Vol.
89,
No.
2, 1979
July
27, 1979
8lOCHEMlCAL
AND
RESEARCH
GALACTOSYL
EMBRYONAL
Prujansky-Jacobovitsl,
G. F.
GLYCOPEPTIDES
CARCINOMA
Gachelin, Jacob
OF
CELLS
T.
Muramatsu2,
N.
Unite de Genetique Cellulaire de 1’Institut Pasteur France, Institut Pasteur, 25 rue du Dr. Roux, 75015 (1) Department of Biophysics, The Weizmann Institute Rehovot, Israel, and (2) Department of Biochemistry, School of Medicine, Kobe, Japan Received
COMMUNICATIONS
Pages 448-455
SURFACE
A.
BIOPHYSICAL
April
Sharon’
et du College de - Paris, France ; of Science, Kobe University
25,1979
SLT%lARY. Surface galactosyl residues of alive embryonal carcinoma cells were m-labeled by the galactose oxidase NaB(3H)4 method. The labeled glycopeptides isolated from Pronase digests of the cells, were found to be similar to those prepared from endogeneously labeled embryonal carcinoma cells. They consisted mostly of high molecular weight material, and contained receptors for peanut agglutinin. It is concluded that at least a fraction of the high molecular weight glycopeptides characteristic of early embryonic cells, is displayed on the cell suface. Teratocarcinomas the
gonads.
chaotic
They
contain
and
clumps
way,
carcinoma
are
(EC) . The
malignant a variety of
latter
cells derive
cell
types
cal
with
multipotential
Several
clonal
early
lines
embryonic in
peptides,
isolated cells
embryonic differentiation
cells
4-6).
A class after
has
been (7). of
are
as of
the
an
cells
as
glycopeptides and
448
from
normal
to
digestion
G. Gachelin,
biologi-
embryos
in
(3).
studies
of
embryos
of of
from
the
fucosyl-glyco-
disappear also
which
normal
weight
a
embryonal
many
early used
characteristic
with in
called
share
widely
Pronase
0006-291X/79/140448-08$01.00/0 @ 1979 by Academic Press, Inc. of reproduction in any form reserved.
stem
molecular
Correspondence should be addressed to Dr. rue du Dr. Roux, 75015 - PARIS, France.
Copyright All rights
type
alternative
high
(7),
cell
of
associated distributed
EC cells
therefore
defined
EC cells
types,
cells
extensive
These
cell
(1,2).
EC are
development,
(reviewed
labeled
of
of
generally
a distinctive
differentiated properties
tumors
13Hl-L-fucose =
EC and during
early in
vitro
normal
embryos
during
Institut
Pasteur,
25
Vol.
89,
postimplantation also of
embryogenesis
been F3Hl
found or
location known,
and
on
cell
high
has
by
here
the
on
of the
of
the cell
agglutinin
that
14
weight
COMMUNICATIONS
glycopeptides
species
EC cells
established
in
(9).
whether
The
digests
subcellular is
or
have
Pronase
glycopeptides
glycopeptides
(PNA),
those
recovered
and a cell
(11).
method
obtained
from Our
Cl-$J-galactose.
molecular
surface,
differentiation-specific cell
[
high
differentiation
the
been
from
presence
part
labelled
RESEARCH
large
galactose-oxidase-NaB[3H14
indistinguishable the
Similar molecular
molecular
not
BIOPHYSICAL
still
not
they
from
cells
(10)
appear
unare
exposed
surface.
We report labelled
(8).
-D-galactose
these it
AND
as a predominant
[14 Cl
of
the
BIOCHEMICAL
No’. 2, 1979
weight that
These
contain marker
results
changes
findings
is binding
that
provide in
labelled show
glycopeptides
they
surface
cells
carbohydrate
at
indeed sites
to
by
that
disappears further
externally
growth
in
least
exposed for upon
evidence moieties
be
peanut EC cell for
exposed
on
surface. MATERIALS
AND METHODS
CELLS. F941 is a clonal line of EC which has lost the capacity to differentiate (12). It11 be referred to it as to “EC cells”. Growth and harvest conditions of the cells .have been published (13). Thymocytes were prepared from 4 week old male 129/Sv mice, syngeneic to the F9-41 EC line (14). REAGENTS.NaB[‘3H]4 was purchased from the Radiochemical Center (8.6 Ci/mM), or from the Cormnissariat B 1’Energie Atomique (10 Ci/mM). The reagent was diluted with 0.01 N NaOH to 10 mCi/ml and kept in 0.1 ml aliquots at -78°C. [14C]-D-galactose, uniformly labeled (300 mCi/mM) was obtained from the Commissariat 2 1’Energie Atomique . Galactose oxidase, a product of Worthington (55 U/mg) or Sigma (50 U/mg) was stored at -78°C as 100 U/ml aliquots in pH 7.0 phosphate buffered saline (PBS,lS). Aliquots were used only once after thawing. Peanut agglutinin (PNA) was isolated as described (16). Anti-PNA serum was raised in rabbits, 300 1~1 of antiserum precipitated 100 ug of PNA. LABELING PROCFDURES. The external labelling of the cells was carried out essentially as described 1(10). Briefly, 0.05 ml of 10 mM unlabeled sodium borohydride in 10 mM NaOH was added to a suspension of 20-30 millions of cells in 0.45 ml of PBS pH 7.4. After 30 minutes at room temperature, the cells were washed three times with PBS pH 7.0, containing 0.001 M phenylmethylsulfonyl fluoride (Sigma). To the washed cells, suspended in 0.5 ml of the same solution, 10 units of galactose oxidase were added. Incubation was continued for 30 minutes at 37’C with occasional shaking. The cells were then washed with the same buffer containing 0.05 M D-galactose and twice with PBS pH 7.4. The washed cells were suspended in 0.45 ml of PBS pH 7.4, and mixed with 0.05 ml of tritiated sodium borohydride (0.5 mCi). After 30 minutes
449
Vol.
89,
No.
BIOCHEMICAL
2, 1979
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
at room temperature, the cells were then washed with growth medium containing 1s : fetal calf serum (Gibco), till no radioactivity was found in the supernatant. Cell viability and recovery were determined after each step of the labeling procedure. [‘4C]-D-galactose labeled 23 hours‘in the presence
EC cells were prepared by culturing of 5 LACi/ml of the raclioact ive s~~gar
F9-41
cells
for
.
;LW,\rTICAL PROCEDURES. Labeled products were directed extensively with F’ronase (Calbiochem, 10 mg/ml, 24 hours, three times, 3: L) as described (7) . Glycopeptides were analyzed by Sephadex G-SO column chromatography in 0.05 M arrunoniaacetid acid buffer pII 6.0 (7). Immunoprecipitation of receptors to PM was carried out on Triton S-100 extracts of I:9-31 cells, as described (17). Briefly, 10 millions of cells were dissolved in 0.S ml of 0.5 “, Triton X-100 in PBS, at 0°C. After- one hour, nuclei and cell debris were removed by high speed centrifugation. Solubilized receptors were complexed with PNA in solution (100 OR/ml) and the comple.xes precipitated with anti-PM .qerum added in slight excess. Innnune complexes were collected and washed by centrifugation. Cell viability was estimated by the Trypan blue exclusion test (13‘1. Raclioactivitv leas measured in an Intertechnique Scintillation spectrometer, using Unisolve ikoch-Light) as the scintillation fllkl. SUG4R ANALYSIS. Glycopeptides prepared from labeled I%41 cells (about 30,000 cpm) were hydrolyzed by a mixture (2.5 mg) of glycosidases from Turbo cornutus (>liles) consistinrr in x and I-‘#galactosidase, u and b-N acctyl galactosaminidase. The reaction was carried out in 0.1 ml of 0.05 M citrate buffer pH 4.0, at 3?‘C, under a layer of toluene. After 24 hours, 0.2 ml of water was added, the solution was boiled for 5 minutes. centrifuged. and the suDernatant was annlied to Whatman 3 !.fi\1paper. The chromatogrk was developpkd in ethyl aceiite-pyridinewater (12:5:4). After drying, the paper was cut into pieces, and counted.
RESULTS
blouse
EC
Thymocytes cells
have
since their
ccl
1 suspension,
were
After
labelling thymocytes
much, activity
thymocytes.
Non
galactose recovered,
washing, ; cell
83
54
was specific
3- 3,5x10 labaling
4
galactosyl ill).
oxidase,
90
and
than
less “, and 55
“, of
In
residues the
were “, of
original viable.
EC cells
5 B of
and
both
cell
95 “, respectively. EC cells
lethality
did
9, respectively. cpm/106
EC cells,
(i.e.
incorporation
450
para
in
differentiated
“, thymocytes
90
recovered “, and
of
EC cells 97
extensive
being
obtained
and
being
were
viability
to
: viability and
labtied
non-reducing
“u EC cells
were
externally
as representatives terminal
with
killed
were
similarly
94
thymocytes
types
chosen
(18))
treatment
75 S of
thymocytes
display
surface
Following
and
been
they
on
the
11s
ce
AND DISCUSSION
not The
and
32
3 of
increase specific
and
?x104 of
cpm/lO’
radioacti-
lel.
Vol.
vity of
89,
No.
in
the
the
2, 1979
absence
total
of
enzymatic
externally
labdled bras
hydrolysis
associated
itosamine.
About
to
emulsin
and
to
the
origin
and
was
The
zed
15
with
given
in
presumably molecular
weight
fractions
70-74
demonstrates
in
these
of well
the
The
was
eluted
elution
near
LC cells
cally
labelled
fully
differentiated order were
two the
was
of thus
EC cells
in
very
similar
(9))
but
to
the
markedly
in
control buffer‘). 1 top)
glycopeptides
:
contained ‘b were
eluted
molecular
weights
labeled (fig.
glysopeptides that
low
(fig.
50-35 lower
are
eluted
and
positions
labelled
analy-
some
EC cells
externally
retarded
estcnsivcly
by
of
obtained
thymo1 bottom).
obtained from
different
at
65-80 to
volume,
from
externally
stayed
profiles
replaced
to
resistant
were
in
other
radio-
also
were
or
classes
corresponding prepared
was
fractions
from
The
the
lOO’C),
found
was
from
N-acetylgala-
NaK[.‘H14
also
main
t).
in
“, with
Elution
excluded
(65-70
exclusively
prof‘ile
from
culture
of
fractions,
eluted
was
prepared
existence
“, of
thymocytes
unreacted
material
of
10
NaR[‘FIJ4,
oxidase
Glycopeptides
were
peak
75
4hrs,
eluted
a)
by
glycopcptides
galactose
the
retarded
In
this
radioactivity
(1500-3500). cytes
which
HCl,
unreacted
9,
prepared
which
resulting
since
b)
and
and
radioactivity
lo-15
released
about
chromatography.
either
and,
sugars
that
(1N
the
for
glycopeptides
EC cells
compound
profile
elution
The to
The
most
identified.
column
(in
of
not
G-50
experiments
one
hydrolysis
and
belong
the
COMMUNICATIONS
types.
radioacitivity,
acid
1.
of
galactose,
the
Pronasc,
figure
cell
the
RESEARCH
accounted
two
revealed
lab&led
Sephadea
by
of
with % of
externally
digested
the
analysis
EC cells
BIOPHYSICAL
oxidasc)
in
chromatography
extensive
AND
galactose
incorporation
Paper
activity
BIOCHEMICAL
from
metabolithat
of
cells. to
tither
substantiate externally
these labelled
findings, with
cells [‘II]
or
from
the
metabolically
same
Vol.
89,
No.
BIOCHEMICAL
2, 1979
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
2wal
2,000
t
l,OOO-
so
40
Y) FRACTION
60
70
60
NUMBER
Figure 1. Elution profiles of glycopeptides prepared from externally labelled F9 cells (Top) and thymocytes (Bottom). The cells were labelled as described in materials and methods with (-0-J or without (-0-J treatment with galactose oxidase. Glycopeptides were prepared from labelled cells by extensive Pronase digestion. Chromatographic analysis was carried out on a column of Sephadex G-50 superfine (27x1.5cm, flow rate of 5 ml/hr). Dextran blue was eluted in fraction 27; IgG ylycopeptides in fraction 52-53; NaBL3H]4 in fractions 70-74.
labelled
with
mixed,
digested
zed
above.
as
I
14
Cl-g-galactose. with
The
Pronase,
The overall
and
profiles
13H]
the
for
452
and
resulting
[
14
C] labelled glycopeptides
the two isotopes
cells
were analy-
were found
Vol.
89,
No.
2, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
I
0000.~
30
40
50
FRACTION NUMBER
Figl.rre 2. Elution profile of the galactosyl-glycopeptides prepared from externally labelled F9 cells (-o-) and endogenously labelled F9 cells (-•-). The experiment is described in the text. Same procedure and legend as in figure 1. very in
similar
(figure
any fraction,
carry
also
on the It
has for
exposed
indicating
to galactose
displayed
on the
surface
extracted
from
purpose,
galactose
the
% Triton
solubilized
oxidase
of
material
could
not
be detected
containing
IJ-galactosyl
on intact
g-galactose,
residues
cells,
and thus
which
are
are
surface. shown
terminal of
that
EC cells,
to
labeled receptors
the of
was
PNA were
immunoprecipitation.
453
nature
PNA with
examined.
EC cells for
14)
disappear
into
interaction
EC cells
by indirect
they
insight
the
PNA (a lectin
residues,
and that
further
labeled
and the
receptors
D-D-galactosyl -
oxidase-NaR["H]4 X-100,
alone
glycopeptides
glycopeptides,
externally
r3H]
proportion
To gain
(11).
labelled
or
that
previously
non reducing
galactose
0.5
cell
been
differentiation
in
[14C]
a significant
accessible
affine
2).
For
were
are
upon
cell
of
the
material this
dissolved
isolated
from
The isolated
Vol.
89, No.
BIOCHEMICAL
2, 1979
AND
BIOPHYSICAL
RESEARCH
, 40
were
the
adorbed
was
recovered
red
in
of
the
weight
in
the
of
and
to
nearly
a part From
Ph’a
of
is
the
large
results
be
concluded
that
peptides,
which
(7)
are
glycopeptides
at
least
are for contain
the
as and
results
the so far
high
showed
on
in
the
molecular
galactosyl
exposed
on and
454
Pronase
digestion
elution
profile
the
cell
molecular
surface,
of and
of
galactosyl early
EC cells. it glyco-
embryonic surface,
N-acetylgalactosaminyl
the
accounts
communication,
weight or
recove-
isolation
a fraction
cell
present
‘b was
large
that
extract
the
glycopeptides
characteristic
a part
mostly
the
weight
described
The
remove
the
0.8
during
extensive
indicate
displayed molecular
only
above.
to
of
added by
analyzed
(2: 1)
while
prepared
prepared labelled
radioactivity
>I g-galnctose,
monophasic,
indeed
qlycopeptides from externally f iqure 1.
precipitate,
These
the
can
0.1
were
glycopcptides.
receptors
13 “, of
were
receptors, was
to
the
chloroform-methanol
immune
presence
3)
with
Ten
Glycopeptidcs
(figure
for
lipids.
the
process.
extracted
60
NUMBER
Figure 3. Elution profile of’ from receptors for PNA isolated F9 cells. Same column as in
receptors
ow
50
FRACTION
COMMUNICATIONS
cells that
these residues
Vol.
in
89,
an
Pl\jA.
No.
external The
BIOCHEMICAL
2, 1979
position,
and
quantitative
peptides
(during
be
attributed
of
the
receptors
rentiation
process
that
decrease in
to
AND
cell to
vitro
of
I’NA
from
contain
the
these
high
molecular
of
changes the
RESEARCH
the>.
differentiation surface
BIOPHYSICAL
cell
reflected surface
binding
during
sites weight
EC cells in
COMMUNICATIONS
(7) the
can
for glyco-
therefore
disappearance the
same
diffe-
(111.
ACIQ’OWLEDGEPLEXS. A. Prujansky-Jacobovits was supported by a short-term BE30 fellowship. The work carried out at the Pasteur Institute was supported in part by grants from the Centre Kational de la Kecherche Scientifique, the Institut National de la Sante et de la Recherche Medicale, the DelGgation G&&ale Li la Recherche Scientifique et Technique, the National Institutes of Health and the Andre Meyer Foundation. REFERENCES : I. 2. 3. 4. .5 . 6. 7. 8. 9. 10. 11. 1' &. 13. 13. IS. 16. 17. 18.
Pierce,, G. and Dixon, F.J. (1959) Cancer, c, 575. Pierce:, G. and Dixon, F.J. (1959.) Cancer, 12, 584. Damjanov, I. and Salter, D. (1974) Current Topics in Pathology, s, 69. Jacob, F. (1977) Immunol. Rev ., 33, 3. i;raham, C.F. (1977) in Concepts inmammalian embryogenesis, MIT press, 315. Gachelin, G. (1973) Biochim. Biophys. Acta, 516, 27. Iluramat:su, T., Gachelin, G., Nicolas, .J.F., Condamine, II., Jakob, Ii., and
89,
No.
2, 1979
July
27, 1979
8lOCHEMlCAL
AND
RESEARCH
GALACTOSYL
EMBRYONAL
Prujansky-Jacobovitsl,
G. F.
GLYCOPEPTIDES
CARCINOMA
Gachelin, Jacob
OF
CELLS
T.
Muramatsu2,
N.
Unite de Genetique Cellulaire de 1’Institut Pasteur France, Institut Pasteur, 25 rue du Dr. Roux, 75015 (1) Department of Biophysics, The Weizmann Institute Rehovot, Israel, and (2) Department of Biochemistry, School of Medicine, Kobe, Japan Received
COMMUNICATIONS
Pages 448-455
SURFACE
A.
BIOPHYSICAL
April
Sharon’
et du College de - Paris, France ; of Science, Kobe University
25,1979
SLT%lARY. Surface galactosyl residues of alive embryonal carcinoma cells were m-labeled by the galactose oxidase NaB(3H)4 method. The labeled glycopeptides isolated from Pronase digests of the cells, were found to be similar to those prepared from endogeneously labeled embryonal carcinoma cells. They consisted mostly of high molecular weight material, and contained receptors for peanut agglutinin. It is concluded that at least a fraction of the high molecular weight glycopeptides characteristic of early embryonic cells, is displayed on the cell suface. Teratocarcinomas the
gonads.
chaotic
They
contain
and
clumps
way,
carcinoma
are
(EC) . The
malignant a variety of
latter
cells derive
cell
types
cal
with
multipotential
Several
clonal
early
lines
embryonic in
peptides,
isolated cells
embryonic differentiation
cells
4-6).
A class after
has
been (7). of
are
as of
the
an
cells
as
glycopeptides and
448
from
normal
to
digestion
G. Gachelin,
biologi-
embryos
in
(3).
studies
of
embryos
of of
from
the
fucosyl-glyco-
disappear also
which
normal
weight
a
embryonal
many
early used
characteristic
with in
called
share
widely
Pronase
0006-291X/79/140448-08$01.00/0 @ 1979 by Academic Press, Inc. of reproduction in any form reserved.
stem
molecular
Correspondence should be addressed to Dr. rue du Dr. Roux, 75015 - PARIS, France.
Copyright All rights
type
alternative
high
(7),
cell
of
associated distributed
EC cells
therefore
defined
EC cells
types,
cells
extensive
These
cell
(1,2).
EC are
development,
(reviewed
labeled
of
of
generally
a distinctive
differentiated properties
tumors
13Hl-L-fucose =
EC and during
early in
vitro
normal
embryos
during
Institut
Pasteur,
25
Vol.
89,
postimplantation also of
embryogenesis
been F3Hl
found or
location known,
and
on
cell
high
has
by
here
the
on
of the
of
the cell
agglutinin
that
14
weight
COMMUNICATIONS
glycopeptides
species
EC cells
established
in
(9).
whether
The
digests
subcellular is
or
have
Pronase
glycopeptides
glycopeptides
(PNA),
those
recovered
and a cell
(11).
method
obtained
from Our
Cl-$J-galactose.
molecular
surface,
differentiation-specific cell
[
high
differentiation
the
been
from
presence
part
labelled
RESEARCH
large
galactose-oxidase-NaB[3H14
indistinguishable the
Similar molecular
molecular
not
BIOPHYSICAL
still
not
they
from
cells
(10)
appear
unare
exposed
surface.
We report labelled
(8).
-D-galactose
these it
AND
as a predominant
[14 Cl
of
the
BIOCHEMICAL
No’. 2, 1979
weight that
These
contain marker
results
changes
findings
is binding
that
provide in
labelled show
glycopeptides
they
surface
cells
carbohydrate
at
indeed sites
to
by
that
disappears further
externally
growth
in
least
exposed for upon
evidence moieties
be
peanut EC cell for
exposed
on
surface. MATERIALS
AND METHODS
CELLS. F941 is a clonal line of EC which has lost the capacity to differentiate (12). It11 be referred to it as to “EC cells”. Growth and harvest conditions of the cells .have been published (13). Thymocytes were prepared from 4 week old male 129/Sv mice, syngeneic to the F9-41 EC line (14). REAGENTS.NaB[‘3H]4 was purchased from the Radiochemical Center (8.6 Ci/mM), or from the Cormnissariat B 1’Energie Atomique (10 Ci/mM). The reagent was diluted with 0.01 N NaOH to 10 mCi/ml and kept in 0.1 ml aliquots at -78°C. [14C]-D-galactose, uniformly labeled (300 mCi/mM) was obtained from the Commissariat 2 1’Energie Atomique . Galactose oxidase, a product of Worthington (55 U/mg) or Sigma (50 U/mg) was stored at -78°C as 100 U/ml aliquots in pH 7.0 phosphate buffered saline (PBS,lS). Aliquots were used only once after thawing. Peanut agglutinin (PNA) was isolated as described (16). Anti-PNA serum was raised in rabbits, 300 1~1 of antiserum precipitated 100 ug of PNA. LABELING PROCFDURES. The external labelling of the cells was carried out essentially as described 1(10). Briefly, 0.05 ml of 10 mM unlabeled sodium borohydride in 10 mM NaOH was added to a suspension of 20-30 millions of cells in 0.45 ml of PBS pH 7.4. After 30 minutes at room temperature, the cells were washed three times with PBS pH 7.0, containing 0.001 M phenylmethylsulfonyl fluoride (Sigma). To the washed cells, suspended in 0.5 ml of the same solution, 10 units of galactose oxidase were added. Incubation was continued for 30 minutes at 37’C with occasional shaking. The cells were then washed with the same buffer containing 0.05 M D-galactose and twice with PBS pH 7.4. The washed cells were suspended in 0.45 ml of PBS pH 7.4, and mixed with 0.05 ml of tritiated sodium borohydride (0.5 mCi). After 30 minutes
449
Vol.
89,
No.
BIOCHEMICAL
2, 1979
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
at room temperature, the cells were then washed with growth medium containing 1s : fetal calf serum (Gibco), till no radioactivity was found in the supernatant. Cell viability and recovery were determined after each step of the labeling procedure. [‘4C]-D-galactose labeled 23 hours‘in the presence
EC cells were prepared by culturing of 5 LACi/ml of the raclioact ive s~~gar
F9-41
cells
for
.
;LW,\rTICAL PROCEDURES. Labeled products were directed extensively with F’ronase (Calbiochem, 10 mg/ml, 24 hours, three times, 3: L) as described (7) . Glycopeptides were analyzed by Sephadex G-SO column chromatography in 0.05 M arrunoniaacetid acid buffer pII 6.0 (7). Immunoprecipitation of receptors to PM was carried out on Triton S-100 extracts of I:9-31 cells, as described (17). Briefly, 10 millions of cells were dissolved in 0.S ml of 0.5 “, Triton X-100 in PBS, at 0°C. After- one hour, nuclei and cell debris were removed by high speed centrifugation. Solubilized receptors were complexed with PNA in solution (100 OR/ml) and the comple.xes precipitated with anti-PM .qerum added in slight excess. Innnune complexes were collected and washed by centrifugation. Cell viability was estimated by the Trypan blue exclusion test (13‘1. Raclioactivitv leas measured in an Intertechnique Scintillation spectrometer, using Unisolve ikoch-Light) as the scintillation fllkl. SUG4R ANALYSIS. Glycopeptides prepared from labeled I%41 cells (about 30,000 cpm) were hydrolyzed by a mixture (2.5 mg) of glycosidases from Turbo cornutus (>liles) consistinrr in x and I-‘#galactosidase, u and b-N acctyl galactosaminidase. The reaction was carried out in 0.1 ml of 0.05 M citrate buffer pH 4.0, at 3?‘C, under a layer of toluene. After 24 hours, 0.2 ml of water was added, the solution was boiled for 5 minutes. centrifuged. and the suDernatant was annlied to Whatman 3 !.fi\1paper. The chromatogrk was developpkd in ethyl aceiite-pyridinewater (12:5:4). After drying, the paper was cut into pieces, and counted.
RESULTS
blouse
EC
Thymocytes cells
have
since their
ccl
1 suspension,
were
After
labelling thymocytes
much, activity
thymocytes.
Non
galactose recovered,
washing, ; cell
83
54
was specific
3- 3,5x10 labaling
4
galactosyl ill).
oxidase,
90
and
than
less “, and 55
“, of
In
residues the
were “, of
original viable.
EC cells
5 B of
and
both
cell
95 “, respectively. EC cells
lethality
did
9, respectively. cpm/106
EC cells,
(i.e.
incorporation
450
para
in
differentiated
“, thymocytes
90
recovered “, and
of
EC cells 97
extensive
being
obtained
and
being
were
viability
to
: viability and
labtied
non-reducing
“u EC cells
were
externally
as representatives terminal
with
killed
were
similarly
94
thymocytes
types
chosen
(18))
treatment
75 S of
thymocytes
display
surface
Following
and
been
they
on
the
11s
ce
AND DISCUSSION
not The
and
32
3 of
increase specific
and
?x104 of
cpm/lO’
radioacti-
lel.
Vol.
vity of
89,
No.
in
the
the
2, 1979
absence
total
of
enzymatic
externally
labdled bras
hydrolysis
associated
itosamine.
About
to
emulsin
and
to
the
origin
and
was
The
zed
15
with
given
in
presumably molecular
weight
fractions
70-74
demonstrates
in
these
of well
the
The
was
eluted
elution
near
LC cells
cally
labelled
fully
differentiated order were
two the
was
of thus
EC cells
in
very
similar
(9))
but
to
the
markedly
in
control buffer‘). 1 top)
glycopeptides
:
contained ‘b were
eluted
molecular
weights
labeled (fig.
glysopeptides that
low
(fig.
50-35 lower
are
eluted
and
positions
labelled
analy-
some
EC cells
externally
retarded
estcnsivcly
by
of
obtained
thymo1 bottom).
obtained from
different
at
65-80 to
volume,
from
externally
stayed
profiles
replaced
to
resistant
were
in
other
radio-
also
were
or
classes
corresponding prepared
was
fractions
from
The
the
lOO’C),
found
was
from
N-acetylgala-
NaK[.‘H14
also
main
t).
in
“, with
Elution
excluded
(65-70
exclusively
prof‘ile
from
culture
of
fractions,
eluted
was
prepared
existence
“, of
thymocytes
unreacted
material
of
10
NaR[‘FIJ4,
oxidase
Glycopeptides
were
peak
75
4hrs,
eluted
a)
by
glycopcptides
galactose
the
retarded
In
this
radioactivity
(1500-3500). cytes
which
HCl,
unreacted
9,
prepared
which
resulting
since
b)
and
and
radioactivity
lo-15
released
about
chromatography.
either
and,
sugars
that
(1N
the
for
glycopeptides
EC cells
compound
profile
elution
The to
The
most
identified.
column
(in
of
not
G-50
experiments
one
hydrolysis
and
belong
the
COMMUNICATIONS
types.
radioacitivity,
acid
1.
of
galactose,
the
Pronasc,
figure
cell
the
RESEARCH
accounted
two
revealed
lab&led
Sephadea
by
of
with % of
externally
digested
the
analysis
EC cells
BIOPHYSICAL
oxidasc)
in
chromatography
extensive
AND
galactose
incorporation
Paper
activity
BIOCHEMICAL
from
metabolithat
of
cells. to
tither
substantiate externally
these labelled
findings, with
cells [‘II]
or
from
the
metabolically
same
Vol.
89,
No.
BIOCHEMICAL
2, 1979
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
2wal
2,000
t
l,OOO-
so
40
Y) FRACTION
60
70
60
NUMBER
Figure 1. Elution profiles of glycopeptides prepared from externally labelled F9 cells (Top) and thymocytes (Bottom). The cells were labelled as described in materials and methods with (-0-J or without (-0-J treatment with galactose oxidase. Glycopeptides were prepared from labelled cells by extensive Pronase digestion. Chromatographic analysis was carried out on a column of Sephadex G-50 superfine (27x1.5cm, flow rate of 5 ml/hr). Dextran blue was eluted in fraction 27; IgG ylycopeptides in fraction 52-53; NaBL3H]4 in fractions 70-74.
labelled
with
mixed,
digested
zed
above.
as
I
14
Cl-g-galactose. with
The
Pronase,
The overall
and
profiles
13H]
the
for
452
and
resulting
[
14
C] labelled glycopeptides
the two isotopes
cells
were analy-
were found
Vol.
89,
No.
2, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
I
0000.~
30
40
50
FRACTION NUMBER
Figl.rre 2. Elution profile of the galactosyl-glycopeptides prepared from externally labelled F9 cells (-o-) and endogenously labelled F9 cells (-•-). The experiment is described in the text. Same procedure and legend as in figure 1. very in
similar
(figure
any fraction,
carry
also
on the It
has for
exposed
indicating
to galactose
displayed
on the
surface
extracted
from
purpose,
galactose
the
% Triton
solubilized
oxidase
of
material
could
not
be detected
containing
IJ-galactosyl
on intact
g-galactose,
residues
cells,
and thus
which
are
are
surface. shown
terminal of
that
EC cells,
to
labeled receptors
the of
was
PNA were
immunoprecipitation.
453
nature
PNA with
examined.
EC cells for
14)
disappear
into
interaction
EC cells
by indirect
they
insight
the
PNA (a lectin
residues,
and that
further
labeled
and the
receptors
D-D-galactosyl -
oxidase-NaR["H]4 X-100,
alone
glycopeptides
glycopeptides,
externally
r3H]
proportion
To gain
(11).
labelled
or
that
previously
non reducing
galactose
0.5
cell
been
differentiation
in
[14C]
a significant
accessible
affine
2).
For
were
are
upon
cell
of
the
material this
dissolved
isolated
from
The isolated
Vol.
89, No.
BIOCHEMICAL
2, 1979
AND
BIOPHYSICAL
RESEARCH
, 40
were
the
adorbed
was
recovered
red
in
of
the
weight
in
the
of
and
to
nearly
a part From
Ph’a
of
is
the
large
results
be
concluded
that
peptides,
which
(7)
are
glycopeptides
at
least
are for contain
the
as and
results
the so far
high
showed
on
in
the
molecular
galactosyl
exposed
on and
454
Pronase
digestion
elution
profile
the
cell
molecular
surface,
of and
of
galactosyl early
EC cells. it glyco-
embryonic surface,
N-acetylgalactosaminyl
the
accounts
communication,
weight or
recove-
isolation
a fraction
cell
present
‘b was
large
that
extract
the
glycopeptides
characteristic
a part
mostly
the
weight
described
The
remove
the
0.8
during
extensive
indicate
displayed molecular
only
above.
to
of
added by
analyzed
(2: 1)
while
prepared
prepared labelled
radioactivity
>I g-galnctose,
monophasic,
indeed
qlycopeptides from externally f iqure 1.
precipitate,
These
the
can
0.1
were
glycopcptides.
receptors
13 “, of
were
receptors, was
to
the
chloroform-methanol
immune
presence
3)
with
Ten
Glycopeptidcs
(figure
for
lipids.
the
process.
extracted
60
NUMBER
Figure 3. Elution profile of’ from receptors for PNA isolated F9 cells. Same column as in
receptors
ow
50
FRACTION
COMMUNICATIONS
cells that
these residues
Vol.
in
89,
an
Pl\jA.
No.
external The
BIOCHEMICAL
2, 1979
position,
and
quantitative
peptides
(during
be
attributed
of
the
receptors
rentiation
process
that
decrease in
to
AND
cell to
vitro
of
I’NA
from
contain
the
these
high
molecular
of
changes the
RESEARCH
the>.
differentiation surface
BIOPHYSICAL
cell
reflected surface
binding
during
sites weight
EC cells in
COMMUNICATIONS
(7) the
can
for glyco-
therefore
disappearance the
same
diffe-
(111.
ACIQ’OWLEDGEPLEXS. A. Prujansky-Jacobovits was supported by a short-term BE30 fellowship. The work carried out at the Pasteur Institute was supported in part by grants from the Centre Kational de la Kecherche Scientifique, the Institut National de la Sante et de la Recherche Medicale, the DelGgation G&&ale Li la Recherche Scientifique et Technique, the National Institutes of Health and the Andre Meyer Foundation. REFERENCES : I. 2. 3. 4. .5 . 6. 7. 8. 9. 10. 11. 1' &. 13. 13. IS. 16. 17. 18.
Pierce,, G. and Dixon, F.J. (1959) Cancer, c, 575. Pierce:, G. and Dixon, F.J. (1959.) Cancer, 12, 584. Damjanov, I. and Salter, D. (1974) Current Topics in Pathology, s, 69. Jacob, F. (1977) Immunol. Rev ., 33, 3. i;raham, C.F. (1977) in Concepts inmammalian embryogenesis, MIT press, 315. Gachelin, G. (1973) Biochim. Biophys. Acta, 516, 27. Iluramat:su, T., Gachelin, G., Nicolas, .J.F., Condamine, II., Jakob, Ii., and
