Biosynthesis in vitro and the pattern of lectin binding receptors in monkey kidney cell surfaces

Vol. 66, No. 4, 1975

BIOSYNTHESIS

IN

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

VITRO

THE

RECEPTORS Manju

Basu,

Department of Notre Laboratories,

Joseph of

Received

IN

R.

August

26,

PATTERN

MONKEY

Moskal,

Chemistry, Notre Dame, Inc., Elkhart,

Dame,

AND

KIDNEY

David

A.

Biochemistry

Bio

46556, 46514

LECTIN

CELL t and hysics P Research

and

BINDING

SURFACES

Gardner

and

Indiana Indiana

OF

Subhash

Basu

Program, Division,

University Miles

1975

SUMMARYThe glycosyltransferase activities involved in the biosynthesis in vitro of neutral blood group-related glycosphingolipids were measured in African green monkey kidney cells (Vero) grown in culture. The a-fucosyltransferases which catalyzed the reaction between GDP-fucose and corresponding acceptors to form H-active and novel Lea-type glycosphingolipids were characterized in membrane fractions isolated from Vero cells and monkey bone marrow. Using lZ51-labeled Ulex europeus and Lotus tetragonolobus lectins the differential binding to Vero cell surface glycoproteins and glycolipids was studied under various conditions. The cytes

which

several

Yang

are

by

as The

Stellner

and

in

Recently

Because

and

glycoproteins

cell

surfaces,

we

distribution

primate

origin work

was

a bovine

a novel

(African aided from

by Miles

spleen gly

concentrated

Research

Grants

Copyright D 19 75 b-v Academic Press, Inc. AN rights of reproductiorr in aiq form reserved.

the

structure

Inc.

1380

has

of a novel

been

biosynthesis

reported

in

membrane

125

been

cell

vitro

line Ulex

but achieved (5)

and

europeus

to

topo-

(Vero(7))

CA-14764-01 Indiana,

an

of primate in

NS-09541-05, , Elkhart,

not

membranes

I-Labeled

of

fraction,

glycosphingolipids

biosynthesis

by

vitro

group-related

a clonal

by was

has

in kidney).

Laboratories,

and

plasma

erythro-

characterized

cosphingolipid

of the

monkey

the

human

glycosphingolipid

Golgi-rich

in

glycosphingolipids

NM

(2),

(4)

blood

green

al.

group

been

adenocarcinoma

investigations

of these

have

blood

et

Lea-type

highly

undertook

Hl

reported

fucose-containing, (6)are

logical

grant-in-aid

of

the

human we

from

isoantigens

Koscielak from

in

vitro

group of

and

isolated (3).

glycosphingolipids

structure

glycosphingolipid

now.

blood

et al.(l)

Hakomori

biosynthesis

This

classified

glycosphingolipid

H-active

until

of fucose-containing

laboratories.

reported Lea

structures

S. B.

of (8) and

a

Vol. 66, No. 4,1975

and

Lotus

ment

tetragor-olobus

of fucose-containing

BIOCHEMICAL

(9)

lectins

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

were

used

glycoproteins

and

MATERIALS

AND

to

study

the

glycolipids

topological

on

the

Vero

arrangecell

surfaces.

METHODS

Cell culture : A clone of African green monkey kidney cells (Vero) was maintained on Eagle’s minimal essential medium (MEM) supplemented with 10% fetal bovine serum (Gibco). Cultures were grown in closed 250 ml Falcon plastic bottles containing 15 ml of medium. The medium was changed twice before harvesti g,and ‘b cells were subcultured when they reached a population density of 3 to 5 x 10 / Tflask. For enzymatic studies in vitro, the monolayers, containing 1 to 5 x lo6 cells/ T-flask, were harvested by scraping. Cell counts were obtained from identical duplicate flasks after harvesting with 0. 25% trypsin in 0.17~ EDTA (pH 7. 0). Donors : Unlabeled GDP-fucose was prepared according to the method of Schachter et al.(lO). Unlabeled UDP-galactose was purchased from Calbiochem. GDP-Lm]fucose (75 mCi/mmole), Na125 I, and UDP-[14C]galactose (282 mCi/mmole) were purchased from Amersham/Searle and New England Nuclear. Acceptors : Lactosylceramide (Galpl-4Glc-cer) and glucosylceramide were isolated from bovine spleen and Gaucher’s spleen, respectively. B active pentaglycosylceramide (lacPent-Cer (.B), Gale.l-3Gal~l-4GlcNAc~l-3Gal~l-4Glc-cer ) and H-active pentaglycosylceramide (lacPent-Cer (H) , Fucal-2Galpl-4GlcNAcpl-3Galpl-4Glc-cer) were isolated fromrabbit and human O-type erythrocytes, respectively. Both lac-nTet-Cer (Gal~l-4GlcNAc/31-3Gal~l-4Glc-cer) and lacTriCer (GlcNAcpl-3GaGl-4Glc-cer) were prepared from the rabbit B-active pentaglycosylceramide by sequential degradation of terminal galactose units with purified fig a-galactosidase (11) and papaya S-galactosidase (12)l. The triglycosylceramide (mTri-cer, GalNAcPl-4GalPl-4Glc-cer) and galactosylceramide were isolated from guinea pig erythrocytes and bovine brain, respectively. The purified glycosphingolipids were analyzed by gas chromatography (13) and gas chromatography-mass spectrometry (14). Reagents : Colcemid was purchased from CIBA Pharmaceutical Company, and dibutyryl cyclic AMP ( (But)2cAMP ) and theophylline, were purchased from Sigma Chemical Co. Borosilicate fiber discs (Whatman GF/A, 1. O/pm porosity, 2.4 cm diameter) were purchased from Scientific Products. Lectins : Ulex europeus lectin was isolated by the procedure of Osawa and Matsumoto (8) and further purified by isoelectric focusing using Ampholine between pH 3. 5 and 10. Purified Lotus tetragonolobus lectin was a enerous gift sample f 251 in the presence from Dr. Marilyn Etzler (9). The lectins were labeled with of Na125 I and Sepharose-4B-bound lactoperoy$yse according to the method of David and Reisfeld (15). The homogeneity of I-labeled lectins was determined by polyacrylamide slab gel electrophoresis at pH 8. 7 in tly5presence and absence of sodium dodecyl sulfate (16). The specific activities of I-labeled Lotus tetragonolobus and Ulex europeus lectins were 4. 6 x 109 and 2. 7 x 108cpm/g, respectively. Preparation of membrane fractions : Confluent monolayers (3 to 5 x lo6 cells/ 250-ml Falcon plastic bottle ) were harvested by scraping for enzymatic studies in vitro. Cells were processed according to our previously published method fractions were isolated at the junction of 0. 32 and 1. 2 M (7) I and membrane discontinuous sucrose gradient. The membrane fraction(VCP-1) isolated at the ’ Chien,

J. -L.,

Basu,

S. , and

Li,

Y.

-T.

1381

(manuscript

in

preparation

)

Vol. 66, No. 4, 1975

junction galactosyl(MBG-2) activities, marrow parations active

from

BIOCHEMICAL

about 60 to 70% of the total glycosphingolipid activities. Isolation of a membrane fraction from containing glycolipid:glycosyltransferase was achieved according to the method published for guinea pig bone (18). The detergent G-3634-A was used to wash these membrane preinstead of Triton X-100 because the glycolipid fucosyltransferases are in its presence (4). and

Vero cells contained fucosyltransferase monkey bone marrow,

RESULTS Among Cer

had

all the

(MBG-2) lyze

the

substrates

highest

activity

membranes the

cantly

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

synthesis low

membrane

or

absent

tested in

(Table of

I).

both

preparation

The

UDP-[14C]galactose cells

(VCP-1)

and

ganglioside It is

high

related

monkey

that

for

the

lacTri-

bone

marrow

which

glycolipids

interesting

activity

donor,

activities

are

cultured

catasignifi-

Vero

cell

UDP-Gal:galactosylceramide

I

: galactosyltransferase

activities

_I_---_-_-_---

-w---P-

as

galactosyltransferase

systems.

Table Glycolipid

Vero

and

contained

DISCUSSION

using

both

ganglioside in

AND

---o------e-

[

Acceptor

14

C] ---_____--_ Galactose

--

VCP-1 ----

7

MBG-2

.----__ pmoles/mg

a

incorporated ---

protein/hr

Galactosylceramide 1080 0 Glucosylceramide 420 0 Lactosylceramide 1120 87 4030 382 --lacTri-Cer lac-nTet-Cer 1060 230 53 GPent -Cer (H) 450 180 12 GglioTri-Cer 0 0 ganglioside -0-GM2 a Complete incubation mixtures contained the following components (in micromoles) in a final volume of 0. 04 ml: acceptor lipids, 0. 05; Triton CF-54, 50 p,g; Cacodylate-HCl buffer, pH 7. 3, 10; MnC12, 0. 25; UDP-[14C]galactose (2,2 x 106 cpm/ pmole), 0. 03; and enzyme fraction, 0. 3 to 0.4 mg of protein. The mixtures and the reactions were stopped by adding 2.5 were incubated for 1 hr at 37O, pmoles of EDTA (pH 7. 0). The incorporation of radioactive galactose units into acceptor glycosphingolipids was assayed by a double chromatographic technique (14) as described in the text. Under these conditions the rate of reaction remained constant up to 1 hr.

1382

Vol. 66, No. 4,1975

BIOCHEMICAL

AND BlOPHYSlCAL

Table Glycosphingolipid

RESEARCH COMMUNICATIONS

II : fucosyltransferase

activities

--

--1__--

[WC]Fucose

incorporated

a

Acceptors VCP-1

MBG-2

pmoles/mg lac-nTet-Cer

protein/

2 hr

2550

GPent-Cer

(B)

950

1300

SA- -al-glycoprotein lacTri-Cer iZPent-Cer

660 0

98 0 0

990 150

a Complete incubation mixtures contained the following components (in micromoles) in a final volume of 0. 025 ml: acceptor lipid, 0. 05; G-3434-A, 0.1 m ; Cacodylate-HCl buffer, pH 6.4, 10; MgCl2, 0. 05; GDP-[14C]fucose (1.44 x 10 E cpm / p.mole), 0. 01; and enzyme fraction, 0.15 to 0. 3 mg of protein. The mixtures were and the reactions were stopped by adding 10 ~1 of incubated for 2 hr at 37O, chloroform-methanol (2:l). The incorporation of labeled fucose into acceptor glycolipids and glycoprotein was assayed by a double chromatographic technique (14) as described in the text. Under these conditions the rate of reaction remained constant up to 2 hr.

a-galactosyltransferase was

absent

from

present

work

transfer

of

same

and bone

marrow

suggests to

was

digalactosylceramide

preparations.

that

galactose

product

the

(19),

Furthermore,

our

a-galactosyltransferase

lactosylceramide

which

and

which

previous

(13)

catalyzes

lac-nTet-ceramide

and

the

might

be

the

enzyme. As

fucose

to

shown both

in

Table

membrane

a-Fucose glucosamine

II,

lac-nTet-Cer

(SA--al-glycoprotein) cell

the

and was

actively (lacTri-Cer)

and

under was

transferred or

was

to to

actively

lacPent-Cer

inactive

preparations was

fucose

the

transferred , whereas

the

present

assay low

substrates

containing N-acetylglucosamine

1383

GDP-[14C]-

desialized

significantly

internal

from

in

orosomucoid

conditions bone

in

marrow terminal

Vero

membranes. N-acetyl-

of

lacPent-Cer

Vol. 66, No. 4, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table Binding

mu

125

of

I-labeled

--

III

Lotus

lectin

to

Vero

cell

surfaces

-------

------e--w

Lotus

[

125

Experiment

I]-lectin wash

----_

bound a

with

to

106

cells

after

l-

5 % --e------m

CC13COOH

5%

cpm Control

CC13COOH

t CHC13-CH30H(2:1)

I

-----------l---__l-ll----------___

x 10

-1

12,600

Colcemid

12,300

11,700

I

i

---

10,700

P---v--

-----__

a Quantitative labeling of control Vero cells grown in the presence of Colcemid (pi55 pg/ml;22 hr) was achieved bq incubation for 15 min at 37O with 2 ml Lotus[ I]-lectin (100 pg/ml, 4.6 x 10 cpm/g) in the presence of MEM (without serum) in 250 ml Falcon T-flasks. After incubation the cells w ,e2%e washed with phosphate buffered saline and harvested by scraping. Aliquots of I-labeled cells (in triplicate) were filtered through glass fiber discs (2,4 cm) in a Millipore apparatus and washed with 10 to 20 ml of cold 5% CC1 COOH or 5% CC13COOH followed by orm-methanol (2:l). The glass fiber3discs were dried at 100’ for 30 min, ch10f2g and I content was quantitatively determined in a toluene liquid scintillation system in the presence and absence of PCS (AmershamfSearle). The results were generally the same with and without PCS.

(H-active)

glycolipids respectively.

lipids, low

or

In the

reported

(SA571)

contained

denced

by

to

form

Lea

bone

marrow

monkey

thin

by more

layer

Steiner than

in

normal

corporation

into infection.

et a1.(5) one

large

chromatography

60 to 707~ incorporation

present

FLV)

VCP-1,

precursor

or

system

b

Le the

-type

blood

latter

group

enzyme

is

absent. It was

for

in

of

SA571 the Glick

cells,

cells and

weight

fucose

whereas

baboon

there after showed

1384

into was

feline that

cell

fucosylglycolipid

radioautography.

3H-labeled

(20)

a nontransformed

molecular and

transformed Buck

that

This total a sharp

as

lipid

decrease

evi-

accounted

fucose-containing

sarcoma-leukemia fucose-containing

line

lipids in

fucose virus(FSV-

glycoproteins

in-

BIOCHEMICAL

Vol. 66, No. 4,1975

present

in

present

after

viral

cell

surface

in

these

cell

be

related

cal

relationships

on

surfaces

primate

presence

of

surfaces,

the

either

Table The

results (2%)

that or

(2:l)

there

was

Vero

cells

after

compared

to

the

of

125

with cells

c

I-labeled

Ulex

Ulex

Experiment

loss

topographipresent

investigate

the

on

were

Vero

cell

performed.

The

bound

to total

of

It

radioactive the

cells of

plus with

(2:l) appears lectin were

in

washed

radioactivity

in

chloroform-methanol

50/c CC13COOH

(Table

IV).

IV

europeus

lectin

with

Vero

cell

surfaces

125

europeus

[

I]-lectin wash 5%

with

CC13COOH

bound a

to

lo6

cells

__--------

after --

t CHC13-CH30H(2:1)

-- L ---*---------M--

A--

t

the

alone.

decrease

50/c CC13COOH only

to

IV

after

3070

washed

may

chloroform-methanol

little

5 “/o CC13COOH

.-

plus

very

was

glycoproteins

Lotus-[1251]lectin

glycoprotein

Table Interaction

the

and

to

There

washing control

III

cells(9‘$)

(2:l).

). Changes

glycoproteins

bound

Colcemid-treated

chloroform-methanol

control

lectin

virus

However,

In order

Table

CC1 3COOH

5%

those

macromolecules

and

represents

to

sarcoma

cells.

unknown.

in

III

glycolipid.

Rous

fucose-containing virtually

radioactive

above

control

with

in

similar

and

neoplastic

summarized

column and

of

glycosphingolipids

experiments

represents

from

are

fucose-containing

glycoprotein

strain

in

various

surfaces

were

glycosphingolipids

expression the

RESEARCH COMMUNICATIONS

mitosis

(Bryan

fucose-containing

cell

57~ CC13COOH

during

transformation

among

the

column

(BHK2l/Cl3)

tophenotypic

the

AND BIOPHYSICAL

cpm

x 10

,

Control

5800

4300

Colcemid

3800

2700

a Ulex

Conditions europeus

were the [125 I]-lectin

same

as those (1. 9 mg/ml;

describe$ 2. 7 x 10

1385

in Table cpm/g)

III, was

except used.

that

Vol. 66, No. 4, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Figure 1. Radioautogram of [14C] glycosphingolipids of Vero cells. [14C]Glycosphingolipids obtained from Vero cells( grown in T-flasks (1 to2 x IO6 cells/flask) after incubation in the presence of the indicat reagents; 14-18,000 dpm/ sample) was applied to silica gel G plates and develop with chloroform-methanol-water, 60:30;6; an x-ray plate was exposed for 30 days. Lane 1. Standard glycosphingolipidsa b, c, d and e are mono-, di-, tri-, tetraand pentaglycosylceramides. Lane 2. [14C]GSL obtained from control Vero cells grown in MEM(2-3 x lo6 cells/ T-flask). Lanes 3,4 and 5 are [14C]GSL obtained from Vero cells grown in similar T-flasks in the presence of 0. 2 mM (But)2cAMP, 0. 2 mM (But)2cAMP plus 1. 0 mM theophylline and Colcemid (0. 2 pg/ ml), respectively.

These

results

suggest

glycoprotein

and

containing

30%

in release

presence

of

cells

with

0.1

after

level

the

treated

M

by of

primary is

L-fucose 0.1

interest related

to

and

cells.

37O

after

The

pattern

5tJ,g/ml)

The at

37O

was

15 min

to

fucose-

15

and

observed

incomplete for

to both

was

. Between

lectins

incubation.

binds

specifically

same

europeus

labeling

probably

binds

(0.

Ulex

the

most

lectin

Colcemid

M fucose

endocytosis

lectin

Lotus of Vero

Lotus

significant

in

cells

europeus

whereas

of 125 I-labeled

lectins

Ulex

glycoproteins

Vero

cell-bound

Our

the

glycolipid

surface

observed

that

in

removal suggests

the of

a

(21). now

a change

is

to

determine

in

surface

1386

whether glycolipid

any or

morphological glycoprotein.

change Signifi-

Vol. 66, No. 4, 1975

cant

morphological

the

changes

presence

plus of

BIOCHEMICAL

of

Colcemid

theophylline Vero

(Fig.

in

The

bands

sylceramides. under

(0.1

(1. 0 mM).

cells 1).

were

the

were

observed

when

Vero

cells

1. 0 pg/ml

(22)),

or

(But)2cAMP

[14C]Galactose

presence

There

these

to

of

was

no The

tri-

and

presence

The

of

the

pentaglycosylceramide(band

changes

in

the

treatment that be

with being

with

[14C]fucose cleaved

content

cell

by of

this

various investigated

Ukena surface

potent is

of

treatment

Colcemid

et

al.

antimitotic

with

agents

glycosphingolipids

reagents

was

cell for

glycopeptide antimitotic

studied

and

were plus 1) is

et a1.(24)

3T3 as

2 mM)

pentaglyco-

glycosphingolipid.pattern

Waddell

such

trypsin

of a potent

(0.

in

into

(But)2cAMP

mouse

a Vero

[14C]fucose-containing concentrations

and

incubated

tetra-,

e , Fig.

of

0. 2570

in

and

(23)

into

tri-,

were

tetraglycosylceramides

properties

incorporated

di-,

change

the

Recently

mono-,

qualitative

in

investigation.

membrane-active

as

quantitatively structure

incorporation

these

identified

conditions.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and

2 min after agent

theophylline. currently

under

have

reported

BHKZ1/C13

Colchicine. surface

increased

We

cells have

glycoprotein (25).

The

observed that

structure

treatment such

after

of as

can and

Vero

Colcemid

cells are

(25). REFERENCES

1. 2. 3. 4. 5. 6. 7. 8.

9. 10. 11. 12. 13. 14.

K. and Hakomori, S. (1973) Biochemistry, 12,656. Stellner, K. , Watanabe, Koscielak, J., Piasek, A., Gorniak, H., Gardas, A. and Gregor, A. (1973) Eur. J. Biochem., 37, 214-225. Yang, H. and HokomGi, S. (1971) J. Biol. Chem. ,246, 1192-1200. Basu, S., Basu, M. and Chien, J. L. (1975) J. Biol. Chem., 250, 2956-2962. Steiner, S. , Brennan, P. J. and Melnick, J. L. (1973) Nat. New Biol., 245 -’ 19- 21. VanBeek, W. P., Smets, L. A. and Emmelot, P. (1975) Nature, 253, 457. Yasumura, Y. and Kawakita, Y. (1963) Nippon Rinsho, 21 , 1209. Osawa, T. and Matsumoto. I. (1972) Methods Enzvmol. 28. 323-327. Etzler, M. (1972) Methods Enzymol., 28 , 340 -344. Schachter, H., Ishihara, H. and Heath-. C. (1972) Methods Enzymol., 2, 285-287. Li, Y. -T. and Li, S.-C. (1972) Methods Enzymol., 28, 714-720. Chien, J. -L. (1975) Ph. D. Thesis, University of Notre Dame, Notre Dame,Ind. Basu, M. and Basu, S. (1973) J. Biol. Chem., 248, 1700-1706. Bjorndal, H., Hellerqvist, C. G., Linderberg,. and Svensson, S. (1970)

1387

Vol. 66, No. 4, 1975

15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Angew. David, Both,

Chem. Int. Ed. Engl., 9 , 610-619. G. S. and Reisfeld, R, A. (1974) Biochemistry, 13, 1014-1021. G. W., Moyer, S. A. and Banerjee, A. K. (1975)Proc. Nat. Acad. Sci. 274-278. USA. , &72 Yeung, K. -K., Moskal, J.R., Chien, J. -L., Gardner, D. A. and Basu, S. (1974) Biochem. Biophys. Res. Commun., 59, 252-260. Basu, M., Chien, J. -L. and Basu, S. (1974) Biochem. Biophys. Res. Commun 60, 1097-1104. Martensson, E., Ohman, R., Graves, M., and Svennerholm, L. (1974) J. Biol. Chem., 249, 4132-4137. Glick, M. C. and Buck, C. A. (1973) Biochemistry , 12 , 85-90. Nicolson, G. L., Lacorbiere, M. and Eckhart, W. (1975) Biochemistry, 214 172-179. Loustalot, P. and Gross, F. (1954) Klin. Wschr., 32, 49 . Schar, B., Ukena, T. E., Borysenko, J. Z., Karnovsky, M. J. and BerG,R. D. (1974) J. Cell Biol., 61, 70-82. Waddell, A. W., Robson, R. T. and Edwards, J. G. (1974) Nature, 248. 239- 241. Basu, S., Basu, M., Moskal, J. R., Chien, J.-L. and Gardner, D. A., Proc. Symp. on Glycolipids, Amer. Oil Chem. Sot., June 6-8, 1975 (in press).

1388