Forms of
<*i-AGP. Disc gel electrophoresis at pH 7.2 and 8.9 showed the
presence of only a single diffuse protein band in the fractions
separated by chromatography on RCA120:Sepharose. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl
sulfate after reduction and alkylation or dissociation with 2mercaptoethanol sodium and dodecyl sulfate still showed only
one diffuse band which stained for both carbohydrate and protein
(Fig. 1, Gels D and E). Samples treated with dilute acid or
neuraminidase to remove sialic acid showed only one somewhat
O
4
3
Fig. 2. Immmunodiffusion pattern of ai-AGP isolated by affinity chromatography
on RCA1M:Sepharose 4B. Central well, antiserum to human plasma a^AGP raised
in rabbits; Wells 1 and 2, samples from «,-AGPc(1 mg/ml) and a,-AGPB (1 mg/ml)
which did not bind to RCA120:Sepharose 4B columns; Wells 3 and 4, samples from
«,-AGPc(1 mg/ml) and a,-AGPB (1 mg/ml) which bound to RCA,M:Sepharose 4B
columns; Well 5, human plasma «i-AGP (1 mg/ml).
sharper band. These results suggest that both forms of ai-AGP
may have only one polypeptide chain and that the polydisperse
appearance of the purified preparations attributable, in part, to
varying amounts of sialic acid.
Purified preparations of «i-AGP and the 2 different forms of
glycoprotein had similar immunological properties. They showed
single discrete bands in double diffusion assays and immunoelectrophoresis against purified rabbit serum antk*,-AGP. As seen
in Fig. 2, both of the purified preparations reacted with rabbit
anti-tt!-AGP serums. Antiserum to human serum ai-AGP also
reacted with the purified samples of «i-AGP yielding single
distinct precipitin lines which fused completely. Immunodiffusion
with antiserum to human plasma showed only a single precipitin
line corresponding to a^AGP. Immunoelectrophoresis with antisera to serum ai-AGP showed single curves when examined
with purified samples of at-AGPt, «i-AGPc, and «i-AGPB. «1AGPc and ai-AGPB showed single precipitin arcs in the a,-a2
region, whereas a,-AGPL showed a single precipitin arc in the ßregion. These results are consistent with the chemical analysis
of these glycoproteins which show that ai-AGPL contained much
larger amounts of sialic acid than did ai-AGPc and ai-AGPB.
These preparations reacted similarly in the standard radioimmunoassays. Binding inhibition studies showed that all of the sam
ples were able to decrease the reaction of 125l-ai-AGP with rabbit
anti-ai-AGP. Similar curves were obtained by plotting percentage
of inhibition versus the concentrations of each preparation.
Molecular Weight and Amino Acid and Carbohydrate Com
position of the 2 Forms of ai-AGP. Both forms showed single
peaks when examined by sedimentation velocity at 60,000 rpm
in 0.154 M NaChO.05 M Tris-HCI, pH 7.5, at 20°.The SÜD.*
of the
Fig. 1. Sodium dodecyl sulfate: polyacrylamide gel electrophoresis. The «,-AGP
samples were incubated at 37° for 4 hr in 0.01 M sodium phosphate, pH 7.0,
containing 1% sodium dodecyl sulfate and 1% mercaptoethanol. After adjustment
to 30% glycerol, the samples were applied to 5% acrylamide gels. The electropho
resis was carried out for 3 hr at 8 amperes/gel. Gels A and B, 75 »<g
of ai-AGPc
stained for carbohydrate (Schiff's reagent) and protein (Coomassie blue), respec
tively; Gel C. 150 pg of «,-AGPc reduced with 2-mercaptoethanol, alkylated with
iodoacetamide. and stained for protein; Gel D, 25 ¿igof the fraction of a,-AGPc
which bound to RCA1M:Sepharose 4B; Gel E, 25 vg of the fraction of «,-AGPc
which did not bind to RCA,M: Sepharose 4B. These gels were stained with
Coomassie blue. Duplicate samples of the last 3 gels showed bands in the same
position as the protein band when they were stained with Schiff's reagent to detect
carbohydrate.
1560
heavy form was 3.4, and the lighter form showed a s20.».
of 3.0.
The molecular weights obtained by exclusion chromatography
on Sephadex G-200 were 44,000 ±2,000 (S.D.) and 36,000 ±
1,500, respectively. The elution volumes of ai-AGP preparations
were compared on columns calibrated with protein and glyco
protein standards (21). The heavy and light forms of ai-AGPL,
«,-AGPc, and «i-AGPBshowed similar molecular weights.
The chemical compositions of the purified preparations were
determined, and the results are summarized in Tables 1 and 2.
The relative amounts of each amino acid in the 2 forms of aiAGP separated by chromatography on RCA! 20:Sepharose are
shown in Table 1. The amino acid contents of these preparations
were nearly identical. The purified preparation obtained by affinity
chromatography on Con A:Sepharose had a similar amino acid
content. The results suggest that fractionation of t^-AGP on
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VOL. 44
ì-AGPfrom Liver Métastases
RCA120:Sepharose into 2 forms results from differences in car
bohydrate composition.
The carbohydrate content of purified preparations of the heavy
form of «t-AGP,calculated from the average of 4 determinations,
sialic acid, 14 mol GlcNAc, 8 mol mannose, 9 mol galactose, and
3 mol fucose per mol glycoprotein, based on a molecular weight
of 37,000. A similar composition was found for the light forms
isolated from oi-AGPc and «i-AGPB,except that they contained
was about 40 ±2% of the total weight, whereas the carbohy
drate content of the lighter form was only 28 ±3%. Based on a
molecular weight of about 27,000 for the polypeptide chain of
the heavy form and a molecular weight of 25,900 for the lighter
form calculated from amino acid analysis, a total molecular
weight of 45,000 and 37,000 for each species, the weight of
carbohydrate in the heavy form is about 18,000 compared to
only 11,000 in the lighter species. The heavy form isolated from
ai-AGPL (Table 2) contained 14 mol of sialic acid, 27 mol of
GlcNAc, 15 mol of mannose, 17 mol of galactose, and 6 mol of
fucose per mol of glycoprotein. The heavy form from ai-AGPc
contained 5 mol of sialic acid, 30 mol of GlcNAc, 19 mol of
mannose, 20 mol of galactose, and 5 mol of fucose per mol. The
heavy form from c*i-AGPB had a similar composition except that
less sialic acid, 3 and 1 mol, respectively.
The differences in the sialic acid content of these preparations
could be due to partial hydrolysis of the acid-labile sialyl residues
during isolation. In order to examine this possibility, 10 mg of
purified a,-AGPL were labeled by treatment with galactose oxi
it contained only 2 mol of sialic acid. The only significant differ
ence in these preparations was the content of sialic acid. No
galactosamine was detected, even when large amounts of acid
hydrolysates were examined by paper chromatography or amino
acid analysis. The lighter form of a,-AGPL contained 8 mol of
dase and sodium borotritide (24). This enzyme oxidizes penulti
mate galactosyl residues when they are substituted by «2,3linked sialic acid. The triantennary oligosaccharide chains in fully
sialylated ai-AGPL contain 2 «-2,3-linked sialic acid residues.
The labeled fully sialylated sample (52,000 cpm/^mol sialic acid)
was added to buffer which was then used to isolate «,-AGPB
from 400 g of metastatic liver. The reisolated t^-AGP contained
39,000 cpm/i/mol of sialic acid. Based on the yield of a^AGP,
24 mg, the dilution of the added sample would have been much
greater if «i-AGPBhad been fully sialylated initially. Within ex
perimental error, the actual dilution (39,000 cpm:52,000 cpm)
would be expected upon mixing 10 mg of a,-AGPL (9 mol of
sialic acid/mol) with 14 mg of ai-AGPB (2 mol of sialic acid/mol).
These results suggest that desialylation does not occur during
isolation of the glycoprotein.
Isolation and Composition of Reduced Oligosaccharides
from 01-AGP. More than 90% of the carbohydrate in <*i-AGP
Table 1
Amino acid composition of 2 forms of cti-AGPBwhich bound and did not bind to
RCA,x:Sepharose 4B
was released as Oligosaccharides containing terminal GlcNAcol
Samples were dialyzed against distilled water, lyophilized, and dried over P20;,. residues after treatment with either 1 N NaOH and 4 M NaBH4
The composition was based on the dry weight of these samples, and average
values obtained after 24 and 48 hr of hydrolysis at 110°in constant boiling HCIare
or hydrazine. The concentration of oligosaccharide chains in the
listed.
heavy and light forms of the antigen was determined by meas
RCA,M-binding
from
uring the amount of GlcNAcol after acid hydrolysis. The light
binding «,- human plasma8
«i-AGPe(mol
form
contained about 3 to 4 chains/mol based on a molecular
(mol %) (resi%) (residues/
AGPB(mol %)
weight of 37,000, and the heavy form contained nearly 5 to 6
mol)Aspartic
Amino acid
(residues/mot)18181623412161610197131471012a,-AGP
dues/mol)221873688910101511101541013
chains/mol. These results support earlier observations which
suggested that differences in the 2 forms of ai-AGP was due to
carbohydrate content. The carbohydrate compositions of ai(glutamine)ProlineGlycineAlanineValineIsoleucineLeucineTyrosinePhenylalanineLysineHistidineArginineMethionineHalf-cystine181918235121817111210161371212RCA,2o-nonacid
AGPL, a,-AGPc, and ai-AGPB and the RCA120:Sepharose 4B
fractions from ai-AGPL are shown in Table 2. Based on a
mannose content of 3 residues, the most significant differences
are in the content of sialic acid. The reduced Oligosaccharides
released from a!-AGPL and ai-AGPc were fractionated by gel
filtration on Bio-Gel P-6 columns (2.2 x 200 cm). The profiles,
shown in Chart 4, indicate that t^-AGP,., the more highly sialy
lated preparation, contains higher-molecular-weight oligosaccha
ride chains than those of ai-AGPc. Two peaks with molecular
weights of about 3400 and 2100 were observed in both prepa
" Hamburger et al. (11).
rations.
(asparagine)ThreonineSerineGlutamic
acid
Table2
Carbohydrate composition of different preparations of «,-AGP
The methods used for analysis of carbohydrate are described in the text. The values are expressed in molar ratios on the basis of a mannosecontent of 3 mol/mol of
oligosaccharide in ,.,-AGP
A
ComponentSialic bound"2.6
(9)"
acid
M un
bound2.4(14) bound2.8
A
bound"0.9
bound0.9
»un
bound1.0
A
bound"0.3
un
bound0.3
(8)
(4)
(5)
(3)
(2)
(2)
(1)
5.4(21)
5.3 (16)
5.3 (26)
5.3(31)
5.3 (27)
5.3(14)
5.3 (30)
5.2 (17)
GlcNAc
5.3(17)
3.5 (14)
3.4(10)
3.4(18)
3.5(11)
3.4(10)
3.4 (17)
3.3 (9)
3.5 (20)
3.5 (22)
Galactose
3.0 (8)
3.0 (17)
3.0(15)
3.0 (8)
3.0(12)
3.0(19)
3.0(21)
3.0 (9)
Mannose
3.0 (9)
0.9 (4)«,-AGPcRCA,»
0.9 (5)RCA, 0.9 (3)Con
0.7 (5)o,-AGP8RCA120bound0.2
0.8 (6)RCA,»0.8 (4)
1.3 (4)«,-AGPtRCA120
1.2 (6)RCA, 1.0 (3)Con
FucoseCon
" The number of sugar residues in Con A-bound «i-AGP,which is a mixture of the heavy and lighter forms, was calculated for the average molecularweight, which is
based on the data that the heavy and lighter forms were 15 and 85% in o,-AGPL,35 and 65% in «,-AGPc,and 65 and 35% in a,-AGPB.
0 Numbers in parentheses, number of sugar residues per mol of glycoprotein.
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E. V. Chandrasekaran et al.
arose have a diantennary structure (1). The compositions of
these oligosacchande chains are shown in Table 3. The binding
of native at-AGP to Con A:Sepharose 4B columns may be due
to the presence of oligosacchande chains with a diantennary
structure which comprise only 15 to 20% of the total carbohy
drate in the glycoprotein. Fractions from ai-AGPL contained
mostly fully sialylated chains, «t-AGPc contained only one-half
the amount of sialic acid as did «^AGPi., and a!-AGPB contained
much less, only 10% of the sialic acid present in ai-AGPL. The
fully sialylated, monosilalylated, and disialylated forms of these
chains were separated by ion-exchange chromatography on
DEAE-cellulose (Chart 6). Each of the fractions was further
purified by gel filtration on Bio-Gel P-6 columns. The elution
300
400
VOLUME (ml)
Chart 4. Isolation of reduced oligosacchandes by gel filtration on Bio-Gel P-6.
The oligosaccharides obtained by hydrolysis of a,-AGP with alkaline borohydride
were passed through a Bio-Gel P-6 column (2.2 x 200 cm) to remove salt and
borate. The fraction containing carbohydrate was concentrated and applied to a
second Bio-Ge) P-6 column (2.2 x 200 cm), and the profiles shown were obtained
by elution with 0.1 M pyridinium acetate, pH 5.2. A, oligosaccharides from a,-AGPL;
B, oligosaccharides from oi-AGPc.
profiles of the asialo, monosialylated, and disialylated reduced
diantennary chains are shown in Chart 7A. Oligosaccharides
which did not bind to Con AiSepharose contained the tri- and
tetraantennary chains. They were converted to the asialo forms
by heating at 80°for 1 hr with 0.05 N H2SO4. These oligosac
charides were readily separated by repeated chromatography
on Bio-Gel P-6 column as described in an earlier report (6). The
elution patterns of the asialo forms of the reduced tri- and
tetraantennary chains are shown in Chart IB.
Structures of the Diantennary Chains Isolated from «i-AGP.
6
FRACTION
12
18
24
NUMBER
Charts. Affinity chromatography
of reduced oligosaccharides
on Con
AiSepharose 4B. The reduced oligosaccharides were acetylated with [14C]acetic
anhydride (0.7 cpm/pmol) as described in the text. The column (0.8 x 11 cm) was
equilibrated with 0.05 M sodium acetate, pH 6.5:1 mw CaCI2:1 rriM MnCb:1 mw
MgCI2, and 2.5 ^mol of each sample were dissolved in this solution and applied to
the column. Fractions of 1 ml were collected; at the volume indicated by the arrow,
elution was initiated with 2 mw EDTA:0.05 M glycine-HCI, pH 2.0. A, Curve O was
obtained with reduced oligosaccharides from .. ,-AGP, ; Curve •was obtained with
a sample from .>,-AGPc; Curve A was obtained with a sample from ,,,-AGPB. B.
Curve •was obtained with the oligosacchande fraction isolated by gel filtration on
Bio-Gel P-6 (Chart 4A, Peak 1); Curve O was obtained with oligosaccharides from
Pea* 2 (Chart 4¿).
The binding of these oligosaccharides to Con A:Sepharose
was examined, and the results are shown in Chart 5. About 15
to 20% of the oligosacchande chains in a^AGPu «i-AGPc, and
ai-AGPB bound to Con AiSepharose (Chart 5A). The profile of
the oligosacchande fractions previously isolated by gel filtration
on Bio-Gel P-6 columns are shown in Chart 58. These results
show that the oligosaccharides which bind to Con A:Sepharose
are present in the low-molecular-weight fraction isolated on BioGel P-6 columns. Oligosaccharides which bind to Con A:Seph-
1562
Carbohydrate analysis showed that the completely sialylated
chains contained galactose, GlcNAc, mannose, fucose, sialic
acid, and GlcNAcol (1.9:2.9:3.0:0.8:1.8:0.9)
(Table 3). NeuNAc
was identified when mild acid hydrolysates were examined by
paper chromatography with 1-butanol:pyridine:water
(6:4:3).
Less than 5% /V-glycolylneuraminic acid could have been de
tected by this method. The anomeric configuration and sequence
of sugars in these oligosaccharides were determined by hydrol
ysis with specific glycosidases and methylation analysis. The
data are summarized in Tables 3 and 4. The intact oligosaccharide yielded alditol derivatives of 2,3,4-trimethylfucose,
2,3,4trimethylgalactose,
3,4,6-trimethylmannose,
2,4-dimethylmannose, 3,6-dimethyl-GlcNAc, and 1,3,5-trimethyl-GlcNAcol in the
ratio of 0.9:1.8:2.0:0.9:2.8:0.7,
respectively. After treatment
with neuraminidase, all of the 2,3,4-trimethylgalactose was con
verted to 2,3,4,6-tetramethylgalactose,
indicating that 2 equiva
lents of sialic acid are attached to galactose by 2,6-linkages.
Treatment of the asialooligosaccharide with ri-galactosidase re
moved all of the galactose, and about 1.9 equivalents of 3,6dimethyl-GlcNAc were converted to 3,4,6-trimethyl-GlcNAc indi
cating that 2 equivalents of galactose are attached by /31,4linkages to 2 GlcNAc residues. Subsequent treatment with ß-Nacetylglucosaminidase released 1.9 equivalents of GlcNAc. This
change was accompanied by the loss of 1.9 equivalents of 3,4,6trimethylmannose and 1.9 equivalents of 3,4,6-trimethyl-GlcNAc
and the appearance of 1.8 equivalents of 2,3,4,6-tetramethylmannose. These alterations indicate that 2 GlcNAc residues are
attached through 01,2-linkages to 2 mannosyl units. When the
resulting glycopeptide was treated with a-mannosidase, about
2.1 equivalents of mannose were released, 2,4-dimethylmannose
was lost, and the amount of 2,3,4,6-tetramethylmannose
de
creased to 0.9 equivalent. These observations are consistent
with the attachment of 2 mannosyl residues to a third mannose
through «1,3-and «1,6-linkages to form a branch point in the
oligosaccharide chain. The reisolated oligosacchande contained
0.9 equivalent of mannose, 0.8 equivalent of fucose, and 0.9
equivalent of GlcNAc per mol of GlcNAcol. Incubation with «-
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VOL. 44
an-AGP from Liver Métastases
Tabte3
Sequentialtreatment of reduced oligosaccharides with specific glycosidases
Assays for sugars and conditions for incubation with glycosidases are described in the text. About 3 ttmtÃ-t
of reduced asialo oligosaccharidewere treated with 0-galactosidase,and 2.2 »imol
of the reisolated glycopeptide, devoid of galactose, were treated with li-W-acetylglucosaminidase.This product was isolated, and 1.6
yjrnolwere treated with «-mannosidase.The product was isolated and treated with fj-mannosidaseand then
with o-fucosidase. The products of the reaction in each case were isolated on Bio-Gel P-6 columns (2.2 x 200
cm), and the residues released were calculated from the amount of mannose present in the isolated product.
Residues released
TreatmentDiantennary
oligosaccharideNeuraminidase
2.0Galactose1.9
,<-Galactosidase
1.8GlcNAc2.9
/3-W-AcetylglucosaminkJase
1.91.1Mannose3.02.10.8Fucose0.8
..-Mannosidase
/J-Mannosidase
a-Fucosidase
0.7GlcNAcol0.91.0
Composition of final productNeuNAc1.8
Triantennary oligosaccharide
Neuraminidase
o'-Galactosidase
,i-N-Acetylglucosamimdase
.,-Mannosidase
a-Fucosidase
Composition of final product
3.1
3.0
2.8
3.9
3.0
0.7
0.9
1.9
2.0
1.0
0.8
0.8
Tetraantennary oligosaccharide
(asialo)
,<-Galactosidase
0-N-AcetylglucosaminkJase
a-MannoskJase
a-Fucosidase
Composition of final product3.92.80.94.82.9
2.1
1.9
2.93.01.0
1.80.8
0.9
0.90.9
0.8
s'6
O
I2
0.3
I
Ul
X
io
UJ
8
0.2
<
ui
O.I
>
0.
O
IOO
ELUTION
200
VOLUME
300
(ML)
Charts. Separation of sialylated reduced oligosaccharides from «,-AGPB,«,AGPc, and a,-AGPL by ion-exchange chromatography on DEAE-cellulose.The
completely salt-free, tritium-labeledoligosaccharidesisolated by affinity chromatog
raphy on Con A:Sepharose 4B, 5 ntnol, were applied to DEAE-cellulosecolumns
(2.7 x 8 cm) which had been washed previously with 2 M pyridineacetate and then
exhaustively with distilled water. The oligosaccharides were eluted with a linear
gradient made up of 250 ml of water in the mixing chamber and 250 ml of 0.25 M
pyridine acetate, pH 5.2, in the reservoir. Fractions of 5 ml were collected, and
aliquots were taken for the measurement of radioactivity. Curve •
was obtained
with the reduced triantennary oligosaccharides from a,-AGPB. Curve O was ob
tained with the reduced triantennary oligosaccharidesfrom <.,-AGPc.Curve A was
obtained with reduced triantennary oligosaccharidesfrom a,-AGPL.
fucosidase released all of the fucose from the glycopeptide. The
disappearance of the 2,3,4-trimethylfucose peak was accom
panied by the loss of 1,3,5-trimethyl-GlcNAcol and the appear
ance of 1,3,5,6-trimethyl-GlcNAcol.
These alterations snowed
APRIL 1984
300
VOLUME
400
(ml)
Chart 7. Gel filtration of the reduced di-, tri-, and tetraantennary oligosaccha
rides on Bio-Gel P-6 columns. The samples, 1 jimoi, were applied to Bio-Gel P-6
columns (2.2 x 200 cm, 400 mesh), and they were eluted with 0.1 M pyridinium
acetate, pH 5.2.A. A, reduceddisialylateddiantennarychain; »,reduced monosialyl
diantennary chain; O, reduced asialo diantennary chain. B. A, reduced trisialylated
triantennary chain; •,reduced asialo tetraantennary chain; O, reduced asialo
triantennary chain. The composite peaks were obtained with different purified
samples in separate runs.
that fucose is attached to the GlcNAcol residue by an «1,6linkage. Subsequent treatment with /3-mannosidaseresulted in
the release of 0.8 equivalent of mannose and the appearance of
peaks corresponding to 3,4,6-trimethyl-GlcNAc and 1,3,5,6-tetramethyl-GlcNAcol. The reisolated reduced disaccharide yielded
equal amounts of GlcNH2 and GlcNH2ol after hydrolysis in 4 N
MCI for 6 hr at 100°.Taken collectively, these results are
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E. V. Chandrasekaran et al.
Table 4
Molar ratio of partially methylated alditol and hexosaminitol acetates in reduced diantennary oligosaccharide
treatment with specific glycosidases
ratioMethylated
after
Molar
derivative2,3,4-TriCHrFuc*
N-acea-man-nosidase0.80.90.91.0After
neura-mmidase0.8 0-galac- tylglucosa-mmidase0.91.8
a-fu-cosidase0.9
oligo
tosidase0.81.91.0
saccharide0.9
2,3,4,6-TetraCH3-Gal
0.2
1.91.70.8
2,3,4-TriCH3-Gal
1.8
3,4,6-TriCH3-Man
2.00.9
2,3,4,6-TetraCH3-Man
2,4 DiCH3-Man
3.4,6-TriCH3-GlcNAc
0.12.8
0.12.9
3,6-DiCH3-GlcNAc
1.S.S-TriCHa-GteNAcolIntact
0.8After
0.7After
" CH3, methyl-; Fuc, fucose; Gal, galactose; Man, mannose.
0.90.8
1.9
0.90.6After
0.8
0.8After
Tables
Oxidation of fully sialylated reduced oligosaccharides with periodate by sequential Smith degradation
The procedures used for periodate oxidation and subsequent acid hydrolysis are described in the text. About 3 umol
of each oligosaccharide were used in these experiments.
Residues remaining
TreatmentDiantennary
oligosaccharidePeriodate
treatmentResidues
hydrolysiswith
released after
acidTriantennary
dilute
oligosaccharidePeriodate
treatmentNeuNAc1.8003.10Galactose1.9002.81.9GteNAc2.92.81.73.93.9Mannose3.00.803.02.0Fucose0.8000.70GlcNAcol1.00.700.90.9
Table 6
ratioAfter
W-acetylgluneuramini(J-galactc-sidase0.80.21.00.80.91.91.10.90.8Molar
a-mannosia-fucosioligosac
cosammidase0.80.91.00.70.80.90.80.7After
derivative2,3,4-TriCH3-Fuc<l2.3,4,6-TetraCH3-Gal2,3,4-TriCH3-Gal2,4,6-TriCrVGal2,3,4,6-TetraCH3-Man2,4,6-TriCH3-Man2,3,6-TriCH3-Man3.4,6-TriCH3-Man3,6-DiCH3-Man2,4-DiCH3-Man3,4,6Methylated
charide0.70.20.91.81.00.80.92.91.00.9After
dase0.82.81.00.80.82.90.90.8After
dase0.80.70.90.80.90.90.8After
periodate1.90.91.10.82.80
dase0.70.90.82.00.7First
,3.5,6-TetraCH3-GlcNAcolIntact
" CH3, methyl-; Fuc, fucose; Gal, galactose; Man, mannose.
consistent with the structure of the A/-asparagine-linked dianten
nary chain shown below.
«26 Gal —*
01 4 GlcNAc —»-Man
01 2
NeuNAc—'•+
N
-»GlcNAc—»GlcNAcol
mannose, GlcNAc, and GlcNAcol. These results further confirm
the structure of the diantennary oligosaccharide shown above.
Structure of the Triantennary Chains Isolated from «,-AGP.
Carbohydrate analysis showed that the completely sialylated
chain which was isolated from ai-AGPL contained sialic acid,
galactose, GlcNAc, mannose, fucose, and GlcNAcol in the ratio
of 3.1:2.8:3.9:3.0:0.7:0.9
(Table 3). Examination by methylation
NeuNAc
Gal - . GlcNAc
Fuc
»Man
analysis showed that the intact oligosaccharide contained 2,3,4trimethylfucose, 2,3,4-trimethylgalactose,
2,4,6-trimethylgalacPeriodate oxidation (Table 5) destroyed all of the sialic acid,
tose, 3,4,6-trimethylmannose, 3,6-dimethylmannose, 2,4-dimethgalactose (Gal), and fucose (Fuc) and about 2 equivalents of
ylmannose, 3,6-dimethyl-GlcNAc, 6-methyl-GlcNAc, and 1,3,5,6mannose (Man), as shown in Table 5. Subsequent reduction with
NaBH4 and mild acid hydrolysis (0.1 N H2SO4 for 1 hr at 80°) tetramethyl-GlcNAcol
(0.7:0.9:1.8:1.0:0.8:0.9:2.9:1.0:0.9)
released 2 equivalents of GlcNAc and a trisaccharide containing
(Table 6). Incubation with neuraminidase resulted in the release
X^1.3
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VOL. 44
a,-AGP from Liver Métastases
of 3 residues of sialic acid (Table 6). The release of all sialic acid
was accompanied by the disappearance of 0.9 residue of 2,3,4trimethylgalactose and 1.8 residues of 2,4,6-trimethylgalactose
and the appearance of 2.8 residues of 2,3,4,6-tetramethylgalac-
residue which was still substituted at position 4.
Hydrolysis with «-mannosidase resulted in the release of only
saccharide product was labeled by treatment with galactose
oxidase and sodium borotritide. Only a very slow release of
galactose was observed when it was incubated with 0-galactosidase a second time. Likewise, incubation with «-galactosidase
cosaminidase released 0.8 equivalent of GlcNAc, and this change
was accompanied by the loss of 0.9 equivalent of 3,6-dimethyl
mannose and the appearance of 0.8 equivalent of 2,3,6-trimethylmannose.
It is evident from these results that the sialic acid residue
which is linked <>2,6 to galactose (Gal) is present in the outer
chain linked 01,2 to the «1,3-mannose (Man) residue. Taken
collectively, the results are consistent with the structure of the
following reduced triantennary chain in which Fuc is fucose.
1.0 residue of mannose (Table 3), and this change was accom
panied by the disappearance of 0.8 residue of 2,4-dimethylmannose and the appearance of 0.9 residue of 2,4,6-trimethylmantose, indicating that 2 sialic acid residues are attached through
«2,3-linkages to 2 penultimate galactosyl units and that one of nose. These observations show that the terminal mannosyl
the sialic acid residues is attached through a «2,6-linkage to a residue is attached through a «1,6-linkage to a branched man
third galactosyl unit. These results were supported by data nosyl unit which is still substituted at position 3.
When the oligosaccharide chain obtained after treatment with
obtained from periodate oxidation studies. Only 0.9 equivalent
neuraminidase, 0-galactosidase, and 0-A/-acetylglucosaminidase
of galactose was destroyed after oxidation with periodate [2.8
was subjected to acetolysis, which is specific for the hydrolysis
residues were reduced to 1.9 residues (Table 5)]. The 2 galac
tose units substituted by sialic acid residues through «2,3- of «1,6-linkages (17), 1 residue of mannose was released. This
result further supported the observation that the branched chain
linkages were resistant to oxidation by periodate.
Treatment of the asialo-reduced oligosaccharide with 0-galaccontaining the 01,4-linked (Fuca1,3) GlcNAc unit is attached to
the «1,3-linked mannose.
tosidase resulted in the release of only 1.8 residues of terminal
galactose. This change was accompanied by the loss of 2.0
One galactosyl residue was destroyed after periodate oxida
residues of 2,3,4,6-tetramethylgalactose
and 1.9 residues of 3,6tion (Table 5). The oxidation product was reduced with sodium
borohydride and isolated by gel filtration on a Bio-Gel P-6 column
dimethyl-GlcNAc (2.9 residues decreased to 1.0 residue) and the
appearance of 1.9 residues of 3,4,6-trimethyl-GlcNAc. These
(2.2 x 200 cm). Methylation analysis showed that the product
contained both 3,6- and 2,4-dimethylmannose and 0.8 equivalent
results show that at least 2 of the galactose units are attached
of terminal GlcNAc. Subsequent treatment with 0-A/-acetylgluthrough 01,4-linkages to 2 GlcNAc units. The reisolated oligo
also failed to release terminal galactose. When this oligosaccha
ride was treated with a-fucosidase and examined by methylation
analysis, the amount of 3,6-dimethyl-GlcNAc increased from 1
residue to 1.9 residues. When the oligosaccharide was treated
with rt-fucosidase and /3-galactosidase, the amount of 3,4,6trimethyl-GlcNAc increased from 1.9 residues to 2.8 residues.
These results indicate that one residue each of galactose and NeuNAc^
fucose are linked to GlcNAc in the outer chain by 01,4- and «1,3linkages, respectively. When the oligosaccharide was treated
with 0-A/-acetylglucosaminidase, 2 residues of GlcNAc were re
leased (Table 3). This alteration was accompanied by the disap
pearance of 1.9 residues of 3,4,6-trimethyl-GlcNAc and 0.8 res
idue of 3,6-dimethylmannose and the appearance of 1.0 residue
of 2,3,4,6-tetramethylmannose
and 0.7 residue of 2,3,6-trimethylmannose. These results showed that both GlcNAc resi
dues were attached through 01,2-linkages to 2 mannosyl units
and that one of them was attached to a branched mannosyl
Gal ^i
GlcNAc —* Man
j»
-* GlcNAc ^GIcNAcol
NeuNACÄGal-^GIcNAc
NeuNAc^i Gal-^t GlcNAc
|a1,3
Fuc
Structure of the Tetraantennary Chains of ai-AGP. Data
presented in Table 3, indicating that these reduced asialo oligo-
Tabte?
Molar ratio of partially methylated alditol and hexosaminitol acetates from reduced asialo tetraantennary
oligosaccharides after treatment with glycosidases
ratioAfter
ß-N-ace-tylglucosamini-dase0.80.91.00.70.80.90.80.7After
o-man-noskJase0.80.70.90.80.90.90.8After
a-fucosi-dase0.70.90.80.20.7
/3-galac
oligo
saccharide0.73.91.00.80.93.91.00.9After
tosidase0.80.81.00.80.92.91.10.90.8Molar
derivative2,3.4-TriChVFuc"2,3,4,6-TetraCH3-Gal2,3,4,6-TetraCH3-Man2,4,6-TriCH3-Man2,3,6-TriCHa-Man3,4-DiCH3-Man3,6-DiCH3-Man2,4-DiCH3-Man3,4,6-TriCH3-GlcNAc3
Methylated
,3,5,6-TetraCH3-GlcNAcolIntact
" CH3, methyl-; Fuc, fucose; Gal, galactose; Man, mannose.
APRIL
1984
1565
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E. V. Chandrasekaran et al.
saccharides contain nearly 4 residues of galactose, 5 residues
of GlcNAc, and 1 residue of GlcNAcol per 3 residues of mannose
in the chain, are consistent with the presence of a tetraantennary
structure in these oligosaccharides. Furthermore, these chains
contain 1 residue of fucose.
As discussed in detail for the triantennary oligosaccharide
chains, the sequential treatment with exoglycosidases (Table 3)
and the permethylation analysis of the remaining oligosaccharide
after each glycosidase treatment, which is reported in Table 7,
showed that the asialo tetraantennary chain had the following
structure.
Gal-^i.
GlcNAc
fli4
Gal—'-+GlcNAc
—1-»Man
a1'6
01 4
01 4
Man-—*GlcNAc —^-»GlcNAcol
014
012
/
the synthesis of these oligosaccharide chains. The synthesis of
the inner portion of A/-asparagine-linked oligosaccharide chains
(31) requires the participation of lipid intermediates (2). The
attachment of 2 /31,2-linked GlcNAc residues to branched mannosyl residues of the processed glycoprotein intermediate leads
to the formation of complex-type diantennary chains. The further
addition of a /31,4-linked GlcNAc unit to the «1,3-linked mannosyl
residue and a /31,6-linked GlcNAc unit to the a1,6-linked man
nosyl residue initiates the synthesis of triantennary and tetraan
tennary chains. Thus, if an intermediate in the synthesis of the
diantennary chain is also a precursor of the triantennary and
tetraantennary chains, the ratio of the 3 types of chains in the
glycoprotein could be constant and independent of the number
of oligosaccharide chains in the glycoprotein. By this mechanism,
the 3 complex-type oligosaccharide chains (di-, tri-, and tetraan
tennary) can be present at any glycosylation site of polypeptide
chain, depending mainly on the interaction of /31,4- and /31,6-
rose columns containing covalently bound lectins.
<ï,-AGPpurified from plasma has been characterized as a
glycoprotein with a molecular weight of about 40,000 and con
taining 40% carbohydrate (33, 34). A number of molecular var
iants of cn-AGP have been reported (25, 32, 39). The present
study clearly establishes at least 3 types of microheterogeneity
in ai-AGP isolated from metastatic livers. First, the number of Nasparagine-linked chains varies. There may be multiple forms
consisting of a mixture of glycoproteins with homogeneous
polypeptide chains containing different numbers of oligosaccha
ride chains. The resolution obtained with RCA!20:agarose col
umns used in the present studies may permit only the separation
of 2 broad fractions of the glycoprotein. These preparations are
still polydisperse, in spite of the fact that the polypeptide chains
are homogeneous. Furthermore, species containing only a few
carbohydrate chains may have been lost during chromatography
on Con A:Sepharose 4B, since they would not be expected to
bind tightly to this column.
Another form of heterogeneity in c^-AGP is related to the
GlcNAc transferases with the glycoprotein intermediates at dif
ferent stages of processing. In this context, it is interesting to
note than an investigation of 16 glycopeptides derived from
human c^-AGP by Foumet ef al. (9) revealed that each glycosy
lation site of pooled plasma a,-AGP possesses different types
of complex-type A/-glycosidic chains. The diantennary chain and
triantennary chain containing fucose were exclusively present in
glycosylation site II, whereas tetraantennary chains with fucose
were not present in this site. These results also indicate that
different types of oligosaccharide chains may be present in a
constant proportion.
A third form of microheterogeneity in a^AGP is the extent of
sialylation of the oligosaccharide chains. This type of heteroge
neity results in the exposure of penultimate galactosyl residues
and dramatically
influences the binding of n,-AGP to
RCA12o:Sepharose columns. The tri- and tetraantennary chains
contain additional galactosyl residues, and the selective desialylation of these chains could lead to increased binding of aiAGP. The number of potential terminal galactosyl residues in an
a, -AGP preparation will depend on the number of oligosaccharide
chains in the glycoprotein. Thus, we were able to separate 2
fractions of ai-AGP containing different numbers of oligosaccha
ride chains on RCAizo: Sepharose columns because the lighter
forms did not contain enough oligosaccharide chains to bind
them to the column. Peripheral microheterogeneity was not due
to the cleavage of sialyl residues during treatment with perchloric
acid. It is possible that the absence of sialic acid in some
preparations of ai-AGP results from incomplete sialylation during
synthesis or from desialylation after secretion.
The exact biological function of the oligosaccharide chains in
ai-AGP is not presently well understood. The intracellular trans
port of glycoproteins depends upon their structural characteris
tics and can be blocked or inhibited by abnormalities caused by
mutation, by environmental modification, by a particular se
quence of amino acids, or by removal of oligosaccharides (28).
Weitzman ef al. (36) have observed in their study on immuno-
structure of the oligosaccharide chains. The glycoproteins iso
lated from tumors of a similar histological type from different
individuals contain di-, tri-, or tetraantennary chains in a constant
proportion of about 20, 50, and 30%, respectively. This ratio
was almost the same in both the lighter and heavier forms of the
«1-AGPisolated from different liver métastases. These results
suggest that this type of microheterogeneity may occur during
globulin glycopeptides of mouse myeloma cells that, in spite of
the fact that the cellular glycosylating apparatus has the potential
of producing a variety of distinct oligosaccharides, the major
factor determining the processing of oligosaccharides is inherent
in the protein structure of a glycoprotein. The at-AGPs isolated
from liver métastases have less tetraantennary complex-type
oligosaccharide chains than does «,-AGP isolated from human
Gal -^-»GlcNAc
-^-
Man
Gal-—-»
GlcNAc
Fuc
DISCUSSION
When tumors are extracted with perchloric acid, glycoproteins,
which have a high carbohydrate content, such as ai-AGP and
carcinoembryonic
antigen, remain in the soluble fraction,
whereas most proteins are precipitated. Subsequent chromatog
raphy on Sepharose CL-6B and Sephadex G-200 columns re
moves high-molecular-weight
components, principally blood
group substances, high-molecular-weight
mucins, and carci
noembryonic antigen. In the present studies, c^-AGP was further
purified by techniques involving affinity chromatography on aga-
1566
CANCER
RESEARCH
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VOL. 44
ì-AGPfrom Liver Métastases
plasma. Foumet ef al. (9) and Yoshima ef al. (38) have reported
that normal plasma «,-AGPcontains more tetraantennary chains,
whereas c^-AGP from metastatic liver has more triantennary
14.
chains. Furthermore, we have identified in liver métastases 2
forms of ai-AGP which have almost identical amino acid com
positions but differ in the number of complex-type oligosacchahde chains. Glycopeptides with tri- and tetraantennary complex-
15.
type oligosaccharide chains pass through Con A:Sepharose
columns, whereas glycopeptides with diantennary complex-type
bind to this lection (16, 27). The t^-AGP isolated from liver
métastasescontains only 15 to 20% diantennary oligosaccharide
chains, and it binds to Con A:Sepharose 4B. These results
suggest that only a small number of the total oligosaccharide
chains in a glycoprotein may be required for specific binding to
some lectins. Furthermore, a^AGP was separated into 2 species
on RCA120:Sepharose 4B, and this separation was dependent
on the density of terminal galactose residues in the glycoprotein.
The binding of this glycoprotein to the lectin required the pres
ence of a minimum number of oligosaccharide chains terminating
in galactose. A number of lectin binding studies (4,5, 8,15) have
shown that a small variation in the pattern of complex-type
oligosaccharide chains, as well as the concentration of terminal
sugars in a glycoprotein, can significantly influence the binding
of the glycoprotein to membrane receptors. In this context, it
may be important to note that the oligosaccharide pattern of aiAGP isolated from liver métastases is different from that of
normal plasma ai-AGP.
17.
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1567
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Structures of the Oligosaccharide Chains of Two Forms of α1
-Acid Glycoprotein Purified from Liver Metastases of Lung,
Colon, and Breast Tumors
E. V. Chandrasekaran, Marco Davila, Daniel Nixon, et al.
Cancer Res 1984;44:1557-1567.
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