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

Cancer-associated glycoproteins defined by a monoclonal antibody, MLS 128, recognizing the Tn antigen.

A murine monoclonal antibody, MLS 128, recognizing the Tn antigen, was established and used for characterization of glycoproteins expressing the Tn an...
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