BIOCHEMICAL
Vol. 75, No. 3, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MANNOSYLTRANSFER FROM GDP-MANNOSE TO OLIGOSACCHARIDE-LIPIDS W. T. Forsee,
J. A. Griffin,
and J.
S. Schutzbach
Department of Microbiology and Institute of Dental Research University of Alabama in Birmingham Birmingham, Alabama 35294 Received
February
24,1977
SUMMARY: Rabbit liver microsomes catalyzed mannosyltransfer from GDP-mannose to oligosaccharide-lipids isolated from porcine liver. The transfer occurred in the presence of 10 mM EDTA, a condition under which the formation of dolichol-P-mannose and other chloroform soluble mannosyl-lipids was almost completely inhibited, indicating that the mannosyl-oligosaccharide linkage was formed by a direct transfer of mannose from the nucleotide sugar. Virtually all of the mannose incorporated into the oligosaccharides was released by a-mannosidase, demonstrating the formation of an a-mannosyl-linkage in the oligosaccharide-lipid product. An enzyme catalyzing the divalent cation independent transfer of mannose from GDP-mannose to exogenous oligosaccharidelipids was solubilized from rabbit liver microsomes and purified over 10 fold. A number mannose
of investigations
is a glycosyl-donor
malian
glycoproteins
transfer
with
from
an enzyme preparation
shown
that
liver
microsomes
and a-1,6-mannosyl-mannose mannosel
under
strated
that
the appropriate formation
inhibited,
acceptor.
Free mannose,
tions
and the physiological
would
now like sugars
catalyzed
in which
to present
directly however,
and aryl-
formation
(8).
and free
of the
was almost
a-linked
sugar
at relatively
demon-
in the presence
of mannosyl-P-lipid
that
a-1,3-,
Our results
of the reaction
demonstrating
of a-1,2-,
occurred
from nucleotide was required
linkages
GDP-[14C]mannose
conditions
significance evidence
the direct
We have recently
the formation
synthesis that
demonstrated
(6,7).
with
reaction
and implied
by mannosyltransfer
All
thyroids
of a-1,3-mannosyl-mannose
occurred
1
have
in mam-
of a-1,2-mannosyl-mannose
when incubated
of 10 mM EDTA, a condition completely
calf
dolichol-P-
to a-methylmannoside
formation from
shown that
of a-mannosyl-linkages
dolichol-P-mannose
using
have
Adamany and Spiro
the resultant
rabbit
years
in the formation
(l-5).
of mannose
mannosides
in recent
disaccharide
to the high
exogenous concentra-
was uncertain. the
formation
We of some
are of the g-configuration.
Copyright 0 1977 by Academic Press, Inc. All rights of reproducrion in any form reserved.
799 ISSN
0006-291X
BIOCHEMICAL
Vol. 75, No. 3, 1977
a-mannosyl-linkages fer
directly
found
AND BIOPHYSICAL RESEARCH COMML’h”CATIOMS
in mammalian
from GDP-mannose
glycoproteins
occurs
by mannosyltrans-
to an oligosaccharide-lipid.
MATERIALS AND METHODS: Oligosaccharide-lipids were isolated from pcrc.ine liver according to the procedures of Forsee and Elbein (9). One un-it of oligosaccharide-lipid acceptor was defined as that amount of oligosaccharide-lipid which had the capacity to accept 40,000 cpm when saturated with [14C]mannose under optimal assay conditions. Dolichol-P was purchased from Sigma Chemical Co. All other chemicals and enzymes were also purchased from connnercial sources. Paper chromatography was carried out on Whatman No. 1 paper in the following solvent systems: (1) 1-butanol-pyridine-water, 40:30:40; (2) 1-propanol-ethyl acetate-water, 7:1:2; and (3) ethyl acetate-pyridinewater, 8:2:1. Carbohydrates were detected with silver nitrate or with p-anisidine phthalate (10) and protein was determined by the procedure of LolArry using bovine serum albumin as the standard. --et al.(ll) ENZYME ASSAYS: Mannosyltransfer from GDP-[ 14C]mannose to oligosaccharldelinid was measured in reaction mixtures containing GDP-[14C]mannose (0.05 &, 246 mCi/mmol, New England Nuclear), EDTA (1 ;mol),-oligosaccharidtelipid (1 unit), and enzyme in a total volume of 0.125 ml of 0.1 M Trisacetate buffer, pH 7.0, containing 5% methanol and 0.8% Triton X-100 (V/V). The amount of enzyme added was 0.5 mg of crude microsomes or 0.05-0.1 mg After 30 min at 25", the reaction was terminated by the of soluble enzyme. addition of 0.05 ml of water, 0.25 ml of methanol, and 0.25 ml of chloroform in succession. The mixture was centrifuged at 8,000 x g for 3 min and the lower chloroform phase was removed and saved. The upper phase and interface were extracted with a second 0.25 ml of chloroform and following centrifugation The combined chloroform layers the upper and lower phases were removed. The pellet were washed with 0.25 ml of CH30H-H20 (l:l), dried, and counted. at the interface of the aqueous and organic phases was washed twice wit:h 0.5 ml of 50% methanol and was extracted with CHCl -CH30H-H20 (10:10:3). The 10:10:3 extract contained the bulk of the [ l4 C1mannosyl-oligosaccharidelipid and the residual pellet contained material tentatively identified as glycoprotein. Mannosyltransfer ing to the procedures exogenous dolichol-P
from GDP-[14C]mannose to dolichol-P was assayed accordpreviously described (8) except that transfer to was assayed in the presence of 0.8% Triton X-100.
Preparation of Rabbit Liver Microsomes: Livers were excised from 1.2-1.5 kg fasted New Zealand White rabbits and a crude microsomal fraction was prepared therefrom as previously described (8). RESULTS Rabbit
liver
microsomes
oligosaccharide-lipid min at 25", chloroform. while all face
the
were
in reaction
the
reaction
The upper lower
of the newly
phase formed
of the two phases.
incubated mixtures
was terminated aqueous did
not product After
phase contain
GDP-[14C]mannose
containing
contained
GDP-[14C]mannose
in the pellet with
30
and
radioactivity.
the pellet
800
After
of methanol
unreacted
significant
and
10 mM EDTA.
by the addition
was retained washing
with
Essentially found
at the inter-
50% methanol,
95%: of
BIOCHEMICAL
Vol. 75, No. 3, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table Stimulation
I
of Mannosyltransfer
by Oligosaccharide-lipids
Reaction mixtures were scaled up 10 fold over standard reactions and the products were fractionated as described for the standard assay except the extraction volumes were correspondingly increased, and Reaction I did not contain added oligosaccharide-lipid acceptor.
Radioactivity Incorporated
Addition to Reaction Mixture Dolichol-Pmannose
I
None
II
the
Oligosaccharidelipids
radioactivity
was extracted
of the fractionation
product into
over the
mannose
10 fold
pellet
that
similar
and related
in the
1,100
760
189,000
10,200
with
and also
CHC13-CH30H-H20 in Table
10:10:3
extract,
in the
while
chloroform
of exogenous
formation
incorporation
of the of
isolated layer,
remained
10:10:3
[14C]mannose
of investigations
procedures
glycoproteins
The results
The addition
A number
fractionation
(10:10:3).
the
the
10 fold.
compounds
I.
stimulated
stimulated
over
Glycoprotein
16,900
as an acceptor
insoluble
demonstrated
Oligosaccharidelipids
150
are presented
oligosaccharide-lipid
(cpm) into
have
dolichol-P-
oligosaccharide-lipids in the residual
pellet
(2,4,5). Solubilization
and Purification
preparation pH 7.0, Triton mixer glycerol
from rabbit and was solubilized
X-100 for
of Mannosyltransferase:
liver
(5 ml) was diluted by the addition
in the same buffer.
1 min,
and was immediately
and mercaptoethanol
(0.1
The mixture diluted ml/liter).
801
1:4
The crude
in 0.1 M Tris-acetate,
of an equal
volume
was gently 1:3
into
After
microsomal
agitated
water
of 2.5% on a Vortex
containing
centrifugation
30% at 100,000
BlOCHEMlCAL
Vol. 75, No. 3, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table Purification
FRACTION
TOTAL ACTIVITY1
4
2.2
SPECIFIC ACTIVITY'
TOTAL RECOVERY (X)
x 103
11
100
Triton X-100 supernatant
120
6.0 x lo3
25
272
DEAR-Cellulose
7
1.5 x lo3
180
68
1.
One unit of enzyme is defined as the amount of enzyme catalyzing the transfer of 1000 cpm of [14 Clmannose to oligosaccharide-lipid under the conditions of the standard assay in 30 min.
2.
Specific
x g for
activity
60 min,
The solubilized that
had been
Triton
X-100
(400 ml)
was found
enzyme was applied
to a column
equilibrated
with
from
the
Mannosyltransfer
from
to enzyme concentration. mannosyltransfer
Characterization prepared
with
standard
(1.5
pH 7.0,
was eluted
with
buffer.
ranged
(Table
x 18 cm)
containing
0.1%
a NaCl gradient
The bulk 0.05
II).
M NaCl,
from ZO-50% with
of the
enzyme
and recovery
an overall
purif-
II).
with
to oligosaccharide-lipid
time
The partially
to any endogenous
or exogenous
supernatant
of DEAR-cellulose
of approximately
GDP-mannose
enzyme was linear
in the
equilibrating
column
(Table
protein.
M Tris-acetate,
The column
concentration
of up to 16 fold
endogenous
0.001
0 to 0.5 M in the
at a salt
ly purified
as units/mg
the enzyme activity
of enzyme activity ication
is defined
and 20% glycerol.
from
was eluted
over
of Mannosyltransferase
VOLUME (ml)
Crude Microsomes
II
for
by the partial-
up to 50 min and was proportional
purified
enzyme did
oligosaccharide-lipids,
not
catalyze
or to either
dolichol-P.
of the Product: solubilized
enzyme
incubations
and the
[14C]Mannoayl-oligosaccharide-lipid in reaction product 802
mixtures was extracted
scaled with
was up 10 fold CHC13-CH30H-
BIOCHEMICAL
Vol. 75, No. 3, 1977
A
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
F b
z t
CA3 &
i,h,
0
10
20
30
CM
Fig.
1.
Paper chromatography of [14 Clmannosyl-oligosaccharide. Carbon-14 labeled mannosyl-oligosaccharide-lipid was prepared in reaction mixtures scaled up 10 fold over standard assay reactions and the product was isolated and hydrolyzed in mild acid as described in the text. The product was applied to paper and the chromatogram was developed descending in solvent 1 for 24 hrs. The figure shows a radioactive strip scan of the chromatogram. Full scale deflection equals 30,000 cpm. The standards are (1) stachyose, (2) maltose, and (3) mannose.
H20 (10:10:3). nitrogen,
The radioactive
dissolved
in 1 ml of 50% 1-propanol
hydrolyzed
at 80"
ed by mild
acid
(Fig.
The major
than
1).
for
15 min.
hydrolysis
the stachyose The
containing
soluble
separated
0.02
under N HCl and
radioactive
products
by chromatography
had an RMan of 0.25
product
as free
was isolated
with
and migrated
that
after
u-mannosidase
[14 Clmannose
2 and 3, establishing mannosyl-linkage
then
was dried
releas-
in solvent more
1
slowly
standard.
was treated
was liberated
The water
were
product
[14C]labeled
and a sample
oligosaccharide-lipid
as judged
the anomeric
chromatography
(Fig.
2).
All
in solvent
of the radioactivity
by chromatography
configuration
1
in solvents
of the newly
formed
was alpha. DISCUSSION
The results microsomes
synthesize
saccharide-lipid the
presented
solubility
the
in this
communication
formation
therefore
could
not
the newly
synthesized
from GDP-mannose.
of dolichol-P-mannose
have been
rabbit
of a cr-mannosyl-linkage
by mannosyltransfer properties
show that
involved
were
as intermediates
u-mannosyl-linkages.
Also, 803
liver
in an oligoMannosyl-lipids
not
synthesized in
with
and
the formation
the reaction
occurred
of at s
Vol. 75, No. 3, 1977
Fig.
2.
maximal
rate
in the presence
of 10 mM EDTA, whereas,
of dolichol-P-mannose
The major distinctly
formed
from
by a direct
acceptor,
saccharide-lipid
divalent
RESEARCH COMMUNICATIONS
product
a previously
obtained
characterized
transfer
into
to contain
of mannose
described
layer
paper
in these
804
studies
was
trisaccharide-lipid
during
was only
the
activity.
from GDP-mannose
mannosyl-unit.
in this
catalyzing
for
(12-14).
the chloroform
a H-linked
enzymes
cations
di-N-acetylchitobiosyl-P-P-lipid
was extracted
was shown
require
oligosaccharide-lipid
different
was also
lipid
AND BIOPHYSICAL
Hydrolysis of [14 Clmannosyl-oligosaccharides by a-mannosidase. The carbon4 labeled product was synthesized by mannosyltransfer from GDP-[ 1t Clmannose to oligosaccharide-lipid using Triton X-100 solubilized enzyme. The labeled oligosaccharide was isolated after mild acid hydrolysis and was purified by paper chromatography as in Fig. 1. Samples of the radioactive oligosaccharide were incubated with either a-mannosidase (B) or with enzyme inactivated by heating at 100" for 3 min (A). Reaction mixtures contained sample (1200 cpm), and a-mannosidase (1 pg; Boehringer Mannheim) in 0.2 ml of 0.5 M ammonium acetate, pH 5.0, and were incubated for 4 hrs at 25'. Reaction products were separated by chromatography in solvent 2. The figure shows radioactive strip scans of the chromatograms. Full scale deflection equals 1000 cpm. The standards are (1) galactose, (2) glucose, and (3) mannose.
formation
linked
BIOCHEMICAL
which to a lipid-
The trisaccharide-
fractionation
(12,14)
and
The [I4 C]mannosyl-oligoextracted
with
CHC13-CH30H-
BIOCHEMICAL
Vol. 75, No. 3, 1977
H20 (10:10:3)
and contained
oligosaccharide
released
than
the
saccharide system
has been
an u-mannosyl-linkage. after
tetrasaccharide, shown
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
mild
acid
stachyose,
upon
to migrate
faster
Additionally,
hydrolysis paper
migrated
the free more
chromatography.
than
stachyose
slowly Free
in the
tri-
same solvent
(9).
This
is
transferase
the first which
report
catalyzes
of the
solubilization
the incorporation
of a mammalian of mannose
into
mannosyl-
an exogenous
acceptor. ACKNOWLEDGMENTS: This investigation was supported by NIH research grants CA 16777 from the National Cancer Institute and DE 2670 from the National Institute of Dental Research. The expert technical assistance of Janet Dodson and Martha Gurley is greatfully acknowledged. REFERENCES 1. 2.
Lennarz, W. J. (1975) Science 188, 986-991. Behrens, N. H., Carminatti, H., Stanelone, R. J., Leloir, L. F., and Cantarella, A. I. (1973) Proc. Natl. Acad. Sci. U.S.A. 70,
3. 4. 5. 6. 7. a. 9. 10. 11. 12.
13. 14.
3390-3394.
Hsu, A. F., Baynes, J. W., and Heath, E. C. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 2391-2395. Lucas, J. J., Waechter, C. J., and Lennarz, W. J. (1975) J. Biol. Chem. 250, 1992-2002. Oliver, G. J. A., Harrison, J., and Hemming, F. W. (1975) Eur. J. Biochem. 58, 223-229. Adamany, A. M. and Spiro, R. G. (1975) J. Biol. Chem. 250, 2830-2841. Adamany, A. M. and Spiro, R. G. (1975) J. Biol. Chem. 250, 2842-2854. Schutzbach, J. S. and Verma, A. K. (1976) Biochem. Biophys. Res. Commun. 71, 203-211. Forsee, W. T. and Elbein, A. D. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2574-2578. Zweig, G. and Whitaker, J. R. (1967) Paper Chromatography and Electrophoresis, V. 1 pp 232-266, Academic Press, New York. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. Levy, J. A., Carminatti, H., Cantarella, A. I., Behrens, N. H., Leloir, L. F., and Tabora, E. (1974) Biochem. Biophys. Res. Commun. 60, 118-125. Wedgwood, J. F., Warren, C. D., Jeanloz, R. W., and Strominger, J. L. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 5022-5026. Chen, W. W. and Lennarz, W. J. (1976) J. Biol. Chem. 251, 7802-7809.
805
Vol. 75, No. 3, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MANNOSYLTRANSFER FROM GDP-MANNOSE TO OLIGOSACCHARIDE-LIPIDS W. T. Forsee,
J. A. Griffin,
and J.
S. Schutzbach
Department of Microbiology and Institute of Dental Research University of Alabama in Birmingham Birmingham, Alabama 35294 Received
February
24,1977
SUMMARY: Rabbit liver microsomes catalyzed mannosyltransfer from GDP-mannose to oligosaccharide-lipids isolated from porcine liver. The transfer occurred in the presence of 10 mM EDTA, a condition under which the formation of dolichol-P-mannose and other chloroform soluble mannosyl-lipids was almost completely inhibited, indicating that the mannosyl-oligosaccharide linkage was formed by a direct transfer of mannose from the nucleotide sugar. Virtually all of the mannose incorporated into the oligosaccharides was released by a-mannosidase, demonstrating the formation of an a-mannosyl-linkage in the oligosaccharide-lipid product. An enzyme catalyzing the divalent cation independent transfer of mannose from GDP-mannose to exogenous oligosaccharidelipids was solubilized from rabbit liver microsomes and purified over 10 fold. A number mannose
of investigations
is a glycosyl-donor
malian
glycoproteins
transfer
with
from
an enzyme preparation
shown
that
liver
microsomes
and a-1,6-mannosyl-mannose mannosel
under
strated
that
the appropriate formation
inhibited,
acceptor.
Free mannose,
tions
and the physiological
would
now like sugars
catalyzed
in which
to present
directly however,
and aryl-
formation
(8).
and free
of the
was almost
a-linked
sugar
at relatively
demon-
in the presence
of mannosyl-P-lipid
that
a-1,3-,
Our results
of the reaction
demonstrating
of a-1,2-,
occurred
from nucleotide was required
linkages
GDP-[14C]mannose
conditions
significance evidence
the direct
We have recently
the formation
synthesis that
demonstrated
(6,7).
with
reaction
and implied
by mannosyltransfer
All
thyroids
of a-1,3-mannosyl-mannose
occurred
1
have
in mam-
of a-1,2-mannosyl-mannose
when incubated
of 10 mM EDTA, a condition completely
calf
dolichol-P-
to a-methylmannoside
formation from
shown that
of a-mannosyl-linkages
dolichol-P-mannose
using
have
Adamany and Spiro
the resultant
rabbit
years
in the formation
(l-5).
of mannose
mannosides
in recent
disaccharide
to the high
exogenous concentra-
was uncertain. the
formation
We of some
are of the g-configuration.
Copyright 0 1977 by Academic Press, Inc. All rights of reproducrion in any form reserved.
799 ISSN
0006-291X
BIOCHEMICAL
Vol. 75, No. 3, 1977
a-mannosyl-linkages fer
directly
found
AND BIOPHYSICAL RESEARCH COMML’h”CATIOMS
in mammalian
from GDP-mannose
glycoproteins
occurs
by mannosyltrans-
to an oligosaccharide-lipid.
MATERIALS AND METHODS: Oligosaccharide-lipids were isolated from pcrc.ine liver according to the procedures of Forsee and Elbein (9). One un-it of oligosaccharide-lipid acceptor was defined as that amount of oligosaccharide-lipid which had the capacity to accept 40,000 cpm when saturated with [14C]mannose under optimal assay conditions. Dolichol-P was purchased from Sigma Chemical Co. All other chemicals and enzymes were also purchased from connnercial sources. Paper chromatography was carried out on Whatman No. 1 paper in the following solvent systems: (1) 1-butanol-pyridine-water, 40:30:40; (2) 1-propanol-ethyl acetate-water, 7:1:2; and (3) ethyl acetate-pyridinewater, 8:2:1. Carbohydrates were detected with silver nitrate or with p-anisidine phthalate (10) and protein was determined by the procedure of LolArry using bovine serum albumin as the standard. --et al.(ll) ENZYME ASSAYS: Mannosyltransfer from GDP-[ 14C]mannose to oligosaccharldelinid was measured in reaction mixtures containing GDP-[14C]mannose (0.05 &, 246 mCi/mmol, New England Nuclear), EDTA (1 ;mol),-oligosaccharidtelipid (1 unit), and enzyme in a total volume of 0.125 ml of 0.1 M Trisacetate buffer, pH 7.0, containing 5% methanol and 0.8% Triton X-100 (V/V). The amount of enzyme added was 0.5 mg of crude microsomes or 0.05-0.1 mg After 30 min at 25", the reaction was terminated by the of soluble enzyme. addition of 0.05 ml of water, 0.25 ml of methanol, and 0.25 ml of chloroform in succession. The mixture was centrifuged at 8,000 x g for 3 min and the lower chloroform phase was removed and saved. The upper phase and interface were extracted with a second 0.25 ml of chloroform and following centrifugation The combined chloroform layers the upper and lower phases were removed. The pellet were washed with 0.25 ml of CH30H-H20 (l:l), dried, and counted. at the interface of the aqueous and organic phases was washed twice wit:h 0.5 ml of 50% methanol and was extracted with CHCl -CH30H-H20 (10:10:3). The 10:10:3 extract contained the bulk of the [ l4 C1mannosyl-oligosaccharidelipid and the residual pellet contained material tentatively identified as glycoprotein. Mannosyltransfer ing to the procedures exogenous dolichol-P
from GDP-[14C]mannose to dolichol-P was assayed accordpreviously described (8) except that transfer to was assayed in the presence of 0.8% Triton X-100.
Preparation of Rabbit Liver Microsomes: Livers were excised from 1.2-1.5 kg fasted New Zealand White rabbits and a crude microsomal fraction was prepared therefrom as previously described (8). RESULTS Rabbit
liver
microsomes
oligosaccharide-lipid min at 25", chloroform. while all face
the
were
in reaction
the
reaction
The upper lower
of the newly
phase formed
of the two phases.
incubated mixtures
was terminated aqueous did
not product After
phase contain
GDP-[14C]mannose
containing
contained
GDP-[14C]mannose
in the pellet with
30
and
radioactivity.
the pellet
800
After
of methanol
unreacted
significant
and
10 mM EDTA.
by the addition
was retained washing
with
Essentially found
at the inter-
50% methanol,
95%: of
BIOCHEMICAL
Vol. 75, No. 3, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table Stimulation
I
of Mannosyltransfer
by Oligosaccharide-lipids
Reaction mixtures were scaled up 10 fold over standard reactions and the products were fractionated as described for the standard assay except the extraction volumes were correspondingly increased, and Reaction I did not contain added oligosaccharide-lipid acceptor.
Radioactivity Incorporated
Addition to Reaction Mixture Dolichol-Pmannose
I
None
II
the
Oligosaccharidelipids
radioactivity
was extracted
of the fractionation
product into
over the
mannose
10 fold
pellet
that
similar
and related
in the
1,100
760
189,000
10,200
with
and also
CHC13-CH30H-H20 in Table
10:10:3
extract,
in the
while
chloroform
of exogenous
formation
incorporation
of the of
isolated layer,
remained
10:10:3
[14C]mannose
of investigations
procedures
glycoproteins
The results
The addition
A number
fractionation
(10:10:3).
the
the
10 fold.
compounds
I.
stimulated
stimulated
over
Glycoprotein
16,900
as an acceptor
insoluble
demonstrated
Oligosaccharidelipids
150
are presented
oligosaccharide-lipid
(cpm) into
have
dolichol-P-
oligosaccharide-lipids in the residual
pellet
(2,4,5). Solubilization
and Purification
preparation pH 7.0, Triton mixer glycerol
from rabbit and was solubilized
X-100 for
of Mannosyltransferase:
liver
(5 ml) was diluted by the addition
in the same buffer.
1 min,
and was immediately
and mercaptoethanol
(0.1
The mixture diluted ml/liter).
801
1:4
The crude
in 0.1 M Tris-acetate,
of an equal
volume
was gently 1:3
into
After
microsomal
agitated
water
of 2.5% on a Vortex
containing
centrifugation
30% at 100,000
BlOCHEMlCAL
Vol. 75, No. 3, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table Purification
FRACTION
TOTAL ACTIVITY1
4
2.2
SPECIFIC ACTIVITY'
TOTAL RECOVERY (X)
x 103
11
100
Triton X-100 supernatant
120
6.0 x lo3
25
272
DEAR-Cellulose
7
1.5 x lo3
180
68
1.
One unit of enzyme is defined as the amount of enzyme catalyzing the transfer of 1000 cpm of [14 Clmannose to oligosaccharide-lipid under the conditions of the standard assay in 30 min.
2.
Specific
x g for
activity
60 min,
The solubilized that
had been
Triton
X-100
(400 ml)
was found
enzyme was applied
to a column
equilibrated
with
from
the
Mannosyltransfer
from
to enzyme concentration. mannosyltransfer
Characterization prepared
with
standard
(1.5
pH 7.0,
was eluted
with
buffer.
ranged
(Table
x 18 cm)
containing
0.1%
a NaCl gradient
The bulk 0.05
II).
M NaCl,
from ZO-50% with
of the
enzyme
and recovery
an overall
purif-
II).
with
to oligosaccharide-lipid
time
The partially
to any endogenous
or exogenous
supernatant
of DEAR-cellulose
of approximately
GDP-mannose
enzyme was linear
in the
equilibrating
column
(Table
protein.
M Tris-acetate,
The column
concentration
of up to 16 fold
endogenous
0.001
0 to 0.5 M in the
at a salt
ly purified
as units/mg
the enzyme activity
of enzyme activity ication
is defined
and 20% glycerol.
from
was eluted
over
of Mannosyltransferase
VOLUME (ml)
Crude Microsomes
II
for
by the partial-
up to 50 min and was proportional
purified
enzyme did
oligosaccharide-lipids,
not
catalyze
or to either
dolichol-P.
of the Product: solubilized
enzyme
incubations
and the
[14C]Mannoayl-oligosaccharide-lipid in reaction product 802
mixtures was extracted
scaled with
was up 10 fold CHC13-CH30H-
BIOCHEMICAL
Vol. 75, No. 3, 1977
A
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
F b
z t
CA3 &
i,h,
0
10
20
30
CM
Fig.
1.
Paper chromatography of [14 Clmannosyl-oligosaccharide. Carbon-14 labeled mannosyl-oligosaccharide-lipid was prepared in reaction mixtures scaled up 10 fold over standard assay reactions and the product was isolated and hydrolyzed in mild acid as described in the text. The product was applied to paper and the chromatogram was developed descending in solvent 1 for 24 hrs. The figure shows a radioactive strip scan of the chromatogram. Full scale deflection equals 30,000 cpm. The standards are (1) stachyose, (2) maltose, and (3) mannose.
H20 (10:10:3). nitrogen,
The radioactive
dissolved
in 1 ml of 50% 1-propanol
hydrolyzed
at 80"
ed by mild
acid
(Fig.
The major
than
1).
for
15 min.
hydrolysis
the stachyose The
containing
soluble
separated
0.02
under N HCl and
radioactive
products
by chromatography
had an RMan of 0.25
product
as free
was isolated
with
and migrated
that
after
u-mannosidase
[14 Clmannose
2 and 3, establishing mannosyl-linkage
then
was dried
releas-
in solvent more
1
slowly
standard.
was treated
was liberated
The water
were
product
[14C]labeled
and a sample
oligosaccharide-lipid
as judged
the anomeric
chromatography
(Fig.
2).
All
in solvent
of the radioactivity
by chromatography
configuration
1
in solvents
of the newly
formed
was alpha. DISCUSSION
The results microsomes
synthesize
saccharide-lipid the
presented
solubility
the
in this
communication
formation
therefore
could
not
the newly
synthesized
from GDP-mannose.
of dolichol-P-mannose
have been
rabbit
of a cr-mannosyl-linkage
by mannosyltransfer properties
show that
involved
were
as intermediates
u-mannosyl-linkages.
Also, 803
liver
in an oligoMannosyl-lipids
not
synthesized in
with
and
the formation
the reaction
occurred
of at s
Vol. 75, No. 3, 1977
Fig.
2.
maximal
rate
in the presence
of 10 mM EDTA, whereas,
of dolichol-P-mannose
The major distinctly
formed
from
by a direct
acceptor,
saccharide-lipid
divalent
RESEARCH COMMUNICATIONS
product
a previously
obtained
characterized
transfer
into
to contain
of mannose
described
layer
paper
in these
804
studies
was
trisaccharide-lipid
during
was only
the
activity.
from GDP-mannose
mannosyl-unit.
in this
catalyzing
for
(12-14).
the chloroform
a H-linked
enzymes
cations
di-N-acetylchitobiosyl-P-P-lipid
was extracted
was shown
require
oligosaccharide-lipid
different
was also
lipid
AND BIOPHYSICAL
Hydrolysis of [14 Clmannosyl-oligosaccharides by a-mannosidase. The carbon4 labeled product was synthesized by mannosyltransfer from GDP-[ 1t Clmannose to oligosaccharide-lipid using Triton X-100 solubilized enzyme. The labeled oligosaccharide was isolated after mild acid hydrolysis and was purified by paper chromatography as in Fig. 1. Samples of the radioactive oligosaccharide were incubated with either a-mannosidase (B) or with enzyme inactivated by heating at 100" for 3 min (A). Reaction mixtures contained sample (1200 cpm), and a-mannosidase (1 pg; Boehringer Mannheim) in 0.2 ml of 0.5 M ammonium acetate, pH 5.0, and were incubated for 4 hrs at 25'. Reaction products were separated by chromatography in solvent 2. The figure shows radioactive strip scans of the chromatograms. Full scale deflection equals 1000 cpm. The standards are (1) galactose, (2) glucose, and (3) mannose.
formation
linked
BIOCHEMICAL
which to a lipid-
The trisaccharide-
fractionation
(12,14)
and
The [I4 C]mannosyl-oligoextracted
with
CHC13-CH30H-
BIOCHEMICAL
Vol. 75, No. 3, 1977
H20 (10:10:3)
and contained
oligosaccharide
released
than
the
saccharide system
has been
an u-mannosyl-linkage. after
tetrasaccharide, shown
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
mild
acid
stachyose,
upon
to migrate
faster
Additionally,
hydrolysis paper
migrated
the free more
chromatography.
than
stachyose
slowly Free
in the
tri-
same solvent
(9).
This
is
transferase
the first which
report
catalyzes
of the
solubilization
the incorporation
of a mammalian of mannose
into
mannosyl-
an exogenous
acceptor. ACKNOWLEDGMENTS: This investigation was supported by NIH research grants CA 16777 from the National Cancer Institute and DE 2670 from the National Institute of Dental Research. The expert technical assistance of Janet Dodson and Martha Gurley is greatfully acknowledged. REFERENCES 1. 2.
Lennarz, W. J. (1975) Science 188, 986-991. Behrens, N. H., Carminatti, H., Stanelone, R. J., Leloir, L. F., and Cantarella, A. I. (1973) Proc. Natl. Acad. Sci. U.S.A. 70,
3. 4. 5. 6. 7. a. 9. 10. 11. 12.
13. 14.
3390-3394.
Hsu, A. F., Baynes, J. W., and Heath, E. C. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 2391-2395. Lucas, J. J., Waechter, C. J., and Lennarz, W. J. (1975) J. Biol. Chem. 250, 1992-2002. Oliver, G. J. A., Harrison, J., and Hemming, F. W. (1975) Eur. J. Biochem. 58, 223-229. Adamany, A. M. and Spiro, R. G. (1975) J. Biol. Chem. 250, 2830-2841. Adamany, A. M. and Spiro, R. G. (1975) J. Biol. Chem. 250, 2842-2854. Schutzbach, J. S. and Verma, A. K. (1976) Biochem. Biophys. Res. Commun. 71, 203-211. Forsee, W. T. and Elbein, A. D. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2574-2578. Zweig, G. and Whitaker, J. R. (1967) Paper Chromatography and Electrophoresis, V. 1 pp 232-266, Academic Press, New York. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. Levy, J. A., Carminatti, H., Cantarella, A. I., Behrens, N. H., Leloir, L. F., and Tabora, E. (1974) Biochem. Biophys. Res. Commun. 60, 118-125. Wedgwood, J. F., Warren, C. D., Jeanloz, R. W., and Strominger, J. L. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 5022-5026. Chen, W. W. and Lennarz, W. J. (1976) J. Biol. Chem. 251, 7802-7809.
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