BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vo1.187, No. 1,1992
Pages 21 7-224
August 31, 1992
CANCER-ASSOCIATED BY A M O N O C L O N A L Hiroshi
Nakada,
ANTIBODY, Mizue
Inoue,
Ikuo Funakoshi, Department
128,
Shigeyuki
University,
Kita-ku,
Pharmaceutical
Numata
Fukui
Faculty
,
THE Tn A N T I G E N Nobuhiro
Tanaka,
and Ikuo Y a m a s h i n a 1
of Engineering,
Kyoto
Company,
DEFINED
RECOGNIZING
Yoshito
of Biotechnology,
*Shionogi
GLYCOPROTEINS
MLS
603,
Japan
Mishima,
Settu
Kyoto
566,
Sangyo
Japan
Received July 15, 1992
SUMMARY: A murine m o n o c l o n a l antibody, MLS 128, r e c o g n i z i n g the Tn antigen, was e s t a b l i s h e d and used for characterization of glycoproteins e x p r e s s i n g the Tn antigen. The Tn antigen was expressed on three p o l y p e p t i d e chains with m o l e c u l a r weights of 250k, 210k and 150k daltons. LS 180 cells were labeled with 3Hglucosamine or 35S-sulfate metabolically, and then the immunoprecipitate derived from the cell lysate was subjected to SDS-PAGE followed by fluorography. It was revealed that these Tn antigen g l y c o p r o t e i n s were p r o d u c e d through the processing of a high m o l e c u l a r w e i g h t precursor. The c a r b o h y d r a t e moieties of the Tn antigen glycoproteins labeled with 3H-glucosamine were released with alkaline-borohydride, and the released sugars were e x a m i n e d by gel filtration and paper chromatography. The c a r b o h y d r a t e s predominantly consisted of GalNAc and sialyl GalNAc (>90%), with a nearly equal distribution. ©1992AcademicPress,Inc.
Various teins
cancer
(mucins)
chains
attached.
diagnosis
ly
(i).
proteins,
produce
large has
antigens
mucins,
In particular, such
as
PMSF,
are
of
useful
structures
occurs
for
(3)
cancer
(i, 2).
For
have
most
and
been
frequent-
of m u c i n - t y p e (T)
often
monoclonal
in g l y c o s y l a t i o n
Gal~l+3GalNAc-Ser/Thr
ITo w h o m c o r r e s p o n d e n c e Abbreviation:
are
progression
glycopro-
carbohydrate
mucins
by a variety
glycosylation
some core
weight
O-linked that
antibodies of cancer
alterations
incomplete
of
shown
recognized
for m o n i t o r i n g
of w h i c h
high m o l e c u l a r
number
been
These m o n o c l o n a l
and
cancer-associated observed,
a It
cancer-associated antibodies.
cells
with
glycoGalNAc-
should be addressed.
p h e n y l m e t h y l sul fonyl fluoride.
217
0006-291X/92 $4.00 Copyright © 1992 by Academic" Press, Inc. All rights of reproduction in any form reserved.
Vol. 187, No. 1, 1992
Ser/Thr known
(Tn)
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(4,5),
and
sialylated
to be c a n c e r - a s s o c i a t e d MLS
128,
cultured teins,
which
LS 180
i.e.,
was
cells,
not with
cytes,
its b i n d i n g
and
surface
glycoproteins,
inhibited
competitively
to r e c o g n i z e
ture
found
reacted
to m u c i n or
we
that MLS
NAc)-Thr
(I0).
antigens
on LS
with
immunizing
glycoproteins
ovine
mice
mucin-type
are
such
submaxillary
128
Tn
(OSM)
which
was
erythro-
as c a n c e r
mucin
a n d C A 3239,
with
glycopro-
MLS
as it a g g l u t i n a t e d
by N C C - L U - 3 5
cell was
are k n o w n
(9).
investigated 128 b o u n d
of G a l N A c - S e r / T h r
by
glycoproteins.
antibody
the Tn a n t i g e n
Recently, and
established
serum-type
as an a n t i - T n
(6-8)
antigens.
only
identified
T or Tn s t r u c t u r e s
the
including
We n o w r e p o r t
epitopic
preferentially
structure to a
for M L S
cluster
128
struc-
(GalNAc)-Ser-(GalNAc)-Thr-(Gal-
the
characterization
of
MLS
128
180 cells. MATERIALS
AND METHODS
Materials. M L S 128 w a s p r e p a r e d as described previously (9). S e p h a r o s e CL 4B, p r o t e i n A and protein A-Sepharose were from Pharmacia, Uppsala. 3H-Glucosamine, 35S-sulfate and 3HENHANCE were purchased from New England Nuclear, Boston. LS 180 cells, a h u m a n c o l o r e c t a l c a r c i n o m a c e l l line, w e r e o b t a i n e d f r o m the A m e r i c a n T y p e C u l t u r e C o l l e c t i o n , R o c k v i l l e . SDS-PAGE and Western blotting. SDS-polyacrylamide slab gel e l e c t r o p h o r e s i s w a s c a r r i e d o u t u s i n g the b u f f e r s y s t e m of L a e m m li w i t h a 6 % g e l (ii). G e l s for f l u o r o g r a p h y w e r e t r e a t e d with 3 H - E N H A N C E , d r i e d and t h e n e x p o s e d to K o d a k X - O m a t A R f i l m with an i n t e n s i f y i n g screen. Proteins separated by SDS-PAGE were t r a n s f e r r e d to a Z e t a - p r o b e m e m b r a n e at 4 v o l t s / c m for 20 hr. The r e m a i n i n g s u r f a c e of the m e m b r a n e w a s b l o c k e d by incubation w i t h 5 % b o v i n e s e r u m a l b u m i n in p h o s p h a t e - b u f f e r e d saline (PBS) at 5 0 ° C for 10 h. The m e m b r a n e w a s i n c u b a t e d w i t h MLS 128 (10 pg/ml) at 4°C for 20 h. A f t e r w a s h i n g w i t h 0.15 M N a C I and 0.05 % T w e e n 20 in i0 m M T r i s - H C l , pH 7.4, the m e m b r a n e w a s incubated w i t h p r o t e i n A - p e r o x i d a s e at r o o m temperature for 1 h. The m e m b r a n e , w a s h e d w i t h the a b o v e b u f f e r again, w a s v i s u a l i z e d w i t h 0.03 % d i a m i n o a z o b e n z e n e a n d 0.003 % H2Oz in 15 mM phosphate buffer, pH 6.8. Metabolic labeling. C e l l s (ixl07) w e r e labeled with 3Hglucosamine-HCl (i0 ~Ci/ml) in M E M c o n t a i n i n g g l u c o s e in a onet e n t h a m o u n t of c o m p l e t e M E M for 20 h. For pulse labeling, the c e l l s w e r e i n c u b a t e d for 5 h in the same m e d i u m a n d then chased in c o m p l e t e MEM. For 3 S S - s u l f a t e p u l s e - l a b e l i n g , the c e l l s were p r e i n c u b a t e d for 1 h in s u l f a t e - f r e e m e d i u m , l a b e l e d w i t h 35Ss u l f a t e (25 ~Ci/ml) a n d t h e n c h a s e d in sulfate-containing MEM. H a r v e s t e d c e l l s w e r e w a s h e d w i t h PBS, a n d t h e n s o l u b i l i z e d w i t h 1 % T r i t o n X-100, 0.2 M NaCI, 1 m M PMSF, in 50 m M T r i s - H C l buffer, p H 7.4, a n d t h e n the l y s a t e s w e r e c e n t r i f u g e d at 1 0 5 , 0 0 0 x g for 1 h. E a c h s u p e r n a t a n t thus o b t a i n e d w a s u s e d for immunoprecipitation. The a n t i g e n on the c e l l s u r f a c e w a s detected according to the m e t h o d of S p i r o et al. (12). Cells labeled with 3H-glucos a m i n e for 5 h w e r e d i s p e r s e d a n d t h e n i n c u b a t e d w i t h the antib o d y at 4°C for 2 h. Excess antibody was washed off with PBS,
218
V o l . 187, No. 1, 1 9 9 2
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS
a n d t h e n the c e l l s w e r e s o l u b i l i z e d as d e s c r i b e d above and the l y s a t e s s u b j e c t e d to i m m u n o p r e e i p i t a t i o n . Inununoaffinity c h r o m a t o g r a p h y . MLS 128-protein A-Sepharose w a s p r e p a r e d b y the m e t h o d of S c h n e i d e r et al. (13). The a f f i n i ty c o l u m n (0.5 x 6 cm) was e q u i l i b r a t e d w i t h 0.2 % T r i t o n X-100, 0.i M NaCI, in 20 m M T r i s - H C l b u f f e r , pH 7.8. The cells were s o l u b i l i z e d w i t h the same s o l u t i o n and then the lysates were c e n t r i f u g e d as d e s c r i b e d above. The s u p e r n a t a n t w a s applied to the i m m u n o a f f i n i t y column. A f t e r e x t e n s i v e w a s h i n g w i t h the same s o l u t i o n , the c o l u m n w a s e l u t e d w i t h 2 M guanidine thiocyanate, 0.i M NaCl, 0.2 % T r i t o n X-100, in 20 m M T r i s - H C l b u f f e r , pH 7.8. Alkaline borohydride treatment. Alkaline borohydride treatm e n t was c a r r i e d o u t a c c o r d i n g to the method of Carlson (14). The r e l e a s e d o l i g o s a c c h a r i d e s w e r e f r a c t i o n a t e d on a S e p h a d e x G15 column. Paper chromatography was performed as described p r e v i o u s l y (15).
RESULTS SDS-PAGE antigen. lowed
by t r a n s f e r
detected found
and W e s t e r n
The LS
180
of 250k,
the m u t u a l performed
A-peroxidase
210k
on t h r e e and
relationship pulse-chase
metabolically
lysate
labeled
o_ff G l y c o p r o t e i n s
was
to a Z e t a - p r o b e
by p r o t e i n
to be e x p r e s s e d
weights
blotting
cell
subjected
membrane. staining.
of t h e s e
with
i).
with
the
mucins
of LS
180
cells
3H-glucosamine-HCl
Tnfol-
antigen
was
Tn
antigen
was
chains
(Fig.
with
SDS-PAGE
The The
polypeptide
150k daltons
analyses
to
with To Tn
investigate antigen,
which
(Fig.
molecular
had
2) or
we been 35S-
250K210K~
150K-
Q
Q
0
3
8
18
28
Fig. i. The Tn antigen glycoproteins. An LS 180 cell lysate (150 pg protein) was subjected to SDS-PAGE followed by transfer to a Zeta-probe membrane. Tn antigens were detected by successive incubation with MLS 128 and protein A-peroxidase, as described under MATERIALS AND METHODS. Fi~. 2. Processing of the Tn antigen glycoproteins. LS 180 cells (i x 107 ) were pulse-labeled with 3H-glucosamine for 5 hr and then chased for various periods, as indicated in the figure. The immunoprecipitates obtained from the lysates were subjected to SDS-PAGE followed by fluorography.
219
Vol. 187, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Fig. 3. The Tn antigen glycoproteins on the cell surface. LS 180 cells (ixl07) were pulse-labeled with 3H-glucosamine for 5 h. After washing with PBS, the cells were incubated with MLS 128 at 4°C for 1 h. Excessive antibody was washed off with PBS. From the lysate, an immunoprecipitate was obtained by adding protein A-Sepharose. The immunoprecipitate was subjected to SDS-PAGE followed by fluorography. sulfate.
When
the e a r l i e s t sponding chased
species
to a v e r y
for
3 h,
be o b s e r v e d . obvious,
shown).
detected
bands
Thereafter the
Thus,
was
cies.
Then,
5 h.
The g l y c o p r o t e i n s were
observed, surface,
suggesting not
during
cells
antigen
were
About
fraction.
borohydride. Sephadex
G-15.
separated III w e r e tively,
carrying
into
moieties
were
into
shown
identified
On
three
with
the
Tn
became
more
labeling
with
weight
not mucin spe-
shown
on in
molecular
occurred
the Fig.
cell 3,
weight
on
for
a was
the
cell
transport.
isolated
glycoproteins. for
20 h.
by i m m u n o a f f i n i t y was
were
oligosaccharides 4,
the
II a n d
as N e u A c ~ 2 + 6 G a l N A c o l chromatography
220
(data
molecular
antigen As
3H-glucosamine-HCl
I,
could
3H-glucosamine-HCl
the h i g h e s t
in Fig.
paper
bands
of the Tn a n t i g e n
fractions,
on d e s c e n d i n g
150k daltons
on c h a s i n g
the
radioactivity
released
As
and
observed
The g l y c o p r o t e i n s
three
When
the p r o c e s s i n g
6 % of the
The
corre-
210k
immunochemically.
that
diffuse
fainter.
pulse-labeled
with
band
(>250k d a l t o n s ) .
210k
and
3H-glucosamine-HCl,
the h i g h m o l e c u l a r
intracellular
glycoproteins
eluted
250k
that
labeled with
raphy.
250k,
were
a
weight
processed
glycoprotein
Carbohydrate 180
were
detected
non-processed
the
it a p p e a r e d
cells
of
changes
with
comprised
1 5 0 k one b e c a m e
similar
the Tn a n t i g e n
surface
pulse-labeled
high molecular
faint
whereas
3SS-sulfate,
with
cells were
were
with
in
and
fractionated
Fractions GalNAcol,
(Fig.
the
alkaline
oligosaccharides III.
5).
Tn
chromatog-
recovered
treated
LS
The
The
on were
II
and
respecmolar
Vol.
1 8 7 , N o . 1, 1 9 9 2
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Fraction
[
Fraction
II
Fraction
TTT
1.0
O.
,2,
Fraction !
_E 8
I
[!
II
I
lit
I
+
2.0
x
)
I0 1D
_>
O
•-~ 6 >.
~ 4
~B
~2 I
1.0