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Demonstration of complexity of the glycosphingolipid fraction of rat small intestine
Vol.
95, No.
1, 1980
July
16, 1980
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages 416-422
DEMONSTRATION OF COMPLEXITY OF THE GLYCOSPHINGOLIPID FRACTION OF RAT SMALL INTESTINE Michael
E. Breimer,
Karl-Anders Department
Received
May
Gunnar
Karlsson
C. Hansson,
and Hakon Leffler
of Medical
Biochemistry,
Box 33031,
S-400
University
33 Gijteborg,
of GGteborg,
Sweden
2,198O
SUMMARY
A total neutra2 (non-acid) gZycoZipid fraction has been isoZated from By siZicic acid column chromatography of the acetyZated rat smaZZintestine. gZycoZipid derivative, 7 different partZy purified fractions were obtained. Thin-Zayer chromatography of both the acetylated and native ~1yeoZipid fractions reveaZed a highi-y compZex pattern uith at least 30 different gZycoZipid bands having a thin-Zayer mobiZity as for mono- to dodecagZycosyZceramides. Mass spectrometry of the permethyZated and permethyzated-reduced ~L~AZH~) derivatives showed the presence of severaZ gZycoZipid species not known before, including oZigohexosyZeeramides with 4, 5, 6 and 7 sugar This residltes and a tetragZycosyZceramide with a bZood group A determinant. is the first report 012such a complex qZycolipid composition of a sir,gZe organ. INTRODUCTION WKibbin small It
(1)
intestine
has later
cells
been
(4,5).
investigation
simple
glycolipid
the earlier
is a relatively
rich
shown
that
techniques complexity revealed
of glycolipids by conventional
(8,9),
cells
small
methods.
not
revealed
(3).
fucose
preparation,
noted
paper
intestine
fucolipids
maturity
of small
to show that
to the epithelial (6)
of different were
first
group
intestine
the present
of rat
the
confined
membrane
and fucolipids
of analysis
of blood
small
cells
on epithelial
probably
are
a microvillus
on epithelial
investigation
source
of rat
from
pattern
(2) were
the fucolipids
a study
fractions
recent
novel
et al.
Although
the glycolipid
systematic
and Suzuki
(7).
will is
a more showed
a very
As a part
intestine
in
of a
including
demonstrate much higher
that than
Vol.
95, No.
BIOCHEMICAL
1 1980
MATERIALS
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
AND METHODS
Small An inbred strain of black and white (hooded) rats was used. intestine was rapidly taken out after ether anaesthesia and sacrificing by decapitation. Lyophilized tissue was cut into small pieces and extracted in one day with chloroform-methanol 2:l (by two steps in a Soxhlet apparatus, The combined vol.) and one day with chloroform-methanol 1:9 (by vol.). extracts were evaporated and subjected to mild alkaline degradation, dialysis, DEAE and silicic acid chromatography as described (10). To free the non-acid glycolipid fraction of alkali-stable phospholipids, acetylation and silicic acid chromatography were used (11). The pure non-acid glycolipids were then fractionated as acetylated derivatives on a column of silicic acid using methanol in chloroform as These were deeluant, and together 7 fractions were eluted (fig. 1). acetylated in KOH in methanol and dialysed and parts of the fractions obtained were subjected to immunological analysis and to direct inlet mass spectrometry after permethylation and after permethylation and LiAlH4-reduction (12). RESULTS At least
30 different
the anisaldehyde small
reagent)
intestine
acetylated
(fig.
1).
in
form
permethylated-reduced
discernable
seven
of the
bands
(BL-B7)
two derivatives,
coloured
derivatives
were
analysed
and the spectra
of two of these
green
chromatography
as acetylated
fractions
derivatives
(all
on thin-layer
fractionated
As shown
by the formulas
glycolipids
containing
tetra-
with of rat
and deby mass
obtained
(B4 and B5) are
on top of the spectrum only
hexoses
and pentahexosylceramides. relatively
(8,9,12),
For the major
1184,
1212 and 1240, 18, 20,
Similarly, 1360,
ions
corresponding
22 and 24 carbon
atoms,
indicated
hexoses,
from
reproduced
by the derivative
formulas,
(spectrum
not
respectively
(compare
produced
acid.
m/e
information supported
417
the
ion
derivative
top
the
formulas
fatty
acids
1156, with
formula).
series
ions
the only
and the
fatty
at m/e 1332, 24:0
1302 may correspond
from
tri-,
at m/e 1128,
with
Some sequence
2),
derivatives
sugars
and normal
fatty
shown)
all
to 4 hexoses
at
and additional
with
these
are shown
The peak
and 16:0
for
B4 (fig.
namely
these
and the trihexosyl
at m/e 1036. one fucose
are obtained
derivative
1416 and 1444,
of fraction
interpreted,
the rule
tetrahexosylceramide
the pentahexosyl 1388,
were
As is
abundant
acid.
is
All
glycolipid
2 and 3, respectively).
three
16,
were
glycolipids,
spectrometry
(figs.
non-acid
fatty
to four are explained
permethylated
proposed.
acid
Vol.
95, No.
1, 1980
BIOCHEMICAL
BI
62
AND
63
64
BIOPHYSICAL
65
66
RESEARCH
67
COMMUNICATIONS
B
Thin-layer chromatogram of acetylated (top) and native (bottom) 1. non-acid glycolipid fractions obtained by silicic acid chromatography of About 40 ug of the total and 10 ug of the acetylated total glycolipids (B). HPTLC (Merck) plates were developed with separated fractions were applied. chlorofonrrmethanol 96:4 (by vol.) for acetylated derivatives and chloroformmethanol-water 60:35:8 (by vol.) for native glycolipids. Anisaldehyde was used for detection (10).
Fig.
Analogously be
interpreted
fig.
3.
and
fatty
Fig.
, the for
at
spectrum least
of four
Oligohexosylceramides acid
2.
fraction conditions: 500 uA,
ion
series
the
reduced
glycolipids with
in
the
six intervals
derivative shown
and
seven at
m/e
in
of the
sugars 1536-1648
fraction
top are
B5 may
formulas shown and
of with
1740-1852,
Mass spectrum of the permethylated-reduced (LiAlH4) derivative Further B4 of Fig. 1. The amount used for analysis was 60 ug. voltage 4 kV, trap current electron energy 36 eV, acceleration ion source temperature 300°C, and probe temperature 260’~.
418
sugar
of
Vol.
95. No.
-- --------. 2N c:I 7 ” 2+ m -m :--=--=--=
BIOCHEMICAL
1, 1980
AND
BIOPHYSICAL
-
-
-
-
-
-
N
m
I
-$
-
-
-
-
-
-
-
-
-
-
-
-
r, N
2 m
-,
COMMUNICATIONS
----_-___
:---o-5 ---------___------_-:: z ::, .ii z2 m-, N c g--=-.-~-~-=
- \D
RESEARCH
N
w
E-z-:-g--=
419
Vol.
95, No.
1, 1980
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
8 !z L
m CI J r
!: 3
:
r r, r.
420
Vol.
95, No.
BIOCHEMICAL
1, 1980
The major
respectively. glycosylceramide
with
glycolipid
3 were
from
shown).
The presence
activity
of the native From the
the chemical Thus,
the
ceramides, bands
fraction
in order
detected
slow-migrating
A tetraan H type
to those
derivative
indicated
(spectrum
was supported
of the mass spectra
of the separate
bands
group
was also
ions
permethylated
COMMUNICATIONS
by blood
in
not
group
A
B 5'
interpretation
three
sequence
of the A glycolipid
identity
and there
additional
the only
RESEARCH
to be a blood
hexosamine,
Again,
obtained
BIOPHYSICAL
appeared
a terminal
pentaglycosylceramide. fig.
AND
bands
of fraction
B 4 were
of decreasing
mobility.
it
was possible
on the chromatogram tri-,
tetra-
identified
(papers
component
of fraction
B 7, which
of fig.
1.
and pentahexosyl-
In an analogous
have been
to ascribe
way all
in preparation),
other
including
was a 12-sugar
the
blood
group
A
glycolipid. DISCUSSION The combined spectrometry apparently
of permethylated a potent
fractions. definitely
part
based
succeeding
spectrometry derivatives
presence
The interesting at present
epithelial
cells
(7,13)
species
of amino
published
the
in
identity
even
of rat
of a more detailed and non-epithelial
of minor
small
residue
ion
of mass
of glycolipid source,
thus
components.
concerning
The composition are being
glycolipids of
421
isolated
compared.
3. Mass spectrum of the pemett~ylated-reduced (I,iAlHb) derivative fraction B5 of Fig. 1. The amount used for analysis was 180 ~lg. Further conditions: electron energy 38 eV, acceleration voltage 4 kV, trap current 500 CA, ion sour'cr temperature 300°C and probe temperature ?95OC.
pii;.
in
advantage
development
separate
intestine
study.
a clear
mixtures the
structures
of glycolipids, is
A further
by distillation
unknown
groups
sugars,
where
is
of glycolipid
several
(8,9),
and mass
derivatives
into
by mass spectrometry.
complexity
subject
known
or absence
fractionated
validating
glycolipids
complexity
The separation
was recently were
revealing
to earlier
analysis
significantly
is
for
established.
on the
of acetylated
and permethylated-reduced
technique
In addition
were
for
use of fractionation
Also, of
Vol.
95. No.
1, 1980
epithelial
cells
BIOCHEMICAL
of different
enzyme and glycolipid
AND
maturity
BIOPHYSICAL
(7,13)
RESEARCH
are being
defined
COMMUNICATIONS
concerning
characteristics.
ACKNOWLEDGEMENTS: We are indebted to I. Pascher, W. Pimlott and B.E. Samuelsson for important help. The work was supported by a grant from Swedish Medical Research Council (No. 3967).
the
REFERENCES 1. 2. 3. 4. 5. 6. 7.
8. 9. 10. 11. 12. 13.
McKibbin, J.M. (1969) Biochemistry 8, 679-685. Suzuki, C., Makita, A. and Yosizawa, Z. (1968) Arch. Biochem. Biophys. 127, 140-149. McKibbin, J.M. (1978) J. Lipid Res. 19, 131-147. Hiramoto, R.N., Smith, E.L., Ghanta, V.K., Shaw, J.F. and McKibbin, J.M. (1973) J. Immunol. 110, 1037-1043. Falk, K.-E., Karlsson, K.-A., Leffler, H. and Samuelsson, B.E. (1979) FEBS Lett. 101, 273-276. Forstner, G.G. and Wherrett, J.R. (1973) Biochim. Biophys. Acta 306, 446-459. J.-F. and Glickman, R.M. (1976) Biochim. Biophys. Acta 441, Bouhours, 123-133. Breimer, M.E., Hansson, G.C., Karlsson, K.-A., Leffler, H., Pimlott, W. and Samuelsson, B.E. (1978) FEBS Lett. 89, 42-46. Breimer, M.E., Hansson, G.C., Karlsson, K.-A., Leffler, H., Pimlott, W. and Samuelsson, B.E. (1979) Biomed. Mass Spectrom. 6, 231-241. Karlsson, K.-A., Samuelsson, B.E. and Steen, G.O. (1973) Biochim. Biophys. Acta 316, 317-335. Handa, S. (1963) Japan. J. Exp. Med. 33, 347-360. K.-A. (1977) Progr. Chem. Fats Other Lipids 16, 207-230. Karlsson, Weiser, M. (1973) J. Biol. Chem. 248, 2536-2541.
422
95, No.
1, 1980
July
16, 1980
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages 416-422
DEMONSTRATION OF COMPLEXITY OF THE GLYCOSPHINGOLIPID FRACTION OF RAT SMALL INTESTINE Michael
E. Breimer,
Karl-Anders Department
Received
May
Gunnar
Karlsson
C. Hansson,
and Hakon Leffler
of Medical
Biochemistry,
Box 33031,
S-400
University
33 Gijteborg,
of GGteborg,
Sweden
2,198O
SUMMARY
A total neutra2 (non-acid) gZycoZipid fraction has been isoZated from By siZicic acid column chromatography of the acetyZated rat smaZZintestine. gZycoZipid derivative, 7 different partZy purified fractions were obtained. Thin-Zayer chromatography of both the acetylated and native ~1yeoZipid fractions reveaZed a highi-y compZex pattern uith at least 30 different gZycoZipid bands having a thin-Zayer mobiZity as for mono- to dodecagZycosyZceramides. Mass spectrometry of the permethyZated and permethyzated-reduced ~L~AZH~) derivatives showed the presence of severaZ gZycoZipid species not known before, including oZigohexosyZeeramides with 4, 5, 6 and 7 sugar This residltes and a tetragZycosyZceramide with a bZood group A determinant. is the first report 012such a complex qZycolipid composition of a sir,gZe organ. INTRODUCTION WKibbin small It
(1)
intestine
has later
cells
been
(4,5).
investigation
simple
glycolipid
the earlier
is a relatively
rich
shown
that
techniques complexity revealed
of glycolipids by conventional
(8,9),
cells
small
methods.
not
revealed
(3).
fucose
preparation,
noted
paper
intestine
fucolipids
maturity
of small
to show that
to the epithelial (6)
of different were
first
group
intestine
the present
of rat
the
confined
membrane
and fucolipids
of analysis
of blood
small
cells
on epithelial
probably
are
a microvillus
on epithelial
investigation
source
of rat
from
pattern
(2) were
the fucolipids
a study
fractions
recent
novel
et al.
Although
the glycolipid
systematic
and Suzuki
(7).
will is
a more showed
a very
As a part
intestine
in
of a
including
demonstrate much higher
that than
Vol.
95, No.
BIOCHEMICAL
1 1980
MATERIALS
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
AND METHODS
Small An inbred strain of black and white (hooded) rats was used. intestine was rapidly taken out after ether anaesthesia and sacrificing by decapitation. Lyophilized tissue was cut into small pieces and extracted in one day with chloroform-methanol 2:l (by two steps in a Soxhlet apparatus, The combined vol.) and one day with chloroform-methanol 1:9 (by vol.). extracts were evaporated and subjected to mild alkaline degradation, dialysis, DEAE and silicic acid chromatography as described (10). To free the non-acid glycolipid fraction of alkali-stable phospholipids, acetylation and silicic acid chromatography were used (11). The pure non-acid glycolipids were then fractionated as acetylated derivatives on a column of silicic acid using methanol in chloroform as These were deeluant, and together 7 fractions were eluted (fig. 1). acetylated in KOH in methanol and dialysed and parts of the fractions obtained were subjected to immunological analysis and to direct inlet mass spectrometry after permethylation and after permethylation and LiAlH4-reduction (12). RESULTS At least
30 different
the anisaldehyde small
reagent)
intestine
acetylated
(fig.
1).
in
form
permethylated-reduced
discernable
seven
of the
bands
(BL-B7)
two derivatives,
coloured
derivatives
were
analysed
and the spectra
of two of these
green
chromatography
as acetylated
fractions
derivatives
(all
on thin-layer
fractionated
As shown
by the formulas
glycolipids
containing
tetra-
with of rat
and deby mass
obtained
(B4 and B5) are
on top of the spectrum only
hexoses
and pentahexosylceramides. relatively
(8,9,12),
For the major
1184,
1212 and 1240, 18, 20,
Similarly, 1360,
ions
corresponding
22 and 24 carbon
atoms,
indicated
hexoses,
from
reproduced
by the derivative
formulas,
(spectrum
not
respectively
(compare
produced
acid.
m/e
information supported
417
the
ion
derivative
top
the
formulas
fatty
acids
1156, with
formula).
series
ions
the only
and the
fatty
at m/e 1332, 24:0
1302 may correspond
from
tri-,
at m/e 1128,
with
Some sequence
2),
derivatives
sugars
and normal
fatty
shown)
all
to 4 hexoses
at
and additional
with
these
are shown
The peak
and 16:0
for
B4 (fig.
namely
these
and the trihexosyl
at m/e 1036. one fucose
are obtained
derivative
1416 and 1444,
of fraction
interpreted,
the rule
tetrahexosylceramide
the pentahexosyl 1388,
were
As is
abundant
acid.
is
All
glycolipid
2 and 3, respectively).
three
16,
were
glycolipids,
spectrometry
(figs.
non-acid
fatty
to four are explained
permethylated
proposed.
acid
Vol.
95, No.
1, 1980
BIOCHEMICAL
BI
62
AND
63
64
BIOPHYSICAL
65
66
RESEARCH
67
COMMUNICATIONS
B
Thin-layer chromatogram of acetylated (top) and native (bottom) 1. non-acid glycolipid fractions obtained by silicic acid chromatography of About 40 ug of the total and 10 ug of the acetylated total glycolipids (B). HPTLC (Merck) plates were developed with separated fractions were applied. chlorofonrrmethanol 96:4 (by vol.) for acetylated derivatives and chloroformmethanol-water 60:35:8 (by vol.) for native glycolipids. Anisaldehyde was used for detection (10).
Fig.
Analogously be
interpreted
fig.
3.
and
fatty
Fig.
, the for
at
spectrum least
of four
Oligohexosylceramides acid
2.
fraction conditions: 500 uA,
ion
series
the
reduced
glycolipids with
in
the
six intervals
derivative shown
and
seven at
m/e
in
of the
sugars 1536-1648
fraction
top are
B5 may
formulas shown and
of with
1740-1852,
Mass spectrum of the permethylated-reduced (LiAlH4) derivative Further B4 of Fig. 1. The amount used for analysis was 60 ug. voltage 4 kV, trap current electron energy 36 eV, acceleration ion source temperature 300°C, and probe temperature 260’~.
418
sugar
of
Vol.
95. No.
-- --------. 2N c:I 7 ” 2+ m -m :--=--=--=
BIOCHEMICAL
1, 1980
AND
BIOPHYSICAL
-
-
-
-
-
-
N
m
I
-$
-
-
-
-
-
-
-
-
-
-
-
-
r, N
2 m
-,
COMMUNICATIONS
----_-___
:---o-5 ---------___------_-:: z ::, .ii z2 m-, N c g--=-.-~-~-=
- \D
RESEARCH
N
w
E-z-:-g--=
419
Vol.
95, No.
1, 1980
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
8 !z L
m CI J r
!: 3
:
r r, r.
420
Vol.
95, No.
BIOCHEMICAL
1, 1980
The major
respectively. glycosylceramide
with
glycolipid
3 were
from
shown).
The presence
activity
of the native From the
the chemical Thus,
the
ceramides, bands
fraction
in order
detected
slow-migrating
A tetraan H type
to those
derivative
indicated
(spectrum
was supported
of the mass spectra
of the separate
bands
group
was also
ions
permethylated
COMMUNICATIONS
by blood
in
not
group
A
B 5'
interpretation
three
sequence
of the A glycolipid
identity
and there
additional
the only
RESEARCH
to be a blood
hexosamine,
Again,
obtained
BIOPHYSICAL
appeared
a terminal
pentaglycosylceramide. fig.
AND
bands
of fraction
B 4 were
of decreasing
mobility.
it
was possible
on the chromatogram tri-,
tetra-
identified
(papers
component
of fraction
B 7, which
of fig.
1.
and pentahexosyl-
In an analogous
have been
to ascribe
way all
in preparation),
other
including
was a 12-sugar
the
blood
group
A
glycolipid. DISCUSSION The combined spectrometry apparently
of permethylated a potent
fractions. definitely
part
based
succeeding
spectrometry derivatives
presence
The interesting at present
epithelial
cells
(7,13)
species
of amino
published
the
in
identity
even
of rat
of a more detailed and non-epithelial
of minor
small
residue
ion
of mass
of glycolipid source,
thus
components.
concerning
The composition are being
glycolipids of
421
isolated
compared.
3. Mass spectrum of the pemett~ylated-reduced (I,iAlHb) derivative fraction B5 of Fig. 1. The amount used for analysis was 180 ~lg. Further conditions: electron energy 38 eV, acceleration voltage 4 kV, trap current 500 CA, ion sour'cr temperature 300°C and probe temperature ?95OC.
pii;.
in
advantage
development
separate
intestine
study.
a clear
mixtures the
structures
of glycolipids, is
A further
by distillation
unknown
groups
sugars,
where
is
of glycolipid
several
(8,9),
and mass
derivatives
into
by mass spectrometry.
complexity
subject
known
or absence
fractionated
validating
glycolipids
complexity
The separation
was recently were
revealing
to earlier
analysis
significantly
is
for
established.
on the
of acetylated
and permethylated-reduced
technique
In addition
were
for
use of fractionation
Also, of
Vol.
95. No.
1, 1980
epithelial
cells
BIOCHEMICAL
of different
enzyme and glycolipid
AND
maturity
BIOPHYSICAL
(7,13)
RESEARCH
are being
defined
COMMUNICATIONS
concerning
characteristics.
ACKNOWLEDGEMENTS: We are indebted to I. Pascher, W. Pimlott and B.E. Samuelsson for important help. The work was supported by a grant from Swedish Medical Research Council (No. 3967).
the
REFERENCES 1. 2. 3. 4. 5. 6. 7.
8. 9. 10. 11. 12. 13.
McKibbin, J.M. (1969) Biochemistry 8, 679-685. Suzuki, C., Makita, A. and Yosizawa, Z. (1968) Arch. Biochem. Biophys. 127, 140-149. McKibbin, J.M. (1978) J. Lipid Res. 19, 131-147. Hiramoto, R.N., Smith, E.L., Ghanta, V.K., Shaw, J.F. and McKibbin, J.M. (1973) J. Immunol. 110, 1037-1043. Falk, K.-E., Karlsson, K.-A., Leffler, H. and Samuelsson, B.E. (1979) FEBS Lett. 101, 273-276. Forstner, G.G. and Wherrett, J.R. (1973) Biochim. Biophys. Acta 306, 446-459. J.-F. and Glickman, R.M. (1976) Biochim. Biophys. Acta 441, Bouhours, 123-133. Breimer, M.E., Hansson, G.C., Karlsson, K.-A., Leffler, H., Pimlott, W. and Samuelsson, B.E. (1978) FEBS Lett. 89, 42-46. Breimer, M.E., Hansson, G.C., Karlsson, K.-A., Leffler, H., Pimlott, W. and Samuelsson, B.E. (1979) Biomed. Mass Spectrom. 6, 231-241. Karlsson, K.-A., Samuelsson, B.E. and Steen, G.O. (1973) Biochim. Biophys. Acta 316, 317-335. Handa, S. (1963) Japan. J. Exp. Med. 33, 347-360. K.-A. (1977) Progr. Chem. Fats Other Lipids 16, 207-230. Karlsson, Weiser, M. (1973) J. Biol. Chem. 248, 2536-2541.
422