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Four antigenic variants of blood group A glycolipid: Examples of highly complex, branched chain glycolipid of animal cell membrane
BIOCHEMICAL
Vol. 49, No. 4,1972
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FOUR ANTIGENIC VARIANTS OF BLOOD GROUP A GLYCOLIPID: EXAMPLES OF HIGHLY COMPLEX, BRANCHED CHAIN GLYCOLIPID OF ANIMAL CELL MEMBRANE* Sen-itiroh
Hakomori,
Klaus
Stellner,
and Kiyohiro
Watanabe
From the Departments of Pathobiology &Microbiology School of Public Health and School of Medicine University of Washington, Seattle, Washington 98195
Received
September
29,1972
Summary - Four variants of blood group A-active glycolipid (Aa,Ab,AC,Ad) have been isolated from lipid extract of human erythrocyte membrane. Aa is a ceraand Ab is a ceramide octasaccharide; these two variants mide hexasaccharide, each contain a straight carbohydrate chain with the same A-determinant group, The Ac variant is a mixture of ceramide deca- to but differ in chain lengths. hendecasaccharide characterized by the presence of two different carbohydrate chains cojointed with a branching structure at the galactosyl residue, which is attached to glycosylceramide. The variant Ad is a mixture of ceramide dodeca, to tettareskaidecasaccharide with branchings and highly complex structure. Blood
group
ABH glycolipids
common carbohydrate and glucose, were
residue
and which
separated
("variants")
same A-specificity structures isolation
gel group
(1,2,4),
were
not
group
variants
consists
known.
to a ceramide
A glycolipid although This
A glycosphingolipid. is also
which
*
forms
the thorough gave four view
the
composition
and
fractionation distinctive
and variants
of the structures
of
presented.
The glycosphingolipid by Dolichos
peaks
each carrying in their
A preliminary
EXPERIMENTAL
sorted
polymorphic
separated,
describes
glycolipids
Two A-active
three
the differences
of a
N-acetylglucosamine,
(1,2).
(3); were
paper
by the presence
of galactose,
chromatography
of human erythrocyte
of blood these
which
is attached
on Silica of blood
have been characterized
biflorus
fraction agglutinin,
PROCEDURE
of membranes
of blood
was prepared
group
Al erythrocytes,
by 90% ethanol
extraction,
This investigation was supported by Research Grants of the National Cancer Institute CA12710, CA10909, and by the American Cancer Society BC-9-B. The generous supply of human erythrocytes through the courtesy of Dr. Eloise Giblett, director of the King County Central Blood Bank, Seattle, and the technical assistance of Ken Russell are gratefully acknowledged.
Copyright 0 19 72 by Academic Press, Inc. AI1 rights of reproduction in any form reserved.
1061
Vol. 49, No. 4, 1972
BIOCHEMICAL
TYPE DEAE
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A, Upper PhaSe - Cellulose Column
CDti&.. CTH-W
A’-..“.
...m... A”-......
...-*
.... p+”*
I
I
0
5000
.... - ..-“..... ,......... ...._. -.“,” ... ... .. f$ .-elunw~__~~~ I
10,000 Eluate
Figure
I
15,000 in
ml
1.
Separation pattern of long-chain neutral DEAE-cellulose and thin-layer of Silica Solvent numbers proportions:
Colunm size 2.5x30 cm. mixtures of the following 4)
15:0.2, 32.5:0.4,
glycolipids gel H.
80:20:0.2, 9) 65:35:0.4,
5)
by chromatographies
on
indicated by chloroform-methanol-water 2) 87.5:12.5:0.2, 1) 90:20:0.2,
77.5~22.5tO.2,
6)
75:25:0.2,
7)
70:30:0.2,
3) 85: 8) 67.5:
10) 60:40:0.5, 11) 57.5:42.5:0.5, 12) 55:45:0.5, 13) 0:100:0. Each fraction (50 ml) eluted from DEAE-cellulose column was concentrated to dryness, and the residue was dissolved in 1 ml of chloroformmethanol, spotted on Silica gelH plate, and developed with chloroform-methanol-water (60:35:8). Spots were revealed with 0.2% orcinol in 2 M sulfuric acid. Thinlayer chromatography patterns of every second fraction are shown in this figure. CDH: lactosylceramide; CTH: Galal+4Gal%l+4GlctCer; Glob: Globoside(CalNAc%1-+4 Galal+4Gal%1+4Glc+Cer); PG: paragloboside (Gal%1+4GlcNAc%1+3Gal%1-+4GlctCer: H,Lea,Leb: glycolipids with those blood group Siddiqui 6 Hakomori, unpublished); activities (Stellner & Hakomori, unpublished); Aa,Ab,AC,Ad: variants of A-active glycolipids. The majority of CDH, CTH, globosides, and PG was found in the lawer phase.
followed
The sphingolipid (2:l);
fraction
insoluble
partitioned
with
water
method
philized.
The residue
cribed
method in the
were
rejected
repeatedly
was then
of Rouser
et al
to Figure
extract
fractionated (7). 1.
in 1 liter
Glycolipids
through
was concentrated,
(1,5).
systems were
"Celite"
according for
and
modification dialyzed
on DEAE-cellulose
eluted
solvents
of chloroform-methanol
to the Svennerholm
The solvent
1062
by organic
by filtration
according
The aqueous
(6).
legend
and fractionation
(10 g) was dissolved
particles
of Folch's
modified
at -2O'C
by precipitation
elution
determined
and lyoto the are desby thin-
BIOCHEMICAL
Vol. 49, No. 4,1972
layer
chromatography
cellulose
column
retained
on Silica
in Figure
on the DEAE-column
neutral
glycolipids
further
separated
after
acetylation
eluted
by the method
at room temperature
freed
were
of elutions
Gangliosides
1.
not eluted
variants
components
and deacetylated
recovered
and were
including into
H, and the pattern
gel
are recorded
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
previously
chromatography
described
(1,2).
essentially
homogeneous
eluted were
on Silica
methoxide
glycolipids
on thin-layer
all
gel
H
Each component
sodium
The free
20 minutes.
that
were
The glycolipids
of A-glycolipid.
in chloroform-methanol-0.5% for
and hematosides
by the solvents
by thin-layer
from the DEAE-
thus
was
(2:1:0.6)
separated
chromatography,
and
having
been
from sialosylglycolipids. Carbohydrate
the modified acid
Sawardeker
(8).
tination,
composition
Blood
method
group
after
A activity
by the use of anti-A
specific
precipitin of the Dolichos
Etzler
(University
dimethylsulfoxide,
Dolichos
used in this
and methyliodide
0.5 -N sulfuric
hydride
of 0.3 acetates
(3% ECNSS' column) chromatography-mass
acid ml
water.
were
The partially
prepared
identified and by their
methylated
retention
mass spectra
spectrometer
"Peak
Identifier"
by Dr. Marilynn out in
acetates
described
(8,11,12).
and
on gas chromatography
obtained with
acid
by hydrolysis
alditol
time
(11,12)
gel.
ml of 95% acetic
followed
by the procedures
by their
diffusion
The permethylated
in 0.3
24 hours,
and by a
was carried
(10).
to acetolysis
at 80°C for
doses
was donated
Methylation
to
of A hemagglu-
on double
study
sodium
mg) was subjected
eventually
biflorus
according
N H2SO4 in 90% acetic
4 hemagglutination
Davis)(g).
containing
They were
with
0.5
by inhibition
of California,
(0.5-l
hexosaminitol
with
was determined
agglutinin
glycolipid
addition
by gas chromatography
hydrolysis
serum with
reaction
A part
after
was determined
by Finnigan
a quadrupole
gas
separator.
RESULTS AND DISCUSSION The carbohydrate indicated
that
and Gal than 1.
composition
Ab contained Ab, and that
of four
variants
more GlcNAc and Gal Ad had more GlcNAc
cyanoethylsilicone-ethyleneglycolsuccinate
1063
than
of A-glycolipid than Aa,
that
AC, although
(Aa,Ab,AC,Ad) AC had more GlcNAc the ratio
of
Vol. 49, No. 4, 1972
Fuc/Glc/GalNAc ration
rate
BIOCHEMICAL
for
all
on Silica
gel
AC, and Ad against and 0.15,
variants
that
biflorus
doses
peus.
Aa should
Glc/Fuc
0.1-0.5
equally
1:2:1:1:1.
serum.
GlcNAc/Gal/GalNAc/Glc/Fuc
1:1.5.
Ad should
showing
an average
of methylation Ab,AC,
with
ratio
study,
it
should
be a ceramide
with Figure
assumed
different
AC and Ad were was absent
by 4 hemagglu--Ulex euro-
showing
a ratio
of ceramide
of deca-
3:4:1.5:
to tettareskaidecasaccharide, The results
5:3:1.5:1:1.5.
and aminosugars
of these
variants
Aa,
1. Smith
and the results
degradation,
Aa should
be a ceramide both
the structures
contrast
to these
characterized
having
of which
of methylation
hexasaccharide a straight were
and that
carbohydrate
postulated
Ab chain
as seen in
therefore
chains
are structure later.
of methylated
have a branching
attached
by branching
was further A similar
with
supported branching in blood
some milk
by Kobata
of ceramide
dodeca-
l-3
and l-6
the
These
Gal.
group
glycoproteins
and Ginsburg
to tettareskaidecasaccharide
1064
studies
structure
(15). with
which
The variants
Aa and Ab.
by degradation with
of methylated
of 2,4-di-O-methylgalactitol,
to a cormnon glycosylceramide
has been well-established oligosaccharide
the hydrolysates
two variants,
by the presence
in the hydrolysates
should
lished
mg
2. In striking
This
and 0.05-0.1
of GlcNAc/Gal/GalNAc/
be a mixture
dodeca-
0.35 with
of GlcNAc/Gal/GalNAc/Glc/Fuc
sugars
octasaccharide,
lengths,
0.50,
with
a ratio
of GlcNAc/Gal/GalNAc/Glc/Fuc of neutral
that
ratio
of Aa,Ab,
precipitin
a precipitin
octasaccharide,
of ceramide
analysis,
strong
caused
having
AC should
an average
shown in Table
Based on sugar
showed
showed
be a ceramide
be a mixture
analysis
and Ad is
is
AC variant
Mig-
was 0.75,
A-hemagglutination
2:3:1:1:1.
to hendecasaccharide
(60:35:g)
value)(l)
variants
hexasaccharide
Ab should
or 1.5:1:1.5).
mg of Aa and Ab variants
inhibit
be a ceramide
(1:l:l
(R-globoside
of the four
agglutinin;
of anti-A
constant
chloroform-methanol-water
of globoside All
of AC and Ad variants tination
was nearly
H with
respectively.
Dolichos
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
AC and Ad
two carbohydrate (see Figure
which
will
2).
be pub-
GlcNAcl-*3(GlcNAc1-*6)Gal by Kabat Ad could a branching
(14)
and in
be a mixture structure.
BIOCHEMICAL
Vol. 49, No. 4, 1972
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE I Methylated Methylated Identified
Sugars
Found in the Hydrolysates of Four Permethylated Variants of Blood Group A Glycolipid
Sugars as Acetates
Aa*
Fraction Ab*
of Variants Ac+
Ad+
4,6-di-O-methylgalactitol
0.97
1.2
0.8
1.3
2,3,4-tri-O-methylfucositol
1.03
1.0
1.6
3.2
2,4,6-tri-O-methylgalactitol
2.3+
3.29
3.31
1.6
2,3,6-tri-O-methylglucitol
1.2
1.1
1.1
1.3
2,3,4,6-tetra-O-methylgalactitol
0.2
0.2
0.8
0.3
2,4-di-0-methylgalactitol
0.0
0.0
1.0
1.0
3,6-di-O-methyl-2-deoxy-2-Nmethylacetamidoglucitol§ll
1.5li
2.37l
4.1ll
3.0n
3,4,6-tri-O-methyl-2-deoxy-2-Nmethylacetamidogalactitol5
1.0
1.0
1.6
0.8
*The ratio of methylated sugar was calculated as 3,4,6-tri-O-methyl-2-deoxy-2-Nmethylacetamidogalactitol 1.0. t These two variants gave 2,4-di-b-methylgalactitol, indicating a branching structure. The ratio of methylated sugar was calculated as 2,4-di-0-methylgalactitol 1.0. 9 The excess proportion of 2,4,6-tri0-methylgalactitol does not agree with the monosaccharide ratio given by direct
The variants was detected
Aa and Ab are blood
in these
variants.
group
The variant
1065
A-specific AC showed,
and no other however,
activity
reactivity
not
Vol.49,No.4,
Figure
1972
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2.
Aa
GalNAcl \3
2Ga11+4G1cNAc1-+3(Ga1)n1+4G1cfCeramide
Fuclf
Ab
GalNAclb 3
AC
Fuel b2
gGalb461 c;iAc
lb
GalNAclf (GalNAc)-,_ f A2Gal l-+461 cMAc1 (Fuel)
Ad
Simjlar to AC but with branching structure.
Possible
differences
~Ga11-+4(G1cNAc1+3Ga1),1+461c+Ceramide
excessive
in structures
GlcNAc
of "variants"
and with
an additional
of blood
group
A glycolipid.
The number of sugar residues corresponding to the structure (Gal), in Aa and Ab and (GlcNAcl+3Gal), in AC has not yet been determined. The excess molar proportion of 2,4,6-tri-0-methylgalactitol found in the hydrolysate of permethylated A",Ab, indicates that q could be 1 to 2 for Aa and and AC (see Table l), however, Ab and 1 to 3 for AC. Assignment of ceramide hexasaccharide to structure Aa (i.e., n=l) was verified by interconversivenesss between Aa and h-active ceramide pentasaccharide, whose structure was recently established (Stellner, Watanabe, and Hakomori, unpublished). The variant AC is a mixture of glycolipids with Methylation or without the terminal sugar residues as shown in parentheses. analysis indicates that some terminals of branched chain are Gal, some are Fucl-tZ~ The structures of and only a small part is in completed form, GalNAc+(Fuc+)Gal. Aa and Ab were supported in an analogy of H-active glycolipid (Stellner, Watanabe. (17). A similar sugar and Hakomori, unpublished), and a long chain Le b glycolipid sequence as in Aa structure was postulated by Iseki on the basis of enzymatic hydrolysis (18).
only
with
Whether
blood
group
the H-active
or one of the branched
A reagents, glycolipid carbohydrate
but
also
is present chains
1066
with
the H-reagent
in these carries
fractions H-active
of Ulex
europeus.
as contaminants terminal
has not
BIOCHEMICAL
Vol. 49, No. 4, 1972
been decided. analysis
The latter
group,
and with
glycoprotein
points
are
tosyl
differing (14),
residue
plexity might
b$ present
only
structural
through
l-3 (chain
chains
provide
with
a long
derived
from
or as membrane organized
with
complex
cell
500 g of packed
increasingly
components
mixtures given
more than
of glycolipids in Figure
complete
ten.
2 are
understanding
It
eight)
they
therefore of this
slightly
that
with
length
tentative.
new molecular
Hakomori, S., and Strych-, Hakomori, S., Chem. Ph Yamakawa, T., Irie, Koscielak, J., Piase ed. D. Aminoff, Aca
D., ids
(1-2
glycolipids",
mg site
when they
of "abundant
glycolipids",
and hematosides
(2,16).
single
carbohydrate
increases,
techniques
variants
structures; Further species
structures
is needed
of membrane.
(1968).
residue
AC and Ad are
those study
chain
and separation
when the sugar
that
Biochem. 1, 1285 5 96 (1970).
1067
distributed
minority
KEFEBENCES 1. 2. 3. 4.
or
as receptor
"minority
therefore, different
only
This
structure
be widely
are an extreme
than
by the existing
with
still
A-determinant.
could
components
is possible,
including
nevertheless,
Nevertheless,
as the chain
impossible
group
complex,
a branching
gangliosides,
into
difficult
is
with
is much higher
of glycolipids
of the com-
functions.
of these
globoside,
at the galac-
Ad is highly structures;
membrane).
the activity
in membrane,
Separation
becomes
cell
group
one A-determinant
the specific
(more than although
to blood
Irrespective
only
The variant
multispecific
of differing
structure
3)
by branching
chain
chains
2) Analogous
or branching)
glycosphingolipid
membranes,
antigen,
by carbohydrate
seemed to carry
such as lactosylceramide,
becomes
length
attached
carbohydrate
various
among
of the sugar
1) The same A-determinant
complexities.
variants.
a ceramide
Such a highly
interest:
and 1% Gal structure.
one of the terminals
would
in view
AC and Ad had a branching
in these
two carbohydrate
into
of special was carried
variants
of molecules
is more plausible
study.
GalNAcal-+3(Fuc1-+2)Gal,
sizes
are
possibility
and methylation
The following
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
for
Vol. 49, No. 4, 1972
5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Siddiqui, B., Kawanami, J., Li, Y-T., and Hakomori, S., J. Lipid Res., September 1972, in press. Svennerholm, L., xcta Chem. Stand. l7, 239 (1963). Rouser, G., Kritchevsky, G., Heller, D., and Lieber, E., J. Amer. Oil Chem. Sot. 40, 425 (1963). Yang, H-J., and Hakomori, S., J. Biol. Chem. 246, 1192 (1971). Etzler, M., and Kabat, E.A., Biochem. 2, 869 (1970). Hakomori, S., J. Biochem. (Tokyo) 55, 205 (1964). Bjorndal, H., Lindberg, B., and Svensson, S., Carbohydr. Res. 5, 433 (1967). BjBrndal, H., Hellerqvist, C.G., Lindberg, B., and Svensson, S., Angew. Chem. Int. Ed. Engl. 2, 610 (1970). Stoffyn, A., and Stoffyn, P., Carbohydr. Res. 13, 251 (1972). Lloyd, K.W., and Kabat, E.A., Proc. Nat. Acad.Sci., USA, 3, 1470 (1968). Kobata, A., and Ginsburg, V., J. Biol. Chem. 247, 1525 (1972). Hakomori, S., VOX sang. l6, 478 (1969). Hakomori, S., and Andrews, H., Biochim. Biophys. Acta 202, 225 (1970). ed. D. Aminoff, Iseki, S., in Blood and Tissue Antigen, Academic Press, New York, 1970, p. 379.
1068
Vol. 49, No. 4,1972
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FOUR ANTIGENIC VARIANTS OF BLOOD GROUP A GLYCOLIPID: EXAMPLES OF HIGHLY COMPLEX, BRANCHED CHAIN GLYCOLIPID OF ANIMAL CELL MEMBRANE* Sen-itiroh
Hakomori,
Klaus
Stellner,
and Kiyohiro
Watanabe
From the Departments of Pathobiology &Microbiology School of Public Health and School of Medicine University of Washington, Seattle, Washington 98195
Received
September
29,1972
Summary - Four variants of blood group A-active glycolipid (Aa,Ab,AC,Ad) have been isolated from lipid extract of human erythrocyte membrane. Aa is a ceraand Ab is a ceramide octasaccharide; these two variants mide hexasaccharide, each contain a straight carbohydrate chain with the same A-determinant group, The Ac variant is a mixture of ceramide deca- to but differ in chain lengths. hendecasaccharide characterized by the presence of two different carbohydrate chains cojointed with a branching structure at the galactosyl residue, which is attached to glycosylceramide. The variant Ad is a mixture of ceramide dodeca, to tettareskaidecasaccharide with branchings and highly complex structure. Blood
group
ABH glycolipids
common carbohydrate and glucose, were
residue
and which
separated
("variants")
same A-specificity structures isolation
gel group
(1,2,4),
were
not
group
variants
consists
known.
to a ceramide
A glycolipid although This
A glycosphingolipid. is also
which
*
forms
the thorough gave four view
the
composition
and
fractionation distinctive
and variants
of the structures
of
presented.
The glycosphingolipid by Dolichos
peaks
each carrying in their
A preliminary
EXPERIMENTAL
sorted
polymorphic
separated,
describes
glycolipids
Two A-active
three
the differences
of a
N-acetylglucosamine,
(1,2).
(3); were
paper
by the presence
of galactose,
chromatography
of human erythrocyte
of blood these
which
is attached
on Silica of blood
have been characterized
biflorus
fraction agglutinin,
PROCEDURE
of membranes
of blood
was prepared
group
Al erythrocytes,
by 90% ethanol
extraction,
This investigation was supported by Research Grants of the National Cancer Institute CA12710, CA10909, and by the American Cancer Society BC-9-B. The generous supply of human erythrocytes through the courtesy of Dr. Eloise Giblett, director of the King County Central Blood Bank, Seattle, and the technical assistance of Ken Russell are gratefully acknowledged.
Copyright 0 19 72 by Academic Press, Inc. AI1 rights of reproduction in any form reserved.
1061
Vol. 49, No. 4, 1972
BIOCHEMICAL
TYPE DEAE
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A, Upper PhaSe - Cellulose Column
CDti&.. CTH-W
A’-..“.
...m... A”-......
...-*
.... p+”*
I
I
0
5000
.... - ..-“..... ,......... ...._. -.“,” ... ... .. f$ .-elunw~__~~~ I
10,000 Eluate
Figure
I
15,000 in
ml
1.
Separation pattern of long-chain neutral DEAE-cellulose and thin-layer of Silica Solvent numbers proportions:
Colunm size 2.5x30 cm. mixtures of the following 4)
15:0.2, 32.5:0.4,
glycolipids gel H.
80:20:0.2, 9) 65:35:0.4,
5)
by chromatographies
on
indicated by chloroform-methanol-water 2) 87.5:12.5:0.2, 1) 90:20:0.2,
77.5~22.5tO.2,
6)
75:25:0.2,
7)
70:30:0.2,
3) 85: 8) 67.5:
10) 60:40:0.5, 11) 57.5:42.5:0.5, 12) 55:45:0.5, 13) 0:100:0. Each fraction (50 ml) eluted from DEAE-cellulose column was concentrated to dryness, and the residue was dissolved in 1 ml of chloroformmethanol, spotted on Silica gelH plate, and developed with chloroform-methanol-water (60:35:8). Spots were revealed with 0.2% orcinol in 2 M sulfuric acid. Thinlayer chromatography patterns of every second fraction are shown in this figure. CDH: lactosylceramide; CTH: Galal+4Gal%l+4GlctCer; Glob: Globoside(CalNAc%1-+4 Galal+4Gal%1+4Glc+Cer); PG: paragloboside (Gal%1+4GlcNAc%1+3Gal%1-+4GlctCer: H,Lea,Leb: glycolipids with those blood group Siddiqui 6 Hakomori, unpublished); activities (Stellner & Hakomori, unpublished); Aa,Ab,AC,Ad: variants of A-active glycolipids. The majority of CDH, CTH, globosides, and PG was found in the lawer phase.
followed
The sphingolipid (2:l);
fraction
insoluble
partitioned
with
water
method
philized.
The residue
cribed
method in the
were
rejected
repeatedly
was then
of Rouser
et al
to Figure
extract
fractionated (7). 1.
in 1 liter
Glycolipids
through
was concentrated,
(1,5).
systems were
"Celite"
according for
and
modification dialyzed
on DEAE-cellulose
eluted
solvents
of chloroform-methanol
to the Svennerholm
The solvent
1062
by organic
by filtration
according
The aqueous
(6).
legend
and fractionation
(10 g) was dissolved
particles
of Folch's
modified
at -2O'C
by precipitation
elution
determined
and lyoto the are desby thin-
BIOCHEMICAL
Vol. 49, No. 4,1972
layer
chromatography
cellulose
column
retained
on Silica
in Figure
on the DEAE-column
neutral
glycolipids
further
separated
after
acetylation
eluted
by the method
at room temperature
freed
were
of elutions
Gangliosides
1.
not eluted
variants
components
and deacetylated
recovered
and were
including into
H, and the pattern
gel
are recorded
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
previously
chromatography
described
(1,2).
essentially
homogeneous
eluted were
on Silica
methoxide
glycolipids
on thin-layer
all
gel
H
Each component
sodium
The free
20 minutes.
that
were
The glycolipids
of A-glycolipid.
in chloroform-methanol-0.5% for
and hematosides
by the solvents
by thin-layer
from the DEAE-
thus
was
(2:1:0.6)
separated
chromatography,
and
having
been
from sialosylglycolipids. Carbohydrate
the modified acid
Sawardeker
(8).
tination,
composition
Blood
method
group
after
A activity
by the use of anti-A
specific
precipitin of the Dolichos
Etzler
(University
dimethylsulfoxide,
Dolichos
used in this
and methyliodide
0.5 -N sulfuric
hydride
of 0.3 acetates
(3% ECNSS' column) chromatography-mass
acid ml
water.
were
The partially
prepared
identified and by their
methylated
retention
mass spectra
spectrometer
"Peak
Identifier"
by Dr. Marilynn out in
acetates
described
(8,11,12).
and
on gas chromatography
obtained with
acid
by hydrolysis
alditol
time
(11,12)
gel.
ml of 95% acetic
followed
by the procedures
by their
diffusion
The permethylated
in 0.3
24 hours,
and by a
was carried
(10).
to acetolysis
at 80°C for
doses
was donated
Methylation
to
of A hemagglu-
on double
study
sodium
mg) was subjected
eventually
biflorus
according
N H2SO4 in 90% acetic
4 hemagglutination
Davis)(g).
containing
They were
with
0.5
by inhibition
of California,
(0.5-l
hexosaminitol
with
was determined
agglutinin
glycolipid
addition
by gas chromatography
hydrolysis
serum with
reaction
A part
after
was determined
by Finnigan
a quadrupole
gas
separator.
RESULTS AND DISCUSSION The carbohydrate indicated
that
and Gal than 1.
composition
Ab contained Ab, and that
of four
variants
more GlcNAc and Gal Ad had more GlcNAc
cyanoethylsilicone-ethyleneglycolsuccinate
1063
than
of A-glycolipid than Aa,
that
AC, although
(Aa,Ab,AC,Ad) AC had more GlcNAc the ratio
of
Vol. 49, No. 4, 1972
Fuc/Glc/GalNAc ration
rate
BIOCHEMICAL
for
all
on Silica
gel
AC, and Ad against and 0.15,
variants
that
biflorus
doses
peus.
Aa should
Glc/Fuc
0.1-0.5
equally
1:2:1:1:1.
serum.
GlcNAc/Gal/GalNAc/Glc/Fuc
1:1.5.
Ad should
showing
an average
of methylation Ab,AC,
with
ratio
study,
it
should
be a ceramide
with Figure
assumed
different
AC and Ad were was absent
by 4 hemagglu--Ulex euro-
showing
a ratio
of ceramide
of deca-
3:4:1.5:
to tettareskaidecasaccharide, The results
5:3:1.5:1:1.5.
and aminosugars
of these
variants
Aa,
1. Smith
and the results
degradation,
Aa should
be a ceramide both
the structures
contrast
to these
characterized
having
of which
of methylation
hexasaccharide a straight were
and that
carbohydrate
postulated
Ab chain
as seen in
therefore
chains
are structure later.
of methylated
have a branching
attached
by branching
was further A similar
with
supported branching in blood
some milk
by Kobata
of ceramide
dodeca-
l-3
and l-6
the
These
Gal.
group
glycoproteins
and Ginsburg
to tettareskaidecasaccharide
1064
studies
structure
(15). with
which
The variants
Aa and Ab.
by degradation with
of methylated
of 2,4-di-O-methylgalactitol,
to a cormnon glycosylceramide
has been well-established oligosaccharide
the hydrolysates
two variants,
by the presence
in the hydrolysates
should
lished
mg
2. In striking
This
and 0.05-0.1
of GlcNAc/Gal/GalNAc/
be a mixture
dodeca-
0.35 with
of GlcNAc/Gal/GalNAc/Glc/Fuc
sugars
octasaccharide,
lengths,
0.50,
with
a ratio
of GlcNAc/Gal/GalNAc/Glc/Fuc of neutral
that
ratio
of Aa,Ab,
precipitin
a precipitin
octasaccharide,
of ceramide
analysis,
strong
caused
having
AC should
an average
shown in Table
Based on sugar
showed
showed
be a ceramide
be a mixture
analysis
and Ad is
is
AC variant
Mig-
was 0.75,
A-hemagglutination
2:3:1:1:1.
to hendecasaccharide
(60:35:g)
value)(l)
variants
hexasaccharide
Ab should
or 1.5:1:1.5).
mg of Aa and Ab variants
inhibit
be a ceramide
(1:l:l
(R-globoside
of the four
agglutinin;
of anti-A
constant
chloroform-methanol-water
of globoside All
of AC and Ad variants tination
was nearly
H with
respectively.
Dolichos
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
AC and Ad
two carbohydrate (see Figure
which
will
2).
be pub-
GlcNAcl-*3(GlcNAc1-*6)Gal by Kabat Ad could a branching
(14)
and in
be a mixture structure.
BIOCHEMICAL
Vol. 49, No. 4, 1972
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE I Methylated Methylated Identified
Sugars
Found in the Hydrolysates of Four Permethylated Variants of Blood Group A Glycolipid
Sugars as Acetates
Aa*
Fraction Ab*
of Variants Ac+
Ad+
4,6-di-O-methylgalactitol
0.97
1.2
0.8
1.3
2,3,4-tri-O-methylfucositol
1.03
1.0
1.6
3.2
2,4,6-tri-O-methylgalactitol
2.3+
3.29
3.31
1.6
2,3,6-tri-O-methylglucitol
1.2
1.1
1.1
1.3
2,3,4,6-tetra-O-methylgalactitol
0.2
0.2
0.8
0.3
2,4-di-0-methylgalactitol
0.0
0.0
1.0
1.0
3,6-di-O-methyl-2-deoxy-2-Nmethylacetamidoglucitol§ll
1.5li
2.37l
4.1ll
3.0n
3,4,6-tri-O-methyl-2-deoxy-2-Nmethylacetamidogalactitol5
1.0
1.0
1.6
0.8
*The ratio of methylated sugar was calculated as 3,4,6-tri-O-methyl-2-deoxy-2-Nmethylacetamidogalactitol 1.0. t These two variants gave 2,4-di-b-methylgalactitol, indicating a branching structure. The ratio of methylated sugar was calculated as 2,4-di-0-methylgalactitol 1.0. 9 The excess proportion of 2,4,6-tri0-methylgalactitol does not agree with the monosaccharide ratio given by direct
The variants was detected
Aa and Ab are blood
in these
variants.
group
The variant
1065
A-specific AC showed,
and no other however,
activity
reactivity
not
Vol.49,No.4,
Figure
1972
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2.
Aa
GalNAcl \3
2Ga11+4G1cNAc1-+3(Ga1)n1+4G1cfCeramide
Fuclf
Ab
GalNAclb 3
AC
Fuel b2
gGalb461 c;iAc
lb
GalNAclf (GalNAc)-,_ f A2Gal l-+461 cMAc1 (Fuel)
Ad
Simjlar to AC but with branching structure.
Possible
differences
~Ga11-+4(G1cNAc1+3Ga1),1+461c+Ceramide
excessive
in structures
GlcNAc
of "variants"
and with
an additional
of blood
group
A glycolipid.
The number of sugar residues corresponding to the structure (Gal), in Aa and Ab and (GlcNAcl+3Gal), in AC has not yet been determined. The excess molar proportion of 2,4,6-tri-0-methylgalactitol found in the hydrolysate of permethylated A",Ab, indicates that q could be 1 to 2 for Aa and and AC (see Table l), however, Ab and 1 to 3 for AC. Assignment of ceramide hexasaccharide to structure Aa (i.e., n=l) was verified by interconversivenesss between Aa and h-active ceramide pentasaccharide, whose structure was recently established (Stellner, Watanabe, and Hakomori, unpublished). The variant AC is a mixture of glycolipids with Methylation or without the terminal sugar residues as shown in parentheses. analysis indicates that some terminals of branched chain are Gal, some are Fucl-tZ~ The structures of and only a small part is in completed form, GalNAc+(Fuc+)Gal. Aa and Ab were supported in an analogy of H-active glycolipid (Stellner, Watanabe. (17). A similar sugar and Hakomori, unpublished), and a long chain Le b glycolipid sequence as in Aa structure was postulated by Iseki on the basis of enzymatic hydrolysis (18).
only
with
Whether
blood
group
the H-active
or one of the branched
A reagents, glycolipid carbohydrate
but
also
is present chains
1066
with
the H-reagent
in these carries
fractions H-active
of Ulex
europeus.
as contaminants terminal
has not
BIOCHEMICAL
Vol. 49, No. 4, 1972
been decided. analysis
The latter
group,
and with
glycoprotein
points
are
tosyl
differing (14),
residue
plexity might
b$ present
only
structural
through
l-3 (chain
chains
provide
with
a long
derived
from
or as membrane organized
with
complex
cell
500 g of packed
increasingly
components
mixtures given
more than
of glycolipids in Figure
complete
ten.
2 are
understanding
It
eight)
they
therefore of this
slightly
that
with
length
tentative.
new molecular
Hakomori, S., and Strych-, Hakomori, S., Chem. Ph Yamakawa, T., Irie, Koscielak, J., Piase ed. D. Aminoff, Aca
D., ids
(1-2
glycolipids",
mg site
when they
of "abundant
glycolipids",
and hematosides
(2,16).
single
carbohydrate
increases,
techniques
variants
structures; Further species
structures
is needed
of membrane.
(1968).
residue
AC and Ad are
those study
chain
and separation
when the sugar
that
Biochem. 1, 1285 5 96 (1970).
1067
distributed
minority
KEFEBENCES 1. 2. 3. 4.
or
as receptor
"minority
therefore, different
only
This
structure
be widely
are an extreme
than
by the existing
with
still
A-determinant.
could
components
is possible,
including
nevertheless,
Nevertheless,
as the chain
impossible
group
complex,
a branching
gangliosides,
into
difficult
is
with
is much higher
of glycolipids
of the com-
functions.
of these
globoside,
at the galac-
Ad is highly structures;
membrane).
the activity
in membrane,
Separation
becomes
cell
group
one A-determinant
the specific
(more than although
to blood
Irrespective
only
The variant
multispecific
of differing
structure
3)
by branching
chain
chains
2) Analogous
or branching)
glycosphingolipid
membranes,
antigen,
by carbohydrate
seemed to carry
such as lactosylceramide,
becomes
length
attached
carbohydrate
various
among
of the sugar
1) The same A-determinant
complexities.
variants.
a ceramide
Such a highly
interest:
and 1% Gal structure.
one of the terminals
would
in view
AC and Ad had a branching
in these
two carbohydrate
into
of special was carried
variants
of molecules
is more plausible
study.
GalNAcal-+3(Fuc1-+2)Gal,
sizes
are
possibility
and methylation
The following
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
for
Vol. 49, No. 4, 1972
5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Siddiqui, B., Kawanami, J., Li, Y-T., and Hakomori, S., J. Lipid Res., September 1972, in press. Svennerholm, L., xcta Chem. Stand. l7, 239 (1963). Rouser, G., Kritchevsky, G., Heller, D., and Lieber, E., J. Amer. Oil Chem. Sot. 40, 425 (1963). Yang, H-J., and Hakomori, S., J. Biol. Chem. 246, 1192 (1971). Etzler, M., and Kabat, E.A., Biochem. 2, 869 (1970). Hakomori, S., J. Biochem. (Tokyo) 55, 205 (1964). Bjorndal, H., Lindberg, B., and Svensson, S., Carbohydr. Res. 5, 433 (1967). BjBrndal, H., Hellerqvist, C.G., Lindberg, B., and Svensson, S., Angew. Chem. Int. Ed. Engl. 2, 610 (1970). Stoffyn, A., and Stoffyn, P., Carbohydr. Res. 13, 251 (1972). Lloyd, K.W., and Kabat, E.A., Proc. Nat. Acad.Sci., USA, 3, 1470 (1968). Kobata, A., and Ginsburg, V., J. Biol. Chem. 247, 1525 (1972). Hakomori, S., VOX sang. l6, 478 (1969). Hakomori, S., and Andrews, H., Biochim. Biophys. Acta 202, 225 (1970). ed. D. Aminoff, Iseki, S., in Blood and Tissue Antigen, Academic Press, New York, 1970, p. 379.
1068