Normal glycosaminoglycan production in a galactosylation-defective lectin-resistant CHO cell variant

Vol. 103, No. 1,198l November

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BIOPHYSICAL

RESEARCH

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16, 1981

NORMAL GLYCOSAMINOGLYCAN PRODUCTION IN A GALACTOSYLATION-DEFECTIVE

LECTIN-RESISTANT

Eve Barak

CHO CELL VARIANT

Briles

Department of Anatomy University of Alabama in Birmingham Birmingham, Alabama 35294 Received

September

14,

1981

Abstract CHO cell variant clone 13 fails to incorporate galactose into either glycolipids or asn-linked oligosaccharides, even though intracellular UDPgalactose pools, galactosyltransferases, and galactose-acceptor macromolecules are normal. Clone 13 is now shown to produce normal amounts of chondroitin sulfate and heparan sulfate, the biosynthesis of which necessarily involves synthesis of a galactose-containing tetrasaccharide sequence. This is the first evidence of galactosyltransfer reactions which proceed normally in clone 13. Based on the known subcellular sites of glycosyltransfer reactions, it is proposed that a Golgi-specific defect involving compartmentalization may be responsible for the clone 13 trait.

Introduction A variety

of lectin-resistant

have well-defined lectins

surface

to the cell.

major

contributions

are

synthesized

from

CHO cells

defective complex

acceptor Since classes

carbohydrate

Characterization

(1,2,3,4). for

asn-linked

the defect

The defect

in clone

other

levels,

of these 13 is

glycoconjugates

are

already

known

glycoconjugates, are

0 1981 by Academic Press, Inc. of reproduction in any form reserved.

also

38

into

affected.

of

to

oligosaccharides was selected Clone

either

glycolipids

13 is

be attributed pools,

in --in vitro to affect

led

(5,6).

cannot

it

the binding

13, which

UDP-galactose normal

OQO6-291X/81/210038-08$01.00/~ Copyright All rights

affect

surface

clone

galactose

oligosaccharides.

as all

is

many of which

has already

germ agglutinin

to incorporate

of galactose-containing

mine whether

of how cell

to wheat

in galactosyltransferase molecules,

which

of such variants

One such variant

resistance ability

have been isolated,

alterations

to our understanding

in its

terations

variants

assays at least

was of interest

or to al-

or galactose(5,6). two distinct to deter-

Glycosaminoglycans

Vol. 103, No. 1,198l

BIOCHEMICAL

CLONE

3

I

AND

BIOPHYSICAL

RESEARCH

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qluc N- 'H

13

qluc N- 'H VI

CHO

II 6 III

0

1

Figure

L ti IO

20

30

40

FRACTION

50

02

60

NO

25

35 45 FRACTION

55 NO

65

1

Bio-Gel P-10 elution profiles of ["HI-glycopeptides prepared by Pronase digestion of [3H]glucosamine-labeled cells. Upper panel, clone 13; lower panel, wild-type CHO cell glycopeptides. Figure

2

Bio-Gel P-10 elution profiles digestion of [3H]galactose-labeled wild-type CHO cell glycopeptides.

were

(GAGs)~

dues

of particular

of proteoglycan

sequence, biosynthetic

prerequisite

the GAGS (7).

In this

normal

of the

the

amounts

first

normally the clone

indication in clone

interest

core

glucuronosyl

of [%I]-glycopeptides cells. Upper panel,

proteins

since

they

through

are

prepared by Pronase clone 13; lower panel,

connected

a specific

to serine

tetrasaccharide

linkage

B1,3-galactosylB1,3-galactosylf3l,4-xyloside, for series

assembly

of the repeating

of experiments,

GAGS, chondroitin of a class

13, and provides

clone

sulfate

a possible

which disaccharide

13 is

and heparan

of galactosyltransfer clue

resi-

shown

units

which

to the metabolic

This

39

HMW, high

is

proceed basis

13 trait.

1 Abbreviations used: GAGS, glycosaminoglycans; Adi-4S, 2-acetamido-2-deoxy-3-0-(B-D-gluco-4-enepyranosyluronic sulfo-D-galactose; HONO, nitrous acid.

of

to produce

sulfate.

reactions

is a

molecular weight; acid)-4-0-

for

BIOCHEMICAL

Vol. 103, No. 1,198l

Materials

AND

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and Methods

Cell culture conditions and the isolation of clone 13 from CHO cells have been described previously (5). Monolayer cultures in 150 mm Falcon dishes were labeled for 48 hr in 25 ml of complete growth medium plus 100 YC of [3H] glucosamine or [3H]galactose (New England Nuclear). The cells were rinsed three times with phosphate-buffered saline, then scraped into 4 ml of 50 mM Tris Cl, pH 8, containing 1 mg/ml Pronase (Calbiochem) and 2.5 mM CaC12. Pronase digestion proceeded at 37" in the presence of a few drops of toluene for 3 to 8 days, with fresh Pronase added daily. The digests were boiled and clarified by centrifugation prior to analysis by gel filtration through a 0.9 cm diameter x 100 cm high column of Bio-Gel P-10, 200-400 mesh (BioRad Laboratories), equilibrated with 0.15 M ammonium acetate, pH 6 or 0.1 M pyridine acetate, pH 5. Column fraction samplings were counted in Scintiverse (Fisher Scientific). Chondroitinase digestion was performed in 800 ul of 12.5 mM Tris Cl pH 8 containing 0.1 U chondroitinase ABC (Miles Laboratories), at room temperature for 24 hr. HONO degradation was performed as described by Shively and Conrad (8). Acid hydrolysis of presumed linkage sequence oligosaccharides was performed in 2 N H2S0,+ for 4 hr at 100". Descending paper chromatography was performed on Whatman 3MM paper in glacial acetic acid : nbutanol : 1 N NH4OH (3:2:1), for analysis of chondroitinase digestion products, or pyridine : ethyl acetate : Hz0 (1:3.6:1.15) (upper phase) for analysis of monosaccharides (5). Samples were mixed with nonradioactive authentic standards before application to the paper. Authentic 4,5-unsaturated disaccharides (Miles Laboratories) were visualized under UV light. Chromatograms were cut into l-cm segments, soaked in 3 ml of water, mixed with 4 ml Scintiverse, and counted in a liquid scintillation counter. Results

and Discussion

Cells

were

and subjected tides rial

were eluting

metabolically to extensive

analyzed

Peak II, 2),

Vi,

(HMW) material

probably

labeled

label

represents

[3H]

to represent

panels,

with

the void from (Fig.

glycopeptides,

either

Figs.

Labeled

mate-

unincorporated 1 and 2),

volume

(V,)

glucosamine

1) and galactose while

glucosamine3

13H] glycopep-

1 and 2).

(lower

glucosamine

complex

(Figs.

was assumed

derived

or

The resulting

column

eluting

by both

[3H]galactose2

digestion. P-10

volume,

+33% of the incorporated

tose. Fig.

on a Bio-Gel

with

In the case of wild-type

molecular-weight for

Pronase

at the salt

metabolites.

labeled

Peak III,

high-

accounted or galac(arrow, labeled

2 UDP-[3H]galactose derived from [3H]galactose is in equilibrium with UDP[3H]-glucose via UDP-glucose-4'-epimerase. In wild-type cells, most of the [3H]galactose supplied in complete medium is incorporated as galactose, but some is incorporated as glucose (e.g., glycolipids), or as other sugars metabolically derived from UDP-glucose (e.g., glucuronic acid, inositol, xylose). In clone 13, supplied [3H]galactose is incorporated mainly as glucose and its derivatives (unpublished data). 3 [3H]glucosamine N-acetylgalactosamine,

is

metabolically and sialic

incorporated acid.

40

as N-acetylglucosamine,

Vol. 103, No. 1,1981

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

VO

3

CLONE

COMMUNICATIONS

13 gal- ‘H

CLONE 13 glcN-‘H

2 8 2 x

’

,I

22-

z u2

I I

0

3

15

25

35

45

55

FRACTION

Figure

00

65

Ii

4 4

NO

I5

25

35 45 FRACTION

55 NO

65

3

~io-~el P-10 elution profiles of [3H]glucosamine-labeled HMWPronase digest products (V, from Figure l), after treatment with chondroitinase ABC. Upper panel, clone 13; lower panel, wild-type. Figure

4

Blo-Gel P-10 elution profiles of [3H]galactose-labeled HMW Pronase digest products (V, from Figure 2), after treatment with chondroitinase ABC. Upper panel, clone 13; lower panel, wild-type.

only

with

glucosamine,

case of clone

probably

13 (upper

panels,

~22% of the incorporated the

remaining

position rich gal

glycopeptides sequences

further.

Peak III,

In the present

mannose-rich

1 and 2), and most

glucosamine

and truncated (5).

Figs.

glucosamine,

incorporated

as wild-type

represents

(arrow,

and probably complex studies,

HMW material of the Fig.

represents

glycopeptides only

In the

glycopeptides. accounted

incorporated

1) eluted

galactose;

in

a mixture lacking

the HMW material

for

the same of mannose-

terminal

sia-

was analyzed

Vol. 103, No. 1,198l

BIOCHEMICAL

0

4

8

AND

12

BIOPHYSICAL

16 20

24

28

RESEARCH

32

36

COMMUNICATIONS

40

44

SEGMENT NUMBER Figure

5 Paper chromatography

"minor peaks" HMW material

by acid hydrolysis

from the

digestion of [3H]galactose-labeled 4). A, clone 13 peak a; B, clone indicate origin peak a; D, wild-type peak a. Arrows sample lanes were cut into l-cm segments for deterPositions of authentic standard monosaccharides,

peak 2; C, wild-type Radioactive positions. mining

of sugars released

obtained after chondroitinase (peaks 5 and a from Figure

radioactivity.

13

chromatographed on the same sheet in separate lanes between the radioactive samples, are indicated: U, glucuronic acid; gal, D(+)galactose; glc, D(+)glucose;

xyl,

D(+)xylose.

The HMW material with

each Pronase

ABC, and the products

chondroitinase

(Figs.

from

Chondroitinase

3 and 4).

and %33% of the wild

type

HMW material,

In the case of galactose-labeled products

was reproducibly

prior

to the major

minor

peaks

represent

were

Paper

sumed linkage tose

sulfate

never

sequences

disaccharides, (Fig.

resolved

observed sequences

chromatographic

and Z3H]-xylose

of A-di-4s

material, into

small-molecular-weight

GAG linkage

peptides.

degraded

by acid (Fig.

5).

digest were

(V,,

Figs.

analyzed

on the P-10

~50% of the

13 HMW material,

to small-molecular-weight

products.

%9% of the two minor

label

peaks,

at Vi

to serine

analysis

of sugars

hydrolysis

revealed

characteristic

peaks digest

6).

42

in

the digest

a and b-, eluting

(arrows,

in glucosamine-labeled

The major

column

clone

peak

attached

1 and 2) was treated

Fig.

material, or small released

products

These

4).

and probably

serine-containing from

the presence

eluting

just

these

pre-

of

[3H]galac-

at Vi mainly

consisted

of chondroitin-4-

Vol. 103, No. 1,198l

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AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

CHO

CLONE

SEGMENT Figure

13

NUMBER

6

Paper chromatography of chondroitinase digestion products (Vi from Figure 4). The chromatogram was cut into l-cm segments, and the radioactivity in each segment was determined. The positions of co-chromatographed authentic 4,5-unsaturated disaccharide standards are indicated: 6S, Adi-6s; 4s. Adi-4s; OS, Adi-OS. Upper panel, wild-type; lower panel, clone 13.

A

CLONE

13 glcN-

B

3H

CLONE

13

gal-

3H

I

500

1,000

500

20

30

40

50

60

20

FRACTION NO Figure

30

40

FRACTION

50

60

NO

7

Bio-Gel P-10 elution profiles of chondroitinase-resistant (Vo from Figures 3 and 4). after treatment with HONO. labeled material; B. [3H]galactose-labeled material. lower panels, wild-type.

43

HMW material A, [3H]glucosamineUpper panels, clone

13;

Vol. 103, No. 1,1981

BIOCHEMICAL

AND

The chondroitinase-resistant treated

with

column

(Fig.

free

HONO cleaves

or N-sulfated

N-acetylated

amino

residues

substituted

charides

residues

a broad

chondroitinase-resistant ly all

type

CHO cells,

of the

total

clude

that

sulfate

incorporated

label

the biosynthesis

droitinase

digestion

amount

of this

fected

by the clone

contains

less

smaller

molecular

solves

the material

is

than

size

by mild

into

it

for

CHO cells

These

asn-linked

reactions

galactosyltransfer of GAGS, which plasmic

reticulum

are

is

likely

that

is

essential-

susceptible. 13 and wildfor

in clone

con-

13 cells.

even after

chon-

Since

no appreciable

biosynthesis

is probably

indicate

little

that

at least

to

on DEAE-Sephacel

Although

af-

the material

can be converted

(9)

~11%

re-

the material(s)

a portion which

consists

has recently

have of the been

(10). reactions

involved

oligosaccharides

occur

to be defective

reactions

involved

(X,13).

its

peaks.

~~50% of the

We therefore

Chromatography

three

only

each account

7).

results

and very

known

proceed

13,

along

of oligosac-

clone

persists

Fig.

polysaccharide

The galactosyltransfer and complex

(V,,

alkali.

at least

both

sulfate

HMW material

acid,

type,

GAGS is unimpaired

Preliminary

10% sialic

distributed

from glucosamine.

in clone

13 defect.

"erythroglycan"-like

described

derived

contains

to HONO, while

for

bearing

the variously-

HMW material

and chondroitin

found

residues

mixture

susceptible

that,

and HONO treatment

been identified,

asn-linked

is

of wild-type

material

on the P-10

also

randomly

In the case of wild

of these

amounts

sulfate

a heterogeneous

can be calculated

heparan

Substantial

not yet

it

analyzed

heparan

13 chondroitinase-resistant

data,

3 and 4) was next

to HONO, and since

HMW material

of the clone From this

Since

are

range.

Figs.

COMMUNICATIONS

at glucosamine

are more-or-less

products

size

(V,,

sulfate

(8).

RESEARCH

was again

are resistant

the degradation with

mixture

heparan

groups

which

,glucosamine

the chain,

HMW material

MONO, and the reaction 7).

BIOPHYSICAL

normally

in the assembly in

in clone

the Golgi

in clone

We therefore

13,

take place

propose

44

apparatus

13 (5).

in the assembly

that

of

of glycolipids (11).

In contrast, the linkage

in the rough there

exists

the sequence endo-

a Golgi-

Vol. 103, No. 1,198l

specific clone

BIOCHEMICAL

compartmentalization

BIOPHYSICAL

problem

RESEARCH

of some kind

which

COMMUNICATIONS

accounts

for

the

13 trait. For example,

UDP-galactose

a specific from

In addition,

(15). removing the

AND

action

subsequent mechanisms

mechanism

the cytosol a further

the inhibitory

could

product

for

into

is

synthesized)

mechanism

of the

has been

galactosyltransfer

nucleotide

of UMP out account

for

it

specific

of a luminally-oriented transport

has been described

(where

of the

the clone

apparatus.

may be due to improper

insertion

of the

the Golgi

membrane,

or there

may be a defect

transports

nascent

glycoproteins

through

reaction,

UDP, by

(16,17), A defect

and in these

the clone

galactosyltranferases

in the mechanism

the Golgi

lumen

for

Alternatively,

13 trait.

13 defect

the Golgi

described

diphosphatase

Golgi

transporting

in

which

apparatus.

Acknowledgements I gratefully acknowledge the generous support, both moral and a portion of this work was of Lennart Roden, in whose laboratory The work was supported by USPHS Grants # 5 T32 AMO7069, DE 02670, AM25822. Some of the results in this report were presented as a the 1980 Annual Meeting of the American Society for Cell Biology

material, performed. and ROl poster at (14).

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

Briles, E.B., and Kornfeld, S. (1978) Trends in Biochem. Sci. 1, 223227. Wright, J.A. (1979) Int. J. Biochem. lo, 951-956. Stanley, P. (1980) in The Biochemistry of Glycoproteins and Proteoglycans (W.J. Lennarz, ed.), pp. 161-189, Plenum Publishing Co., New York. Briles, E.B. (1982) Int. Rev. Cytol., in the press. Briles, E.B., Li, E., and Kornfeld, S. (1977) J. Biol. Chem. 252, 1107-1116. Briles, E.B., Li, E., and Kornfeld, S. (1976) Fed. Proc. 35, 1642. Rod&, L. (1980) in The Biochemistry of Glycoproteins and Proteoglycans (W.J. Lennarz, ed.), pp. 267-371, Plenum Publishing Co., New York. (1976) Biochemistry 15, 3932-3942. Shively, J.E. and Conrad, H.E. (1978) J. Biol. JIrnefelt, .I., Rush, J., Li, Y-T., and Laine, R.A. Chem. 253, 8006-8009. Li, E., Gibson, R., and Kornfeld, S. (1980) Arch. Biochem. Biophys. 199, 393-399. in The Glycoconjugates (M.I. Horowitz and Schachter, H. (1978) W. Pigman, eds.), Vol. II, pp. 87-185, Academic Press, New York. (1968) J. Cell. Biol. 38, 358-368. Horwitz, A.L., and Dorfman, A. Sugahara, K., Cifonelli, J.A., and Dorfman, A. (1981) Fed. Proc. 40, 1705. (1980) J. Cell Biol. 87, 3UOa. Briles, E.B. Kuhn, N., and White, A. (1975) Biochem. .I. 148, 77-84. Kuhn, N., and White, A. (1977) Biochem. J. 168, 423-433. Brandan, E. and Fleischer, B. (1980) J. Cell Biol. 87, 200a.

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