Ganglioside headgroup disorder as a sequel to lectin binding

Vol.

95, No.

August

3, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

1299-1305

Pages

14, 1980

GANGLIOSIDE HEADGROUP DISORDER AS A SEQUEL TO LECTIN BINDING Pat M. Lee and Chris

W.M. Grant

Department of Biochemistry University of Western Ontario London, Ontario, Canada N6A 5Cl Received

June

24‘1980

SUMMARY: Evidence is presented that gangliosides show a measurable tendency to exist in clusters in lipid bilayer systems; and that these clusters are subject to disruption by a lectin binding event.

INTRODUCTION The events

that

made the subject presumed that

surround

of considerable

significance

lectins

bind

with

high

events

to a metabolic

focus in

headgroup

with

minimal

study

employed

events

gangliosides lectin

were

(reviewed

in

with

Abbreviations:

species,

and glycophorin high

affinity BSA, bovine

for

able

serum albumin;

of their

is well

known and

a chain

cell.

of

We have in an attempt

of early

structural family

glycoprotein

is widely therein).

as receptors

to

changes of glycolipids

of specific work

which

(6 and ref.

to function

NANA residues

N-Acetylneuraminic

of the

glycophorin,

glycoprotein

are

initiate

in a variety

For our

because

of glycolipids

as a suitable

involvement

1 - 5).

of a transmembrane

role

have been

It

and glycoproteins

chosen

documented

the human erythrocyte

as an example

on the part

on the possible

of their

residues

can sometimes

gangliosides

Gangliosides

because

recognition

labeled

ambiguity

such phenomena.

for

response

years

processes.

to sugar they

cells

in recent

recognition

by so doing

spin

to eucaryotic

investigation

selectivity

and that

leading

binding

to physiological

glycoproteins;

employed

lectin

for

we have accepted Both WGA, a

(6 - 8). WGA, wheat

germ agglutinin;

NANA,

acid. 0006-291X/80/151299-07$01.00/0 1299

Copyrrghr Gj 1980 by Academic Presr. Inc. All rights of reproducrion rn any form reserved.

Vol.

95, No.

3, 1980

Given

BIOCHEMICAL

that

interaction

binding

with,

one might

Indeed

However,

we demonstrate

mobile

here

in lipid

- presumably

The concept

a cell

receptor

surface

the role

at low

the rule lectin

a disruption that

lectin

at least

can actually

event

might

initial

concentrations.

concentrations,

of potential

as recognition

macromolecule;

for

become more

of pre-existing,

a binding is

oligosaccharide

to be a basic

at high

the headgroups

aggregate

of gangliosides

MATERIALS

that

COMMUNICATIONS

a multivalent

immobilization

is

reflecting

RESEARCH

headgroup by,

this

bilayers,

interactions.

involves

chain

in our hands

BIOPHYSICAL

crosslinking

oligosaccharide

result.

gangliosides

of a lectin

and perhaps

expect

AND

non-covalent

lead

to disruption

interest

in

of

understanding

sites.

AND METHODS

L-a-dimyristoyl and L-a-dipalmitoyl phosphatidylcholine, cholesterol and egg phosphatidylcholine were obtained from Sigma. The latter (Type III-E) was further purified by column chromatography on silicic acid. Dioleoyl phosphatidylcholine was obtained from Serdary Res., London, Canada. All phospholipids were pure, as judged by thin layer chromatography on silica gel G (Stahl). Gangliosides were isolated from beef brain by a modification of the method of Kanfer (9) in which gangliosides obtained from the initial Folch extraction were purified by chromatography on silicic acid (Bio Rad 200-400 mesh eluting with CHC13/MeOH) and checked for purity by thin layer chromatography. Ca2+ and Mg2+ were added as their chlorides, and EDTA as the HEPES, WGA and globulin free BSA were obtained from Sigma. disodium salt. Spin labeled derivatives of gangliosides and glycophorin illustrated in Figure 1 were prepared as described by us previously (10-13). Lipid mixtures were made by dissolving appropriate amounts of each in chloroform/methanol and pumping extensively under vacuum to remove traces of solvent. Dried lipid mixtures were suspended in buffer by vigorous vortexing. EPR spectra were recorded on a Varian El2 spectrometer equipped with a TM110 cavity. Probe temperature was measured with a copper constantan thermocouple and was 21°C unless otherwise specified. RESULTS AND DISCUSSION The characteristics have been work

described

described

residues: la),

number

of labels in the

randomly

have

randomly

or restricted

Figure

that

by us previously

here

either

of headgroup

per

nitroxide

labeled

(e.g.

10-13).

rings

distributed

ganglioside

amongst

the 16 available

oligosaccharide

1300

attached

(NH2-linked,

labels

is are

and glycophorin

The derivatives

the various

or glycoprotein these

gangliosides

covalently

to NANA residues

case of glycophorin

amongst

spin

to headgroup sugars

Figure on the order

presumably chains

used in the

(COOH-linked, lb).

The

of 1.

distributed (12).

sugar

We have

Note

Vol.

95, No.

BIOCHEMICAL

3, 1980

AND

iiI dH

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

CH3-i=O

K\

CHUH H

N

t

0 H OH

H

H

H

con-

Ctl,-;-NH 0

Figure

1.

(a)

Mode of attachment glycoprotein

The COOH-coupled functions and

(b)

The

is

NH2-coupled

of

headgroup

spin

label

which

is

ranging

such labeled

studied

from

organic

solvent

to investigate

their

of WGA binding

on the

It

is very

bilayers. glycophorin the

spin

free label

as described motion. increased

in

specific

for

headgroup

cells

to lectin

similar

(15);

and is

with

a fraction labels,

have

Figure

increased

the

Y-axis

of Keith

to headgroup what

lectin

WGA binding on

by the method

the effect

in lipid for

plotted

related

begun

2a typifies

previously

parameter

in systems

recently

of glycophorin

essentially

1301

Only spin

(lo-13),and

inversely 2a is

by

components

~c , measured

in Figure

immobilization

The

and

alcohol chains

bearing

reported

(12,13). time,

purposes. occupied

headgroup

to plots

solution

shown

glycolipid

NANA residues

binding.

oligosaccharide

previously

The result

carbohydrate

to intact

response

correlation

to

label which has a prediliction for primary scattered throughout the oligosaccharide

A typical ganglioside is shown for illustration of the possible attachment points are actually especially when using the type in la.

extensively

labels

freedom

one might binding

et al

predict:

- the

only

to is

(14) of

Vol.

95, No.

BIOCHEMICAL

3, 1980

AND

BIOPHYSICAL

,FJ .-----•/i a Li

RESEARCH

COMMUNICATIONS

r

16

14

12

10

0

.05

.l 0

0

.05

16

.l 5

.l 0

.l 5

/--*.

./ e

12. I!---0

.05

.l 0

.I 5

0

.05

.l 0

.l 5

0

.05

.l 0

.l 5

17

13

(mM)Figure 2. a variety

Effect of of different

lectin binding systems

on headgroup

oligosaccharide

dynamics

in

The Y-axis parameter (spin label correlation time, Tc,) is inversely related to headgroup motional freedom. EPR samples were made by adding 20 p of various stock WGA solutions to equal volumes of the glycolipid or glycoprotein specimens. Lipid concentrations were 40 umollml for Za, 10 umol/ml Spectra were for 2d, and 28 umol/ml for Zc,e,f,g (all samples unsonicated). recorded 3 min after mixing (a)

Typical data obtained using the spin labeled qlycoprotein, glycophorin, for comparison. Clycophorin was assembled at a lipid/protein molar rat of 18O:l into 1:l egg phosphatidylcholine/dipalmitoyl phosphatidylcholine bilayers by dialysis from detergent solution (dodecyltrimethylammonium bromide) as described elsewhere (12,16). The bilayer structures were harvested by centrifugation. Glycophorin was spin labeled on random sugar residues (Figure la). Medium 0.1 N KC1 containing 2 mM Ca2+ and Mg2+, 0.3% BSA, and buffered with 5 mM HEPES pH 7.3

(b)

Ganglioside labeled on random sugars (Figure at 0.7 mg/ml. Medium 0.145 N NaCl buffered and containing 5 ml+ EDTA

(c)

Ganglioside labeled on NANA residues (Figure lb), Medium 0.145 layers of egg phosphatidylcholine. EDTA and 0.3% BSA, buffered with 5 mM HEPES

(d)

Ganglioside choline. fered with

labeled on random Medium 0.145 N NaCl 5 mM HEPES pH 7.3

sugars, containing

1302

at

la) but free in solution with 5 mM HEPES pH 7.3,

at 4.8 mol % in N NaCl containing

5 mol % in 5 ml EDTA,

egg phosphatidyl0.3% BSA, and

bi5 ti

buf-

Vol.

95, No.

3. 1980

BIOCHEMICAL

noteworthy

aspect

phenomenon

is

However

being

seen

for

the effect

remarkable

(Figures

increased

in response

ganglioside of the This

is

gangliosides

exist

in

although toward

tetravalent

lectin.

Hence

clusters

which

we have

claimed

that

small

extensively

in simple are various clustering.

been mentioned

above.

(c-g))occur is

at

not bound.

to a small

in motional

should

the gangliosides

event.

However,

be a certain

chains

(10,15),

would

fraction

freedom.

that

binding

of gangliosides

of maximal

curves

one assumes

oligosaccharide

of

- presumably

The point

binding

increase

there

may

WGA to available

by an initial that

mobility

concentration

the ganglioside

that

if

shows a

decreases

in

Zb).

tendency we have

disperse

until

quite

bilayers.

mechanisms

which

Attractive Probably

and phase

separation

phenomena

gangliosides

appear

(Figure

bilayers

as the

available

understood

quantitites

of hydrated

ferences,

would

perturbed

nature

headgroup

of total

lipid

dips

of

in a general

semi-crystal

differences,

it

amongst

lipid

(the

the majority

previously

forces

now assumed

ganglioside

are

cases,

mobilization

can be readily

attractive

There

mobility

by the

A similar

cooperative.

the headgroup

headgroup

can result

observation

concentrations,

2c the ratio

1 : 35.

in

levels,

such that

in Figure

(+)

COMMUNICATIONS

in solution

to gangliosides

lectin

oligosaccharide

For instance

is

In all

to high

concentrations

obtained

RESEARCH

2c - g).

to crosslinking

lectin-induced lectin

at low

BIOPHYSICAL

to gangliosides

of WGA binding

increase is

the curve

WGA binding

difference:

actually lectin

that

AND

differences, may fail

might

forces more

amongst

important

membrane (e.g.

lipids

17-19). or glycerol

to fit

be expected

optimally

to contribute

oligosaccharide

though with

is

On the basis vs. into

length

backbone

dif-

a phosphatidylcholine

Ganglioside labeled on NANA residues, at 5 ma1 % in dioleoyl choline. Medium 0.145 N N&l containing 5 mM EDTA, buffered HEPES pH 7.3

(f)

Ganglioside labeled on NANA residues, at 0.5 ml % plus 4.5 ganglioside in egg phosphatidylcholine. Medium 0.145 N N&l 5 m?l EDTA and buffered with 5 mM HEPES pH 7.3

(g)

Same as (f) but in bilayers cholesterol as well

1303

transition

of chain

ceramide

have

known

phase

(e)

of egg phosphatidylcholine

chains

the well

resultant

to

phosphatidylwith 5 mu mol % containing

containing

unlabeled

10 mol %

Vol.

95. No.

3. 1980

lattice.

BIOCHEMICAL

Probably

observation

chain

described

C20 sphingosine

length

here

with

AND

BIOPHYSICAL

differences

since

Cl8 fatty

beef acids

are

brain

RESEARCH

not a key factor

gangliosides

are

choline

the melting mixtures

words,

behaviour

some mixing

must occur.

of ganglioside-ganglioside 'clusters'

are

separation workers

is

poorly

gangliosides in rigid

(11).

matrix

lead

to closer

patches

occurs

the

the above

observation

oleoyl

phosphatidyl-

(20).

In other

that

the ganglioside

of laterally theory

of bilayer

headgroups

If

can be readily

of incorporated

are more mobile

in

fluid

understood

point

of the

surrounding

are

should

segregated

on the basis

into

of high

concentrations.

CONCLUSIONS The observations (i)

that

gangliosides

phospholipid (ii)

if,

presented

have

show a striking

two implications:

tendency

to cluster

spontaneously

in

bilayers

by whatever

(10,22), against

here

mechanism,

the possibility these

the original

exists

gangliasides receptor

gangliosides

for

that

can disrupt subsequent

1304

occur a specific

in aggregates binding

the aggregate, redistribution.

than

in ganglioside

2-b of Ca ) an ion which

gangliosides

to

temperature

increase

by the presence (10)).

phase and co-

mobility

the melting

gang-

clustering

transition

on headgroup

own evidence

dispersed

of ganglioside

temperature-induced

packing

30 mol %

seem likely

phase

some 5 - 6°C below

ganglioside

2). below

our

the

bilayer

effects

(and can be abolished

egg

by Chapman and McConnell

ganglioside

however,

mobility

host

temperature

Briefly,

bilayers;

headgroup

the host

in

with

pool from

Clg/

observation

the same phenomenon

that

with

a sizable

originally

invoked

our observation

would

to be expected

proposed

dependence

this

it

with

probably

behaviour

correlates

local

proximity,

(17,21). We have recently

explain

Combining

in equilibrium

This

lioside.

concentration

the

largely

(Figure that,

of ganglioside/stearoyl

has a measurable

in

and we see the phenomenon

phosphatidylcholine and dioleoyl phosphatidylcholine Bunow and Bunow have shown by scanning calorimetry ganglioside,

COMMUNICATIONS

event thereby

in real directed freeing

cells

Vol.

95, No.

3, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

ACKNOWLEDGEMENTS This

research

and the Medical

was supported Research

Council

by grants

from

the National

Cancer

Institute

of Canada.

REFERENCES 1. 2. 3. 4. 5. 6. 7. a. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

Edelman, G.M. (1976) Science 192, 218-226. Fishman, P.H. and Brady, R.O. (1976) Science 194, 906-915. "Ganglioside Functionll Porcellati, G., Ceccarelli, B. and Tettamanti, G. eds. Plenum Press (1976). Yamakawa, T. and Nagai, Y. (1978) Trends Biochem. Sci. 3, 128-131. Critchley, D.R., Ansell, S. and Dilks, S. (1979) Biochem. Sot. Trans. 7, 314-319. Tomita, M., Furthmayr, H. and Marchesi, V.T. (1978) Biochemistry 17, 4756-4770. Boldt, D.H., Speckart, S.F., Richards, R.L. and Alving, C.R. (1977) Biochem. Biophys. Res. Commun. 74, 208-214. Bhavanandan, V.P. and Katlic, A.W. (1979) J. Biol. Chem. 254, 4000-4008. Kanfer, J.N. (1969) Methods Enzymol. 14, 660-664. Sharom, F.J. and Grant, C.W.M. (1978) Biochim. Biophys. Acta 507, 280-293. Lee, P.M., Ketis, N.V., Barber, K.R. and Grant, C.W.M. Biochim. Biophys. Acta (in press). Lee, P.M. and Grant, C.W.M. (1979) Biochem. Biophys. Res. Commun. 90, 856-863. Lee, P.M. and Grant, C.W.M. Can. J. Biochem. (in press). Keith, A.D., Bulfield, G. and Snipes, W. (1970) Biophys. J. 10, 618-629. Sharom, F.J. and Grant, C.W.M. (1977) J. Supramol. Struct. 6, 249-258. Grant, C.W.M. and McConnell, H.M. (1974) Proc. Natl. Acad. Sci. USA 71, 4653-4657. Shimshick, E.J. and McConnell, H.M. (1973) Biochemistry 12, 23512360. Chapman, D. (1975) Quart. Rev. Biophys. 8, 185-235. Lee, A.G. (1977) Biochim. Biophys. Acta 472, 237-344. Bunow, M.R. and Bunow, B. (1979) Biophys. J. 27, 325-337. Phillips, M.C., Ladbrooke, B.D. and Chapman, D. (1970) Biochim. Biophys. Acta 196, 35-44. Lee, G., Consiglio, E., Habig, W., Dyer, S., Hardegree, C. and Cohn, L.D. (1978) Biochem. Biophys. Res. Commun. 83, 313-320.

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