Specific binding of sulfated proteoglycans to Concanavalin A-Sepharose 4B

Vol. 83, No. 1, 1978 July 14, 1978

SPECIFIC

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Pages 241-246

BINDING OF SULFATED PROTEOGLYCANS TO CONCANAVALIN A SEPHAROSE 4B

Jochen Institute

Schwermann,

Roswitha

of Physiological

Received

Prinz

Chemistry,

von Figura +)

and Kurt

Waldeyerstr.15,

D-44 Miinster,BRD

May 22,1978

Cl

35 Summary: More than 90 % of S proteoglycans isolated from the secretions of human skin fibroblasts bind to Concanavalin ASepharose 4B (Con A:Sepharose) in the presence of 1 M NaCl. Above pH 5.0 1 M concentrations of methyl-a-D-mannoside and other ha tenic inhibitors for Con A-sugar interaction prevent binding of 1x5sP proteo glycans, whereas equimolar concentrations of non-haptenic carbohydrates do not effect binding. Below pH 5.0 [35S]proteoglycans bind to Con A-Sepharose in the presence of both methyl-a-D-mannoside and galactose. About 60 % of the proteoglycans bound at pH 4.0 are 35 eluted at pH 7.5 in the presence of 1 M methyl-a-D-mannoside. S Glycosaminoglycans prepared from P5S]proteoglycans do not bind to Con A-Sepharose in the presence of 1 M NaCl. These results indicate a P5S]proteoglycan-Con A interaction via the protein core of the proteoglycan and the sugar binding sites of Con A.

[I

Introduction: occuring

Sulfated cell

surface

The amount of sulfate with

It

is generally

established

of

type,

cell

for

that

sulfated sites

+) To whom reprint

with

surface

Several

of Con A (9-11).

requests

with

sulfate

the cell

241

Con A is

the membraneous have

do not However,

varies

carbohydrates.

investigations

Con A has so far

should

(4,6-8).

with

(l-6).

and heparan

surface

associated

glycosaminoglycans

proteoglycans

mammalian cells

dermatan

Con A interacts

and glycolipids.

to be generally

and transformation

cell

assumed that

sugar binding sulfated

cycle

appear

on cultured

as proteoglycans

used as a probe

glycoproteins

the

constituents

chondroitin-4/6-sulfate,

associated the cell

widely

glycosaminoglycans

interact

the not

with

interaction

been studied.

be addressed.

Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

8lOCHEMlCAL

Vol. 83, No. 1, 1978

We examined secretions

the

(3,4)

interaction

of human skin

proteoglycans

that

this

non-proteolytic

of

least

with

since

isolated

Con A.

in part

study,

RESEARCH COMMUNICATIONS

proteoglycans

fibroblasts

stem at

were used for

mild

AND BIOPHYSICAL

from

they

from

r35S1

Secretory

the cell

the

surface

can be isolated

under

conditions.

Materials and Methods: The monosaccharides were obtained from Serva, Heidelberg, Con A-Sepharose from Pharmacia, Freiburg. [35S]proteoglycans were isolated from the secretions of human skin fibroblasts as described (4). The relative amounts of chondroitin-4/6-sulfate, dermatan sulfate and heparan sulfate in this preparation were 11:83:6 (4). [35S]glycosaminoglycans were prepared from these proteoglycans by treatment with 0.15 M NaOH for 4 h at 37OC and subsequent neutralisation. Radioactivity was determined in a liquid scintillation counter (Packard, Model1 3390/TT) using Unisolve (Zinsser, Frankfurt) as scintillation medium. Results:

Proteoglycans

fibroblasts

5

35

in

the applied less

The latter

1).

into

be quantified

carbohydrates

the

desorbed

more than

70 % of

eluent from

the Con A-Sepharose

ClS proteoglycans 35

of

time

solutions

and with

for

A interaction.

ClS proteoglycan 35

the

binding

sugar

with

higher

binding

Only

242

earlier

M methyl-u-D-

columns

the

applied

binding.

carbohydrates that

could when

equilibrated

effect

to Con A-Sepharose sites

bound under

reproducibility,

inhibitory those

whereas

Con A-Sepharose

or preventing

their

1

with

90 % of

column.

to Con A-Sepharose allowing

were tested

proteoglycan-Con

with

in agreement of

in less

in

is

Inclusion

samples were applied

and eluted

finding

equili-

More than

( 9-11).

The interaction

interact

(Table

secreted

columns

literature

['" S1proteoglycans

the

from the

on Con A-Sepharose

at pH 7.5

human skin

35

in the

mannoside

the

prepared

of

[I S proteoglycans bound to Con A-Sepharose, 5 % of the [I S glycosaminoglycan chains were

conditions.

reports

secretions

35

than

these

were loaded

M NaCl buffered

1

from the

ClS glycosaminoglycans

and

[ S1proteoglycans brated

isolated

Several on the

[35S1

inhibited are known to

of Con A. Thus glucose,

Vol. 83, No. 1, 1978

Table

Elution

1:

BIOCHEMICAL

of

AND BIOPHYSICAL

P5S]proteoglycans

and

es S] glycosaminoglycans at pn 7.5

p5S]Proteoglycans mM Tris/IiCl,

RESEARCH COMMUNICATIONS

eluted (% of

pH 7.5)

from

Con A-Sepharose

p5S]Glycosaminoglycans applied)

eluted

(in

50

1.0

M

NaCl

1.0

M

Galactose 1.0 M N&l

1.0

M Methyl-a-D-mannoside in 1.0 M NaCl

56.1

1.8

1.0

M Methyl-a-D-mannoside in 1.0 M N&l

15.2

1.3

0.1

M Sodium in 1.0

7.6

in

92.7

1.9

borate, M N.&l

pH 7.5

1 .o

2.3

0.4

P51

Samples of 0.2 ml (containing 20 000 cpm S radioactivity) 2.0 cm Con A-Sepharose columns, equilibrated in 1 M NaCl After incubation for 2 h at room temperature the columns fractions of 3 ml with the eluents listed above. Between elution with methyl-a-D-mannoside the column was kept at

mannose, not to

their

a-methyl

galactose, inhibit

glucuronic

the binding

The effect

was studied

galactose

in

(Table

L-rhamnose

1

between

of

(Fig.

A).

pH 4.0

M NaCl more than

buffered

Elution

with

at pH 7.5 desorbed

at pH 4.0 in

the presence

galactose.

At high

chains

did

not

tested

(Fig.

Discussion: binding

of

binding

sites

1

salt

bind

were found

35 S proteoglycans Cl

of

to 8.0.

90 % of over

the applied

prevented

binding

1

concentrations

to Con A-Sepharose

only in

1

above M NaCl

M methyl-a-D-mannoside

bound or

[I35S glycosaminoglycan over

the whole pH range

B).

The present results clearly demonstrate the specific 35 S proteoglycans to immobilized Con A via the sugar

[I

of the

lectin.

Interaction

243

with

1 M

tested.

[I35S proteoglycans

60 % of

of either

of

[I35S proteo-

pH range

1 M methyl-a-D-mannoside up to

to Con A-

In the presence

the whole

1 M methyl-a-D-mannoside

1

and xylose

but

2).

bound to Con A-Sepharose

Presence 5.0

acid,

and N-acetylglucosamine,

of pH on the binding

Sepharose

glycans

derivatives

were applied to 0.5 x in 50 mM Tris/HCl, pH 7.5. were stepwise eluted in the first and the second 200C for 16 h.

the Con A sugar

1

M

BIOCHEMICAL

Vol. 83, No. 1, 1978

Table

2:

Sugar

Sugar

specific

inhibition of the Con A interaction

M)

(1

AND BIOPHYSICAL

Proteoglycans

RESEARCH COMMUNICATIONS

pS]proteoglycan-

eluted

(% of

applied)

12.9

Glucose

104.4

Mannose

102.8

N-Acetylglucosamine

104.5

Galactose

1 4.7

Glucuronic

acid

6.0

L-Xhamnose

5.9

Xylose

1

Methyl-a-D-mannoside

96.5

Methyl-a-D-glucoside

9.3

103.5

from fibroblast secretions (20 000 cpm) were applied P5 S3 Proteoglycans to 0.5 x 2.0 cm Con A-Sepharose columns, equilibrated and eluted with solutions containing the indicated sugars (1 Ml in 1 M NaCl, 50 mM Tris/HCl, pH 7.5. The elution volume was 3 ml.

binding

sites

residues

requires

that

are present

with

in proteoglycan unsubstituted

molecules

hydroxyl

C-4 and C-6 configurated

as in a-D-glucopyranose

glycosaminoglycan

chains

prepared

not

interact

the

that

the

with

carbohydrates

Con A are

located

Below pH 5.0 appears

that

of

interaction

above

pH

5.1

the

sites

between

the pH to 7.5,

may strengthen

Since

(12).

proteoglycans

of Con A, it of

is

do likely

proteoglycans

proteoglycans

to

and Con A

The binding thus

at pH 4.0

excluding

irreversible

on the Con A-Sepharose

to be around

attractive

at C-3,

core.

above pH 5.0.

negative

groups

secreted

the binding

proteoglycans

reported

Con A may weaken the charge

than

by raising

of the

Con A is

opposite

mediating

to be stronger

denaturation

binding

on the protein the

can be reversed

p1

sugar

from

sugar

charge forces the

244

pH 5.1 of

both

(13).

below

The

may indicate,

proteoglycans

, whereas

interaction.

This

column.

pH

Involvement

and 5.1

their of

BIOCHEMICAL

Vol. 83, No. 1, 1978

=9

bo-

2 0 0 E

40-

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

: I -z

20cl Z 5m oL

4

I

I

I

5

6

7

5I

a

I

a

,

I

6

I

a

7

PH

Fl-

. 1: Effect of pH on the interaction of S glycosaminoglycans with Con A-Sepharose.

[ S1proteoglycans 35

and

[ 1 35

A: S Proteoglycans from fibroblast secretions (20 000 cpm) were ioaded onto 0.5 x 2.0 cm Con A-Sepharose columns equilibrated and eluted with 1 M methyl-a-D-mannoside, 1 M NaCl in 50 mM buffers (O--O) or with 1 M galactose, 1 M NaCl in 50 mM buffers (M). was used for pH 4.0-5.5, The elution volume was 3 ml. Sodium acetate sodium phosphate for pH 6.0-7.0 and Tris/HCl for pH 7.5-8.0. S Glycosaminoglycans [Iequilibrated and eluted 35

B: columZs,

electrostatic

forces

suggested

earlier

The specific from the raises

number of Con A with to cell

interaction of

the question of

in Con A-polyelectrolyte

surface

(SFB

cell

has been

receptors exerted

isolated

at physiological

surface

receptors

associated

proteoglycans

fibroblasts

effects

surface

ACKNOWLEDGMENTS:This gemeinschaft

cultured

the biological

surface

of Con A with

whether

the cell

cell

interaction

(14).

secretions

are part

were loaded onto Con A-Sepharose as described above.

associated for

pH

proteoglycans

Con A. By inference

by the

may be mediated

interaction

a of

by Con A binding

proteoglycans.

work was supported

104). 245

by the

Deutsche

Forschungs-

Vol. 83, No. 1, 1978

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

References: 1. Dietrich, L.P. and Montes de Oca, H. (1970) Proc. SOC. Exp. Biol. Med. 134, 355-962. 2. Kraemer, P.M. (1971) Biochemistry lo, 1437-1444. 3. Neufeld, E.F. and Cants, M., in: Lysosomes and Storage Diseases (1973) (Hers, H.G. and von Hoof, F. ed.) pp 261-275, Academic Press, New York and London. 4. Kresse, H., von Figura, K., Buddecke, E. and Fromme, H.G. (1975) Hoppe-Seyler's Z. Physiol. Chem. 356, 929-941. 5. Sjoberg, I. and Fransson, L.A. (1977) Biochem. H. 167, 383-392. 6. Dietrich, C.P. and Montes de Oca, H. (1978) Biochem. Biophys. Res. Cormnun. 80, 805-812. 7. Kraemer, P.M. and Tobey, R.A. (1972) J. Cell. Biol. 55, 713-717. 8. Underhill, C.B. and Keller, J.M. (1975) Biochem. Biophys. Res. Commun. 63, 448-454. 9. DiFerrante, N. and Hrgovcic, R. (1970) FEBS Lett. 2, 281-283. 10. Buonassisi, V. and Colburn, P. (1977), Arch. Biochem. Biophys. 538, 571-579. 12. Goldstein, I.J., Hollermann, C.E. and Smith, E.E. (1965) Biochemistry 4, 876-883. 13. Entlicher, G., Kosti?, J.V. and Kocourek, J. (1971) Biochim. Biophys. Acta 236, 795-797. 14. Doyle, R-J., Woodside, E.E. and Fishel, C.W. (1968) Biochem. J. 106, 35-40.

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