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Gangliosides and Associated Enzymes at the Nerve-Ending Membranes G. TETTAMANTI, A. PRETI, B. CESTARO, M. MASSERINI, S. SONNINO, and R. GHIDONI Department of Biological Chemistry, The Medical School, University of Milan, Milan, Italy

Gangliosides are characteristic glycolipid components of the plasma membranes of mammalian cells. They are particularly abundant in the nervous tissue, specially the grey matter, where their concentration is about one tenth that of total phospholipids. The evidence concerning the high content of gangliosides in the neuronal membranes, and of their peculiar location in the outer membrane surface, stimulated research and speculation on the possible involvement of gangliosides in brain specific functions. However, in order to provide a plausible working hypothesis for such involvement a more precise knowledge on the contribution given by gangliosides to the local environment of the neuronal membrane is required. Chemical and physico-chemical properties of gangliosides: a molecular introduction to ganglioside behavior in cell plasma membranes. Gangliosides are a family of glycosphingolipids which contain at least one residue of sialic acid. The number of sialic acid residues per ganglioside molecule varies from 1 to 7, with an average content of 2-2.5 in the brain gangliosides of most v e r t e brates (1). The sialic acid residue(s) is(are) attached to the neutral oligosaccharide core which may contain glucose, galactose, N-acetylhexosamine (generally N-acetylgalactosamine) and fucose. The most abundant oligosaccharide core occurring in brain ganglio sides is ganglio-N-tetraose, gal(β, 1->3)calNAc(β, 1->4)gal (β, 1->4)glc. The acidic oligosaccharide is β - g l y c o s i d i c a l l y linked to ceramide, formed by a long chain fatty acid (primarily C 18:0) and a long chain, mainly unsaturated, base (C 18 and C 20) linked together by an amide bond.

0-8412-0556-6/80/ 47-128-321 $5.75/ 0 © 1980 American Chemical Society Sweeley; Cell Surface Glycolipids ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

322

CELL SURFACE

The the of

oligosaccharide portion

high

hydrophilicity

of gangliosides,

of the molecule, displays

interactions: hydrogen bonds

These

bonds

chains ules

of adjacent

than

within

important NMR

are more

role

studies

likely

and ion bonds

gangliosides

(or other

the same ganglioside

in any process

performed

binding

glycoconjugates) Thus

molec

they

association.

play^gn The

et a l . ( 2 ) on ganglioside

— -in

micellar

this

form-

ganglioside

sialic

acid

explain

as oxygen

(with

galactosamine molecule.

lead to visualize rich

its carboxylic

and the terminal

surfaces

why the affinity

of G.

binding

involving

but also

galactose

The occurrence of these

KAtt

sites of

not only the

the N - a c e t y l -

residue

additional

for cations

C

G

M ]

the cation

group),

sites).

the saccharide

molecule.

of ganglioside

by S i l l e r u d

responsible for

a double potentiality

(cation

to occur between

GLYCOLIPIDS

present

oxygen

is much

in

the

ligands may

larger than

Μ1 that

exhibited by free

The

oxygen

rich

and β-methyl

α - ,

surfaces

described

glycoside

of sialic

in ganglioside

G

à

J

acid.

are e x -

4

Mi pected ral

to be present

feature The

in all gangliosides

and to constitute

a gene-

of ganglioside chemistry.

apolar chains of the ceramide portion

of gangliosides are

responsible for the hydrophobic properties of gangliosides and for of

their

availability

sine

and the carbonyl

spread mutual with

association,

other

range

In the presence :

18-50

gliosides from

the

were

core

10 A(see

20 A

weight

10 ,

10

M.

-10

properties showed

and leads

in which

with

water.

molecule

groups

about

solutions

The literature

of gangliosides,

that

ganglioside

G

K i M

on the cylinder

30

(10),

that

of the

groups of

large

for the c r i t i c -

are in the range works

of S c h w a r z -

reporting

investigations

recently performed

A ,

head

micelles

values

for the recent

scattering

( hydration

tFje radius of

by the sugar

( 9 ) and of Formisano et a l . light

gives

structures of gan-

structures

at 3 7 ° C ,

In dilute aqueous

except

volume

the apolar chains radiate

the sugar

In these

is,

are formed.

Laser

chain

them to associate in

quantities of water

packed cylinder

( 4 )

M (4,5,6,7,8),

maj^ri et ^

of each

of an hydrophi lie

concentration (cmc) of gangliosides

rrîcellag -

with

l).

tendency for

association

molecules

and the annulus formed

Figure

molecular

of small

of the rods,

all ganglioside

lipid

properties

described

in contact

formation

would tend to

their

of approximately equal

% ) hexagonally

the center

surface

10

in the ganglioside

strong amphiphilic

water.

acid,

reducing

to promote

The

group of the sphingo-

( 3 ).

an hydrophobic portion

them

of the fatty

chains

and thus

molecules

presence

the 3-hydroxyl

oxygen

the two hydrocarbon

The and

al

to hydrophobic interactions.

an hydrogen bond between

a cmc

of

on the micellar

in our laboratory,

and G ^ ^ in the concentration range

Sweeley; Cell Surface Glycolipids ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

18.

TETTAMANTI

ET

Nerve-Ending

AL.

Membranes

323

Figure 1. Dimensions of the saccharide and lipid portion of a ganglioside in a cylinder structure (adapted from Curatolo et al (4))

1

GM1

3x10"

2

CONCENTRATION, M

Figure 2. Laser light scattering of ganglioside G

Mt

TURBIDITY

•—• A—A ·-· Δ-Δ

40

ο 30

in aqueous solution

GM1 GDla GQlb GTIb

20 10

20

40

60

Figure 3. Physicochemical features of mixed aggregates of phosphatidylcholine, phosphatidylethanolamine (PE, used as surface marker), and gangliosides (G i, G , G τ ib, G ) at increasing propor tions of ganglioside. Highest value of the outer PE/total PE ratio corresponds to liposomes. Lowering of turbidity and concurrent enhancement of ratio indicate presence of micelles. "Break" point is in dicated as the "transition ganglioside/ phospholipid molar ratio." M

Dla

ο 20 40 60 GANGLIOSIDE IN THE MIXTURE MOLAR %

Q]b


CELL SURFACE

324 10

-10

Figure to our G

M,

2).

which

was

examined,

Thus the

cmc

should be

measurements tê micelles,

doubles the

that

like

While

micelles,

low

( J_0 ).

are

formed

and

other

chains,

the

This (on

ellipsoids

weak bonds

Ganglioside

The earliest

These

authors

mixtures

ganglioside that

it

is

At

studied the

sediments

of

A

the

ratios

was

recently

technique

unilamellar

face

sided

sulfonic

proper

the

and till

on the

ity

molar

is

of

to

of

be

TNBS)

in the

micelles

saccharide

and L e s t e r

ganglioside-phospho-

value

of

solvent 0,

the

leads

(over

in our

by

4)

as

mixed

mixtures

laboratory.

phases

F o r this

integrity

al.

(used

the

(

12

as

liposomes.

The

shown

turbidity

In fact

remains are

sur-

dissolved with

type pf

the

aggre-

ratio

the

level

and the

unchanged

being formed

and increase

of

ratio

in Figure

the

gangliosides,

micelles

).

a

were mixed

residue

ganglioside/phospholipid

mixed

At

6-trinitrobenzene

employed.As

absence of

the

micelles.

of

ganglioside/phospholipid molar

to

low

phosphatidyl-

gangliosides

and sonicated.

sided phosphatidylethanolamine

of

at

indicating

Barenholtz et

with 2, 4,

Over

value

that

than 0.05)

and monitoring the

removed,

species

in the

decrease

ratio

undertaken

preparing

outer

that

ratios (lower

on ganglioside-phospholipid

and the various

ganglioside

same as

gradual

of

phases or

60%. by

the

investigation on gan-

done by H i l l

supernatant

study

revealed

at pH 7.

aggregation

the

to

very

phosphatidylcholine bilayer.

defined

depends on the

a certain

cess

is

hydrogen

adjacent

and phosphatidylethanolamine

organic

buffer

gate formed

between

behavior

liposomes described

marker

acid,

together,

are

less

more detailed

Phosphatidylcholine

a

into the

and ganglioside

present.

used

once

with the phosphatidylcholine,

incorporated

interactions we

that

upon ultracentrifugation and observed

intermediate

are

associate

micellar structure.

was

ganglioside/phosphatidylcholine

choline

not

better,disk-

monomers

interactions)

systematic

interactions

ganglioside/phophatidylcholine

high

the

are

or,

interactions

Phospholipids.

lipid

established

which

may support

rapidly to

see

and

a value

This

(

According

the

A ,

micelles)

micelles

hydrophobic

M.

micelles

monomers

stability of

glioside-phospholipids ( JJ_ ).

of

are

enhancing the

60

interpreted assuming

basis

as. micelles

10

structures.

(rodlike

ganglioside

can be

is

ganglioside

dissociation of

the

than

c

molarity,

solutions

but prolate

present

hyo^rodynamic radi^s of

-10

in dilute

micelles.

form

10

exhibited by cylinder

idea that

spherical

at

are

lower

GLYCOLIPIDS

as

of

3 proturbid-

% of at about shown

aminogroups


18.

available for med

TNBS.

appears

to

be

side/phospholipid ratio",

varies

to

for

0.

10

acid do

G

content

not

larly

of

,

G_ , Tib

This

.

ecular

the

it:

from

to verify

was

that

(

in artificial

phospholipids, cooperative mation of

hydrogen

,

at

phorin,

enhances

the

glycophorin

Proteins. on the

with

microbial

nin,

deal

with

ganglioside ly

exceeded

the

of

Gangliosides

used

that

general binding

the

monomeric model has

for

not

yet

to

The

effect.

levels

immobilizaof

glyco-

Probably

leading to a

these evidences 4)

that

are

gangliosides

the

proteins.

magnitude and/ agents.

A l l published

either pure or mixed, wheat

interactions.

germ aggluti-

Considering

studies,

which

the

abundant-

interactions pertain

gangliosides.

gangliosides

As

in

glycoprotein,

by crosslinking

Of

cannot

protein-ganglioside been

for-

and d e c r e a -

addition

process

All

described

toward

interactions, by

ganglioside

Figure

in these

the

gangliosides

membrane

bind

of

through the

gangliosides,

interferon,

monomeric

%

tendency

physiological

gangliosides,

hormones,

molarity,

1.5

"clusters",

easily

ganglioside-protein

mean

monomeric)

layers,

interactions of

(

divalent cations

see

and recording

saccharide chains.

of

crosslinking

(

for

in phospholipid

likely

causes

gangliosides.

lipid

does

A

the

them being enhanced

rather than to

not

of

hypothesis

toxins,

of

erythrocyte

in the

micellar

proteins.

groups

0. 2

supramol-

(at physiological

tendency

concentrations.

concentrations 10

the

range,

1 to

investigation indicate

mobility of

The presence

mobility or

rich

on fluid

stability of

studies

acid

assembly

with

this

adjacent

increases

cations

gangliosides

gangliosides

of

G ^ sialic

a micellar organization.

gangliosides

the

for the

particu-

liposomal

amounts of

themselves,

between

head

magnitude

tend to form, or

sugar

takes part

more packed consistent

ions

ganglioside

a sialic

a

0.

show a measurable

interaction

ganglioside

lower

of

results

among

bonds

this

,

the

further

tion

of

or Mg

crosslinking ses

determine

would give

terms)

0.25

monovalent

from

low concentrations

toîminuish.

+

Ca

at

interaction

layerg tends of

even

in molar

consequence

;

phosphatidylcholine bilayers

pHs) gangliosides,

"transition

from

while

for-

ganglio-

by decreasing

G

spin-labeled

The

being

the

the physiological

dynamics of

.

\3_)

,

or

of

divalent cations,

for

presence

made by using

E P R signals

ions

Ca

the

rises

ratio",

0. 45

Ca

of

transition,

it

start

value

gangliosides:

within

0. 2 ^ o

in the

absence

micelles

In addition,

"transition

organization

attempt

for

words

gangliosides.

means that

which, in the

layers

ratio

In other

which

325

The c r i t i c a l

different

at concentrations

double

An

-

the

Membranes

at

sharp.

molar

in||uence the

Ca

4

The point

quite

with

^ lib

almost

a

Nerve-Ending

TETTAMANTI ET A L .

worked out.

course

to

this

interact with (micellar and

In a recent


our

CELL SURFACE

326

investigation

on the

binding

of

G ^

ganglioside

with

GLYCOLIPIDS

bovine

serum

Mi albumin,

differential

fugation wed

studies,

that

at

UV

absorption,

associated

least

three

G

k

-albumin

J

fluorescence

and u l t r a c e n t r i -

with chromatographic evidences, complexes

are

formed,

sho-

dif-

M1 fering tion. ^M1

markedly

One form m

o

n

o

m

e

r

s

in their

is

molecular

result

The other

#

./albumin

the

ratio of

one

of

the

weight

and molecular

interaction

two complexes

ganglioside

between

are

conforma-

albumin and

characterized by a

micelle

per

albumin polypep-*

Ml tide chain:

one

other

which

from

one,

complex

hydrophobic

bumin been

micelles,

Monomeric with

monomers ces,

high

The

gangliosides to

the

sugar

the

apolar portion

for

the

surrounding

be

of

capable

to

to

nervous

be

only

are

nerve

to c a r r y

(

likely

endings

large

content

V7_) .

acceptable .

a 5-fold

The

all

cell

on

to

has

ganglio-

inspect

at

Ganglioside to

all

surfa-

these processes

surface,

to

be

indicating

responsible

(

( JJ3 ).

sialic

either

acid from

intrinsic

to

the

plasma

was

of

but

in the

to have a

nervous

sialidase

membranes,

side

similar-

to expose (

Y7J*

(neuraminidase)

or

makes

on these membranes

sialylglycoconjugates membrane

tis-

synaptosomal

appear

membrane

it

shown

separation procedure,

1_9,_20 ) ,

outer

ver-

( 25-30 nmoles of

of

gangliosides

synaptosomal

membranes c a r r y

remove

included-

the

neuronal

the portion

( J J > ),

on the yield

all

displayed by

membrane)

than elsewhere

glycolipids (

chains to

all

gangliosides

in a conventional

the

is

The

mg protein)

based

of

membranes of

However

synaptosomal

enrichment of

surface

in the membranes

membranes )

content

the neurons.

much higher

gangliosides

Synaptosomal to

In all

molecule

in the plasma

amounts of

An evaluation,

oligosaccharide

able

Ul ).

exposed

ganglioside

gangliosides

membranes obtained

to

(

synaptosomal

present

cells,

contains

ganglioside

18_)

form,

and sialyltransferase

endings

bound N-acetylneuraminic acid /

ly

labelled

adhere potentially

ganglioside

The highest

tissue

surrounding

(

of

enabled

monomeric

included

appear

the

sialidase

nerve

cells.

membrane

(

to the

result

ganglioside-al-

conformation

availability

radioactivity

in the

walls

groups

Gangliosides tebrate

sue

mixed

protein

which

interaction.

Gangliosides,

not

and i r r e v e r s i b l y

complexes,

actually

original

specific

and plastic

head

two

rearranged.

appear

glass

the

gangliosides.

of

slowly

These

interactions are

a very

behavior

polymerizes

a dimer.

in which

extensively

sides the

is

21

their )·

activity,

-gangliosides

added ( _22,


23, *2A) ·

18.

In parallel sent in

Nerve-Ending

TETTAMANTi E T A L .

with

along

the

the

gangliosides

all

neuron

synaptosomal

occurrence

of

a

sialidase.

location branes paratus the

In fact

site for

to

be

:

experimental no

to

possible endings.

previous on

5

diffrential

of

red

to

the

no

lowered

(d)

a

specific

of

of

the

Then,

The

same enrichment

as

exposed

confirmed

with the

obtained

by

of

(

on removing

(

26

As

)

shown

appeared,

submitting 28

ner-

perfecting

).

,

the

and the

density

see

biochemical

showed

gradient

activities

; of

(c)

low

membranes of

parallel

to

C

in the

This

Table this

I ): ex-

markedly

this

membrane reductase origin

), ;

gangliosides and

that

of

authentic

synaptosomal activity,

evidence, by other

nervous

),

absolute c o n -

other

and sialidase, This

and corroborated that

(b)

intracellular

sialy transferase

6.

;

NADPH-Cyt.

gangliosides

see

S'-nucleotidase very

same preparation of

in Figure

(

).

membrane markers

concentration of

sialidase

5

and compa-

activity;

endings

plasma

esterase,

reductase,

Figure

analyses

( LDH )

nerve

authentic

-Ach-

hypothesis

ending

light

when preparing

( 27,

a series

inside

nerve

homogenization.

alsewhere

shock

membranes

substantial

properties be

to

specific

activity of

the

course

was

unruptured

C

complex

occur

which came out,

ending fraction,

specific

the

of

to

approached

to c a r r y

during

memap-

a highly homogeneous preparation

low contamination of

contains

consists

material

plasma

membrane markers. branes

hypoosmotic

nerve

( NADH-Cyt.

enhancement the

homogeneous.

to

acetylcholine as

is

than that

Golgi

)

to

and efforts

membranes

described

activity of

tent,

of

light

submitted

presence

this proving

of

formed

attention

the

the main

the Golgi

evidences

material

of

strategy of

possibility for

lactate dehydrogenase

11

them

and

origin,

membranes

when

qualifying

markers

complex

our

been

treated

specific

ATP-ase,

only

( 26

up the

that

pre-

plasma

the

ending preparation we obtained

fairly

starting

the

enhanced (

,

"trapped

cludes

the

be

the

of

recently

considered

centrifugations,

synaptosomal

(a)

has

nerve

ending fraction

preparation

we

difficulties

known to

In setting

is

be enriched

synaptosomal

Therefore

We

which

to

assessment

in the

fragments

The preparation procedure

hypoosmotically and

task.

Therefore

contamination

the

The

is

and cytoenzymatic

we focused

morphologically, nerve

devoid of

contain Golgi

methods,

Figure

).

activity

cortex.

intracellular

on this

( J22, 23

activity,

appears

apparatus

an easy brain

approach

endings.

membranes of Relying

ve

not

morphological

preparations

all

should be

is

has

nerve

,

much more technical

the Golgi

with calf

our

the

)

glycosyltransferases.

used

which

problem

sialidase ( 25

sialy(transferase

membranes encountered of

this

surface

membranes

327

Membranes

mem-

displaying

and bearing which should

proofs,

tissue

strongly

sialyltrans-


CELL SURFACE

GLYCOLIPIDS

Figure 5. Electron microscopic examination of the "nerve ending fraction" (Xl3,000) and of the "synaptosomal membrane fraction" (χ7150)


18.

Nerve-Ending

TETTAMANTi ET A L .

Membranes

TABLE

Biochemical the

sides

are

enzyme med C

characteristics

"Synaptosomal

expressed

substrate

tissue,

in

cases

all

as

the

"Nerve

min"

at

are

the

lower

ending fraction"

from

calf

brain.

International

37°C The

-

30°

data

for

1 0

Nerve

%

of

of the

(

NADH-,

shown,

mean values

than +

Units

6

1 nmole and

referred

ending

acid

;

transfor-

NADPH-Cyt

to

experiments;

mean

and of

Ganglio-

nmoles bound N-acetylneuraminic

in milli

and L D H ) .

fresh

I

f r a c t i o n " , obtained

activities

reductase

of

329

1 g starting

the

S. E .

was

values.

Synaptosomal

fraction

membrane

fraction

Parameter Activity (or

total

"Occluded" L D H

specific

4.4

ATP-ase

149

Ach-esterase

Activity

concentration)

1.0

0.

226.

(or total

67

0

0.

specific

003

161.

concentration)

0.O16

865.6

3. 83

18.4

3. 41

4.

5.4

3. 4

5 -nucleotidase

101.2

15.3

13. 2

71.

Gangliosides

171.6

26.

16. 83

90.5

0. 43

2.3

Neuraminidase

NADH-Cyt.

3.

C

165.

17

0

0.48

0

26.0

0. 024

0

34.2

f

Enrichment

3. 50

19.

1

65

3. 48

4.

79

0. 73

1

reductase

NADPH-Cyt.

C

15.

18

2.3

0. 316

1.7

0. 74

reductase

S i a l y l transferase

+

+

Sialyltransferase

119.9

activity

19.0

14.

expressed

as

1

76.3

c.p.m.

4.

min

ôf

02

incub-

14 ation

using

C-NeuAc-CMP

and lactosylceramide

as

substrates


CELL SURFACE GLYCOLIPIDS

330

100

300

500 PROTEIN, pg

700

900

30 60 90 INCUBATION TIME, min

120

Figure 6. Effect of CMP-NeuAc concentration (V/S), of pH (V/pH), of enzymatic protein concentration (V/protein), and of incubation time (V/t) on the activity of synaptosomal membrane-bound sialyltransferase. Calf brain cortex. Acceptor substrates for sialyltransf erase: (jç) lactosylceramide; (f) desialylated fetuin; (φ) endogenous glycoprotein; (±) endogenous glycolipids.


ferase

has,

tosomal the of

Nerve-Ending

TETTAMANTI ET A L .

18.

as one of its sites

membranes .

biochemical

gangliosides

cycle

potentiality

is schematically

other

plasma

transmission. ization mes ion

capable

The

fact

transferase

A biochemical

of

fluctuations

ganglioside,

membranes sialidase support.

at

sialidase,

very

purified

cortex,

1/8-

branes,

were

since

reasonably

ratio

ring of

of c e l l s

in the nerve

to

ending and

has a consistent

has been

occurrence

of a number

of

not yet a s -

sialyltransferase

( 29, 3Q, 31 ) makes

organization of

membranes,

the specific in the brain

low range.

should

molar

the chemical

ratio,

non neural

of general

cells

value

concentration of different

as much

nature

of both

greater,

layer

l/4.

and

locaof the

This

discussed

molar

above.

phospholipids

the presence

This mem-

within a

asymmetrical

i.e.

ratio"

mat-

in p l a s -

gangliosides

varies

in the outer

can be accounted

of gangliosides

observed

of vertebrates.

animals

of the individual

membranes,

protein.

in this

for synaptosomal

Due to the ganglioside

of the "transition

mg of total

per mg

established

) the highest

phospholipid ratio

be twice

in synaptosoaml

quantities

prepared from rat

0.73-0.93

and 0.073-0. 125 mg of gangliosides

hydrophobic proteins,

tive

functionali-

of gangliosides

surface

shown ( J_6 ) to contain

is in the range

Likely

The location

membrane

synaptosomal

the ganglioside/

membrane

chains.

and s i a l y l -

probable.

be considered

phospholipids

tion

( 4. 0 ),

sialyltransferase 8.

is by far ( 5O-100 fold

might

hence the

picture of the location

of sialyltransferase

ma membranes obtained from figure

pHs

the optimal

A schematic

and

ganglioside/phospholipid -

ratio;

membranes

phospholipids

erial

correlate

the reported

surface

special-

of the saccharide

at acidic

from

neuro-

and in the enzy-

ganglioside

contribution to the supramolecular

synaptosoaml

Highly

acid /

may

plasma

However,

Ganglioside

tissue for enabling

the sialylation-desialylation cycle,

in Figure

The sidedness

assignement

The

pHs,

7.

in gangliosides

best

of pH value.

is shown

the external

brain

works

hence

in the outer

certained.

this

the sialic

11

This

membranes differentiate

and crosslinking capacity

at neutral

).

correlation to this functional

enrichment

sialidase

the synap-

sialylglycoconjugates

in F i g u r e

the synaptosomal

ty of these enzymes, local

location,

membranes feature

for a "sialylation-desialylation c y c l e

as of other

membranes of brain

to modify

that

of subcellular

depicted

is the striking

complexing

331

Thus the synaptosomal

( as well

In conclusion, the

Membranes

occur-

of cholesterol and

for enhancing the r e l a -

required for causing

a bilayer-


332

CELL SURFACE

Figure 7.

I

t

I

t

Sialylation-desialylation cycle of gangliosides

C2> sialyltransferase

Figure 8.

GLYCOLIPIDS

ο

Location of gangliosides, sialidase, and sialyltransferase at the nerveending membrane


18.

micellar

transition.

Gangliosides membrane is

mainly

presence with (

tend

),

ve.

the

ganglioside

crosslinking agents,

stay

Over

the

to

in equilibrium diffusing, critical

greater

Now,

as

being

(

recorded

respect

exceeded

in the

linking

agents.

any

Sharom signals

Therefore

sites, we

or

remind that

interactions affinity)

the

in

to

to

be

the

clustering

appropriate

viceversa.

of

stress

In other

displays

sible

phase transitions. (

Several cluster

the

are

the

thermo-

already

in the

quoted started

layer

is

of

Ca

serve

ions

as

point

ganglioside

of

view,

is

In this

interact

high

either

or

ganglioside

a defined

with

binding

location on the

which direct

In conclusion

clustering. is

that

non-covalent

and reform

See

as

clusters.

would occur

membrane. can

points

sites.

this

are

cross-

membranes appear

clusters

%

carbohydrate-carbohydrate

in addition, are

1.5

abundantly

ganglioside

the

or

survive.

( hydrophobically ),

focal

the

proteins

9

involved

is

easily

removed,

supramolecular

great flexibility

would

signi-

important

the stress

organiz-

and of

rever-

).

which can

carbohydrate

An

the forces

and could break

when

kind of of

Figure

consequences of

of

mob-

ions

these, to

Moreover

stable

sites

of

immobilization

a physiological

characteristics

organization

of

( carrying

as

size;

dissol-

and giving a possible functional

words

ation

in their

gangliosides

in given

in mind,

stable

which could

which have

serve

governing

process

kept

ganglioside

under or

may easily

gangliosides

molecules

ficance point

of

ganglio-

interactions,

concentration

mutual

Thus proteins

and glycoproteins,

membrane, packing

).

( by

presence

formed:

ganglioside

membrane embedded proteins

membrane glycoproteins

be

from

in preferential

should

are

synaptosomal

stable

of

a certain

and should

mutual

membranes.

the

essential of

in the

concentration

the formation

the formation

or

ganglioside

surface,

receptors

gangliosides,

process than

molecules,

size

This

In analogy

surrounding solution of

sufficiently

a ganglioside

for

the

of

on the

clusters

and on the

membrane

less

and Grant, of

synaptosomal

problem,

respect

critical

would be

membrane

candidates

One

with

at

,

,

is

which facilitate

to phospholipids.

on the

whether

by

1 _ 3 _ )

present

in

ganglioside

than the

reported

investigation

which

with the

such

cations.

concentration

concentration,

dynamically favoured,

ideal

a patch

crosslinking agents

clusters

with

"critical"

of

concentration

capping of

made.

distribution

The formation

crosslinking agents

leads

should be

their

like divalent

suggested for

absence of

laterally

other

even.

thus

dependent on

below a certain

cannot

:

be

been

333

a consideration

clusters

not

of

in the

Membranes

may

side clustering

ile,

However

to form

surface

what has

32

or

it

Nerve-Ending

TETTAMANTI ET A L .

be

carrying

expected from

molecules


the

in the

CELL SURFACE

334

membrane

surface.

have

indicated

been

First, as

carbohydrate portions ificity. not

The

of

mutual

perative

would cause easier pid

composition

the

rearrange tion,

in

this

bilayers

toxin,

(

33

of

exposed

of

the

the

as

the

F o r this

evidence

from

This

some

yielded as

both ly

sides.

similar

remainder cal

As

being

model

the

the

the l i -

ganglio-

reaching,

This

). An

by

of

of

or

to

organiza-

basis

for

indication

Tosteson

and

channels when were

above view ,

cholerae

leads

rearrangement

to the of

the

channels.

ganglioside

behavior in

of

of

great

ganglioside

model,

about

as

of

of

gangliosides much more

by

vesicles,

a study The

the

In fact

et

al.

(

gangliosides

inner

side.

Of

the

monolamellar

34j

mimic-

which

lipid

authors

layers ( J^) ,

liposomes gangliosides

are

on

on a substantialouter

course

sided,

the

an asymmetri-

layer

would

above purposes. phospholipid

prepared according

containing

on

sur-

exper-

gangliosides

liposome

the

model

model

quoted

carrying

on the outer

size,

of

help.

on the

precise

phospholipids and

suitable for

using

course,

immobil ization

used

limitations.

60%

distribution

of

availability

would be

and homogeneous

These

enabling

at

ganglioside

the

ganglioside

located on the

on this direction, small

of

determined by Cestaro

ked

V2 ).

till

agent for

study

dispersions

this

(

containing

multilamellar vesicles,

system,

location of

of

important

der

of

report

to

look

Here

coo-

glycocalyx

areas,

molecular

development

membranes needs,

membrane

were prepared from and

recent

the

the

hypothesis

liposome.

suffers

the

the

would be forced

Figure 10

According

for

modulating the

us

aggregation. be

see

a crosslinking

model

supports. cell

provided

(

in the

toxin.

synaptosomal

imental

may

lipids

spec-

might

membranes

cluster

of

proteins,

increase

The

G. clusters. The following MI would result in the formation

matrix

The

value.

let

a cluster.

would greatly

seen

by

surface

binding kinetics a

Finally

to

their

determining

oligosaccharide-free

describing

functioning

experimental

face

is

,

also,

the

ligands.

glycerolmonooleate,

synaptosomal

ing

)

surfaces,

patch organization of

polar channels

to cholerae

formation

An

of

and glycoproteins

cell

but,

to

a micellar kind of

sense can be

Tosteson

lipid

ratio

involving

the formation

of

apolar

transition

toward

likely

give

in correspondence

side/phospholipid

at

instruments for

binding

the

the formation with

sites

carbohydrate chains on

receptors

Second,

collision

exceeding,

of

interactions,

nature.

gangliosides

being the

aggregation

only facilitate

extent

both

receptor

GLYCOLIPIDS

We

renwor-

vesicles,

to Barenholtz

phosphatidylcholine (


carrying

TETTAMANTI ETA L .

Nerve-Ending

Membranes

335

GANGLIOSIDE GLYCOPROTEIN CLUSTER

GANGLIOSIDE CLUSTER

OUTER MEMBRANE LAYER

GLYCOPROTEIN

Figure 9.

GANGLIOSIDE

Formation of stable ganglioside clusters: role of proteins (hatched irregular circles) and of glycoproteins as focal points of clustering

GANGLIOSIDE CLUSTER

CHANNEL

Figure 10. Formation of a polar channel in correspondence of a ganglioside cluster. Note the presence of proteins and glycoproteins (hatched irregular circles).


CELL SURFACE

336

C

choline)

incubation

and phosphatidylethanolamine

in the

presence

tritium ι labelled The process cles

is

ionic

ganglioside

ganglioside

rate

is

as

G

with G

( .

see

equal

(

would be

0.6-0.7

cess of

μιηοίβε )

dependent

the

as

it

upon the

saccharide

than

chain.

This

transition

ratio

glioside

molecules

to vesicles

of

maintain at

the

by

ganglioside

The

to

the

of

phospholipid vesicles

at

37°C

sialidase

lerae

a

D1

the

m

c

e

activity.

,

,

e

»

s

which

is

found

34^)

values

this

substrate for As

incorporated

isolated

and

into

with

ganglio

the

acid

are

on G

incubated

cholerae )

Vibrio

hfgher the

is

after

submitted

than on

present

the

action

of

in

the

ganglio

vesicle

rate of

when phospholipid

incubation a certain

to

Cho

neuraminidase

therefore

proof,

is

expression

yielding a mixed

enzyme;

matrix

-phospholipid

record of

this

loss

mixtures

( NeuAc

Since

likely

lipid

with phospholipid vesicles

the

in and

observed.

when

Vibrio

ganglioside

a further

gan

the

hours,

was

micelles

are

into vesicles,

increases.

alter

lower

of

significant

In fact,

than 50-fold

all

to a

much

with phospholipid v e s i

kinetics.

lowest

interaction

inserted

which

Initially

no

molecules

display

more

is

association

vesicles

treated

to

pro

quality

corresponds

vesicles,

kinetics

yielding the

allowing

a better

.were

(

the

phosphatidylethanolamine

process.

sigmoidal

a

release

vesicles,

a

not

stable for

N-acetylneuraminic

the™ gmoidal

form, By

then

a max reached

incubation with cold

and ganglioside

phenomenon.

become

NeuAc

of

amount of

Vibrio

Cholerae

a

sialidase G

of

liposomes V '

micellar

G^^ JD1

was

a fusion

incorpora

saturation

significantly

the

0.5-

with time

cases,

which

The

micelles

ganglioside

times,

release

following

sides

by

which follows

sialidase

mixed G

likely

different

,

recorded,

the

a

level

sided

tem 0.9

and from

The

in all

0.07,

remain

upon

ganglioside

of

for

not

radioactivity from

insertion of

vesicle,

outer

of

).

moiety,

above.

these

separation

of

).

incorporation

incorporation was

lipid

ratio

does

Moreover,

interaction of

lead

since

11

expected for

discussed

proportion of

value.

followed

the

cles

vesicles,

%

a constant

sides, of

the

of

saturation

molar

the

tegrity

level

)

the

proportionately

Figure

ganglioside

ganglioside/phospholipid

),

Interestingly,

imum and approximately

( 35

starting from

vesicles

G^.

proceed

) upon

concentration,

instance,

,

mol

(containing

gangliosides

ganglioside

For

by

into phospholipid v e s i

monolamellar

concentration

highest

incorporate

pH,

(

into vesicles

ganglioside

tion

strength,

phospholipid (

μηιοίβε of

and

do

( 95/5,

micelles

incorporation

dependent phenomenon.

μη-ιοίβε of

of

ganglioside

gangliosides)

ganglioside

a time,

perature

2

of

of

GLYCOLIPIDS

the

kinetics

are

hyperbolic,

-phospholipid vesicles

prepared

the by

same exhibited

sonication.

This


by

mixed

means

TETTAMANTI ET A L .

Nerve-Ending

Membranes

337

Figure 11. Effect of incubation time (at 37°C) and of ganglioside concentration on the incorporation of gangliosides (G , G , G ib) into phosphatidylcholine monolamellar vesicles. Phosphatidylcholine (as vesicles): 9 μmol. Ganglioside: from 0.5 to 2 μ/nol. After incubation the mixtures were passed through a 1 X 20 cm Sepharose 4B column to separate vesicles from ganglioside micelles. M1

Dla

T


CELL SURFACE

338

Figure 12.

GLYCOLIPIDS

Time course of NeuAc release from liposome-associated ganglioside G by the action of Vibrio cholerae sialidase Dla

Incubations done at 37°C in 0.05M Tris-HCl buffer, pH 6.8, with 1 IU of enzyme (Behringwerke). Released NeuAc determined by method of Warren (37); available amino groups (carried by phosphatidylethanolamine) by TNBS method (12). Arrow indicates addition of detergent (Triton X-100, 0.5%). Ganglioside pattern during enzyme hydrolysis was monitored by TLC (silica gel plates; solvent: chloroform/methanol/ 0.3% aqueous CaCl , 60/35/8, by vol, 2-hr run; spots detected by spraying with Ehrlich's reagent and heating at 110°C for 10 min). 2

(A) :

liposomes containing phosphatidylcholine, phosphatidylethanolamine, (90/3/7, by mol) and prepared by the sonication method (12)

and G

D l a

(B) : liposomes containing phosphatidylcholine and phosphatidlyethanolamine (90/5, by mol), prepared by sonication, were incubated in 0.05M Tris-HCl buffer (pH 6.8) with G micelles for 1 hr, then separated by Sepharose 4B column chromatography. These liposomes contained about 5% (by mol) of incorporated ganglioside. D l a


TETTAMANTI ET A L .

Figure 13.

Nerve-Ending

Membranes

339

Time course of oxidation of the terminal galactose residue of liposomeassociated ganglioside G by the action of galactose oxidase M1

Incubations done in 0.05M Tris-HCl buffer (pH 6.8) at 37°C, with 1 IU of enzyme (Kabi). Oxidation was followed by the coupled o-anisidine peroxidase procedure. Formation of oxidized G was also monitored by thin-layer chromatography, under the conditions described in Figure 12. Note that oxidized G could be reduced to the starting G by NaBH treatment. All other conditions as described in Figure 12. Mt

M1

h


Mi

CELL SURFACE

340

that

in both vesicle

tion )

the

insertion

bohydrate chains As

shown

troduced tion

of

vior

about ter

60

%

sided

the

by

which

aminogroups lipids,

the

their

in the

G

à

J

as

available

integrity

Of

as

)

becomes

available

second

results,

(

by

species

this

the

of

sialidase

showing

containing

to

are

terminal

13

).

All

introduced

The

mined

by

insertion

as

outer

layer

the of

carried

enzyme

the

in which

to

of

measure-

the

0.5

%

by

behavior of

inner

is

100

G

which

Triton

split

of

the

sided off.

gan-

Con-

%

of

releasable

addition

of

Triton

à

was

i

X-100.

were obtained

monitored

as

the

oxidation (

in which

ganglioside

An

initial

see

gangliosides units only in

latter

layer,

the

a certain

the vesicle

structure.

prevent

by

entry the

micelles,

this

leads

of

of

other

mimicking

(

suggested

followed lipid

units

deter-

by

been

resulting hand the

the

stable

matrix of

diffusion

into the

lipid

on

inside

requirements for

across

the

layer.

incorporated into

are

being formed

with-

in a stabilization of acquired

and incorporation

a saturation

is

mainly

ganglioside

weak bonds

this

adhesion

has

is

into the

energetic groups

ganglioside

On the

to

carrying

side,

adhesion,

ganglioside

high

and other

further

or

moieties

polar head

amount

hydrogen

lipid

process

the

large

contact,

vesicles,

outer

carbohydrate chains,

ganglioside

This

monolayer

located on the

the oligosaccharide chains,

side

of

bevavior,

liposomes

phospholipid

being prevented

charge

TNES

galactose-oxidase

absorption c a r r y

ganglioside

lipid

the vesicle, in

( the o u -

treatment

aminogroups,

and

liposomes

differential

means that

asymmetrically

the

movement of After

by

follows.

the v e s i c l e . the

galactose

this

formation

occur

by

that only

layer.

gangliosides to

releasable

enzyme

by

ac-

beha-

JVl 1

available

the outer

of

sialidase

shown

IV1 1 Figure

%

micellar dispersions

before

liposomes,

60

in-

the

differential

on addition

the one

car-

^ was a

the

indicated

( as

and forms

absorpthe

submitted to

expected,

to

during

course,

liposome

TNBS

released

4

is

remainder NeuAc

versely,

Identical

about

meaning,

Noteworthy,

destroys

with

)

is

only

by

leads

in which G are

the following

case

sialidase,

layer

surface.

sided phosphatidylethanolamine

gliosides

NeuAc

layer

liposomes

sialidase

).

sonication or lipid

absorption,

ganglioside

maintain

outer

by

In the first

split

of

when

or

unchanged r e c o r d s .

X-100,

with

12

Cholerae

ganglioside

liposomes ments of give

in Figure

is

in the

to protrude on the

observed.

acid

( prepared by

gangliosides

sonication,

Vibrio

is

sialic

by

species of

GLYCOLIPIDS

)

surface of

process.


ganglio-

18.

Nerve-Ending

TETTAMANTi E T A L .

Membranes

341

Conclusion

The of

studies on the

gangliosides

these on

the

recent

years,

involvement

portant

to

expect

in

different

ligands

fields

-

gangliosides

near

out

the

by

developed.

future,

interactions played

by

-

of

at

will

of

biologically and

it

is

reasonable

progress

membrane

provide enough

gangliosides

im-

adequate

gangliosides

the

in

increasing evidences

Thus

integrated

and behavior

more frequent

sophisticated

physico-chemistry

role

the

in a number

more

enzyme events occurring

-glycocalyx

to figure

stimulated

Moreover

in the

properties

membranes became

models have been

that,

membranes;

surely

of

phenomena.

experimental

physico-chemical

in artificial

of in

research artificial

surface; information

in synaptosomal

mem-

branes.

Symbols

The was

used

ganglioside

nomenclature

proposed

by

Svennerholm

(

36

)

fol lowed.

Acknowledgements

The

experimental

supported

by

(

),

C.N.R.

grants

data

from

reported

in this

paper

the Consiglio Nazionale

pertain

to

work

del le Ricerche

Italy.

Literature cited 1. Wiegandt, H. Advances Lip.Res., 1971, 9, 249. 2. Sillerud, L.O.;Prestegard,J.H.;Yu,R. K.;Schafer, D.E.; and Konigsberg, W.H. Biochemistry, 1978,17,2619. 3. Howard, R.E.;and BurtonR.M.Biochim.biophys.Acta, 1964, 84, 435. 4. Curatolo W.;Small D.W.;Shipley G.G. Biochim.Biophys.Acta 1977, 468, 11. 5.Gammack,D.B. Biochem. J., 1963, 88, 373. 6. Rauvala, H. FEBS Lett.., 1976, 65, 229. 7. Yohe, H. C.; and Rosenberg, A. Chem. Phys. Lipids, 1972, 9, 279. 8. Yohe H. C.; Roark, D.E.; and Rosenberg, A. J. Biol. Chem. 1976, 251, 7083. 9. Schwarzmann, G.; Mraz, W.; Sattler, J.; Schindler, R.; and Wiegandt, H. Hoppe-Seyler's Z.Physiol. Chem.1978, 359, 1277


342

ti,

cell surface glycolipids

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