Immunological identification of the human erythrocyte monosaccharide transporter

Vol.

94, No.

June

30,

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4, 1980

AND

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RESEARCH

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Pages

1980

1401-1408

IMMUNOLOGICAL IDENTIFICATION OF THE HUMAN ERYTHROCYTE MONOSACCHARIDE TRANSPORTER Stephen

A. Baldwin

and Gustav

E. Lienhard

Department of Biochemistry Dartmouth Medical School Hanover, New Hampshire 03755 Received

May

13,198O

to the purified cytochalasin B binding component of the SUMMARY. Antibodies They human erythrocyte glucose transporter were prepared in rabbits. and partially inhibited its precipitated detergent-solubilized transporter, were used to locate the transporter binding of cytochalasin B. The antibodies polypeptide in SDS-polyacrylamide gels of erythrocyte membranes prepared from freshly drawn blood in the presence of protease inhibitors. They labelled only the region of the gel corresponding to that occupied by the purified transporter, with an apparent molecular weight range of 45,000-75,000. These findings indicate that the isolated transporter does not arise by proteolytic either during the storage of blood or degradation of a larger polypeptide, during purification of the transporter. -INTRODUCTION Several putative

groups,

monosaccharide

The purified

with when

catalyzes

the

Lienhard,

unpublished

glycosylated

into

stereospecific

SDS-polyacrylamide

electrophoretic

characteristics

transport

system

membrane

(6,7).

maltosyl

isothiocyanate,

a 100,000 that

dalton

the purified

However,

55,000

Mullins

a reagent

that

identified

transporter

1401

and G.E.

with

weight these

as a component

glucose

a proteolytic

of

erythrocyte

have reported

membrane is

molecular

of the

inactivates

In

heterogeneously

apparent

and Langdon

of the erythrocyte dalton

is

(2,4). it

Baldwin

A protein

radiolabelling

recently

polypeptide

which

been

vesicle, 3.M.

(1,2,3).

inhibitor

amount

(1,

electrophoresis.

has also

by differential

reversible

of a phospholipid of D-glucose

of a

membrane

stoichiometric

band of average

gel

isolation

B, a potent

The protein,

as a broad

upon

the

the bilayer transport

the

the human erythrocyte

and in near

results). runs

reported

cytochalasin

affinity

inserted

(5),

own, have from

binds

high

addition,

our

transporter

glycoprotein

of transport,

55,000

including

transport, (8,9).

that labels

They propose fragment

of the

Vol.

94, No.

native

4, 1980

transporter

upon storage

(9).

antibodies

to the

only

membranes inhibitors.

produced

of blood

detergents

bind

BIOCHEMICAL

AND

by the action

or during

to investigate

isolated

transporter.

prepared

of from

We conclude

proteolytic

fragment

MATERIALS

AND METHODS

molecular

weight

that

the purified

of a larger

proteases,

possibility,

We report

drawn

COMMUNICATIONS

of the membrane this

freshly

RESEARCH

of endogenous

solubilization

In order

to proteins

BIOPHYSICAL

blood

55,000

here in

in the transporter

either in nonionic

we have prepared that

the antibodies

SDS gels

of erythrocyte

presence is

of protease probably

not

a

polypeptide.

Freund's complete and incomplete adjuvants were obtained from Difco Laboratories. [ '251]-protein A (80-90 pCi/ug) was purchased from New England Nuclear. Protein A-Sepharose (C1)4B was from Pharmacia. Nikko1 (octaethylene glycol dodecyl ether) was supplied by Nikko Chemicals Ltd., Tokyo. Antiserum against glucose transporter that had been purified in Nikko1 and reconstituted in dioleoyl phosphatidylcholine (5) was raised in a New Zealand White rabbit. The transporter (250 ug) in 1.1 ml 10 mM sodium phosphate/l45 mM NaCl, pH 7.2 (phosphate-buffered saline) was emulsified with an equal volume of complete Freund's adjuvant and then injected subcutaneously along the back. Additional injections of antigen in incomplete Freund's adjuvant were made after 4 and 6 weeks. The rabbit was bled at intervals during a period of 2 weeks after the last injection, and the resultant antiserum was stored at -70°C. Control serum was obtained from the same rabbit prior to the first injection. IgG was isolated from sera by chromatography on protein A-Sepharose (C1)4B as described by Goding (10). Erythrocyte membranes were prepared from outdated units of blood, or from blood freshly drawn into citrate, by the method of Steck and Kant (11). Protein-depleted membranes were prepared from them by alkaline extraction, as previously described (2). Erythrocyte membranes were also prepared from freshly drawn blood by the method of Bennett and Stenbuck (12), in which proteolysis is minimized through removal of protease-rich white cells by gravity sedimentation, lysis of the red cells in the presence of protease inhibitors, and washing of the membranes in the presence of these inhibitors. The membranes were immediately prepared for electrophoresis as described below. Samples SDS gel electrophoresis was performed in 10% microslab gels (13). prepared in 2.5% SDS/O.8 mM EDTAi54 mM dithiothreitolf42 mM TrisCl, pH 6.8. Immediately after addition of the SDS, they were held at 100°C for 5 minutes, and then made 6% in sucrose. Gels were stained by the method of Antigens were localized on gels after the methods of Steck and Yu (14). Fixed, washed, unstained gels were Burridge (15) and of Adair et al. (16). They equilibrated in 50 mM TrisC1/150 mM NaCl/O.l% NaN3, pH 7.5 (buffer A). were then incubated for 24 h in 6 ml of buffer A containing 1 mg/ml gelatin After two days of washing with and 120 ul of antiserum or control serum. buffer A, they were incubated for 24 h in 6 ml buffer A containing 1 mg/ml a further two days of gelatin and l-2 uCi of iodinated protein A. After washing, the gel slices were dried down and autoradiographed for 2-7 days on Cronex 2: X-ray film (DuPont).

were

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AND

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protein-depleted erythrocyte For immunoprecipitation experiments, in 0.5% Nikkol. membranes (2 mg/ml) in 50 mM TrisCl, pH 7.4, were solubilized Insoluble material was removed by centrifugation at 130,000 x g for 1 hour. Incubation of the detergent extract with antibody was carried out for 1 hour at room temperature, in the presence of 2% polyethylene glycol 6000 (17). Immunoprecipitates were collected by centrifugation at 27,000 x g for 10 minutes, and then washed in buffer several times before being assayed for protein by the method of Peterson (18). In some experiments membranes were reconstituted in the supernatant from immunoprecipitation by the addition of phospholipid and removal of detergent in order that cytochalasin B binding activity could be measured (5). RESULTS The rabbit

antiserum

purified

transporter

precipitin

line

not

react

form

with

of

the

detergents

An

experiments, minor

which

to give

pre-immune

both

serum

evident

corresponded

to aggregate

with

the

same intense

especially

probably

is known

the

whereas

band,

transporter,

reacted

in to

partially

did

an

oligomeric

in nonionic

(5).

to ascertain

quantitative

precipitin

of purified

antibody

membranes

(Fig.

the antigen-binding curves

were

different

2).

The antibody

of protein

precipitation

of transporter-associated

experiments,

was used

purified

capacity

constructed

with

precipitation

these

IgG fraction

membranes

additional,

the purified

transporter,

In order

cyte

1).

purified

and erythrocyte

in immunodiffusion

(Fig.

reactions

and its

amounts

by

of the antiserum,

incubating

a fixed

of detergent-solubilized

to antigen

in experiments

ratio designed

cytochalasin

IgG was used because

giving

erythromaximal

to examine

B binding unfractionated

amount

the

activity. serum binds

B

A

Figure 1. Immunodiffusion reaction of the erythrocyte glucose transporter with rabbit IgG. The agar Ouchterlony plates were equilibrated with phosphate-buffered saline, 0.1% Nikkol. Wells (A) and (B) contained 74 pg rabbit anti-transporter IgG and rabbit pre-immune IgG, respectively. Wells (1) and (2) contained samples (17 up) of protein-depleted and of normal erythrocyte membranes, respectively; the membrane samples were dissolved in 2% Nikko1 and then centrifuged to remove insoluble material before use. Well (3) contained 2 ug purified glucose transporter dissolved in 2% Nikkol. After incubation for 48 hours at 22"C, the plates were extensively washed and then stained with Coomassie Blue.

1403

In

Vol.

94, No.

4, 1980

BIOCHEMICAL

AND

Membrane

Protein

Added

Figure 2. Immunoprecipitation of protein membranes. Samples (0.48 mg) of rabbit anti-transporter IgG (0) were incubated solubilized protein-depleted erythrocyte protein that precipitated was measured.

cytochalasin the

B.

solubilized

tion,

control

Antibody

so the antibody linear

greater

with

than

transporter quantity

of complete

cytochalasin

completely

as measured

partially

inhibited

Gorga,

sides.

capable

precipitated

after

reconstitu-

the binding

of

membranes. personal

The extent

B binding

communication), of inhibition

and an amount precipitation

These

was less

of IgG ?.5-fold

of the

by only

and

solubilized

40% in an equivalent

of membrane.

In experiments

designed

was used to identify active

ligand

55,000

on microslab

labelling

to both

concentration,

inhibited

almost

activity,

(F.R.

antibody

that

from solubilized erythrocyte pre-immune IgG (0) and of rabbit with various amounts of detergentmembranes, and the amount of See text for details.

in protein-depleted

to be unsealed

has access

COMMUNICATIONS

none of the activity.

also

transporter

RESEARCH

(fig)

antibody

IgG precipitated

B to the are known

the

B binding

to the transporter

cytochalasin

than

1 shows that

cytochalasin

whereas

membranes

Table

BIOPHYSICAL

bound

labelled

closely

to locate rabbit

a broad gels

of the

corresponded

antigen

on SDS gels,

anti-transporter zone of average purified to the

antibodies. apparent

transporter typical

1404

[1251]-protein The radio-

molecular (Fig.

Coomassie

Blue

A

3).

weight The pattern

staining

profile

of

Vol.

94, No.

4, 1980

BIOCHEMICAL

AND

TABLE Immunoprecipitation from

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

1

of Cytochalasin Detergent-solubilized

B Binding Membranes

Activity

Rabbit IgG (4.8 mg) was incubated with 80 pg detergent-solubilized, Experimental details are protein-depleted erythrocyte membranes. After immunoprecipitation, the proteins that given in the text. remained in the supernatant were reconstituted into membranous form by removal of the detergent, and then glucose transporter-specific cytochalasin 3 binding was,measured with 4 x 1O-8 M cytochalasin B (CB), according to the method previously described (23). The slightly higher recoveries of activity obtained in the presence of control relative to the case of no addition, probably resulted from a I&, stabilizing effect of the protein on the solubilized transporter. CB Binding Activitya in Supernatant after Immunoprecipitation

Additions

None

% CB Binding Activity Precipitated

the

1.72

Pre-immune

1.88

IgG

Anti-transporter

IgG

'expressed

as

0

[Bound

0.14

CB]/[Free

CB]

92

(23)

T-9 ?f

95K72K-

iii T;

L3K35K-

A

B

35K-

C

D

E

I=

G

H

Figure 3. Antibody labelling of SDS gels of purified transporter (A,B, 0.3 ug protein), of erythrocyte membranes prepared from outdated blood (C,D,E, 4.5 ug protein), and of erythrocyte membranes prepared from freshly drawn blood in the presence of protease inhibitors (F,G,H, 4.5 ug protein). Gels C and F were stained with Coomassie Blue; the molecular weights of some of the major protein bands are given in kilodaltons (22). Gels A, D, and G were incubated with rabbit anti-transporter serum followed by [125 II-protein A. The labelled bands were detected by autoradiography. Gels B, E, and H were treated in a similar fashion, but pre-immune rabbit serum was used. The presence of some radioactivity at the very top of gels incubated with either pre-immune serum or anti-transporter serum probakt\S resulted largely from the nonspecific trapping of immunoglobin and [ ' II-protein A in fragments of the stacking gel adhering to the separating gel.

1405

Vol.

94, No.

4, 1980

of tube could

gels

BIOCHEMICAL

of the purified

be applied

Samples amount

of transporter,

gels.

Whether drawn

gave rise

blood

purified

nor

of other

the

antiserum method

in its

staining

from

corresponding

drawn

inhibitors, closely

and intensity

(Fig.

with

the same

blood,

or from

these

samples

resembled

that

3).

to a molecular

incubated

pattern).

on microslab

freshly

that

that

approximately

to electrophoresis

labelling

Gels

of transporter

a faint

of protease

of the gel.

COMMUNICATIONS

There weight

all of the

was little of 100,000,

pre-immune

serum

showed

out with

mouse

3).

experiments

against

the

presented

described purified

of Tung --et al.

to those

amount

to contain

blood,

position

of the region

(Fig.

All

outdated

of antibody

regions

no labelling

subjected

also

RESEARCH

(the

gave only

were

in the presence

transporter

or no labelling

gels

calculated

from

to a pattern

(2)

membrane,

prepared

BIOPHYSICAL

transporter

to the microslab

of erythrocyte

freshly

AND

glucose

(19).

above

here

for

have also

been

transporter,

The results

obtained

the rabbit

antibodies.

carried

raised were

in mice

essentially

by the identical

DISCUSSION The almost from it

detergent

contains

extracts

erythrocyte

SDS gels

membranes,

Membranes

the

to that prepared

proteolytic

from

purified

55,000

dalton

It

is

erythrocytes

with

a 100,000 impermeant

under

is

a single 55,000

conditions

It

our dalton

is

results membrane

reagent

protein

1406

broad

those

protein

region

transporter. to minimize

unlikely arising

of higher

with

maltosyl

or unfractionated

of labelling

product

that

Furthermore,

dalton

therefore

activity

IgG shows

designed

a degradation

on a membrane

rabbit

transporter only

blood.

to reconcile

the

labels

B binding

transporter.

to the same pattern

outdated

proteases

difficult

9 who labelled

rise

transporter

of endogenous

the glucose

by the isolated blood

by the

purified

antiserum

fresh

from

membranes

of either

give

prepared

of cytochalasin

against

occupied

degradation

by membranes

action

directed

with

corresponding

precipitation of erythrocyte

antibodies

when incubated

(8,9)

quantitative

given

that

the

from

molecular

of Mullins

by incubation

isothiocyanate,

as is

the weight

and Langdon of intact a putative

Vol.

94, No.

affinity

BIOCHEMICAL

4, 1980

label

X-100,

the

for

the

labelled

presumably

action

species

was suggested

results

show that

that

Mullins

was partially

of endogenous to correspond

this

in the

protected

the transporter

from

cyanate.

We suggest

in

may have which

been

component it

presence

incorporated

of the

has been

55,000

dalton

externally

shown that range

the

considerable

labelling

their

of 100,000,

the

transporter

this

by endogenous

weight Our

is worth

noting

B, that

dalton largely

isothio-

amount anion

protein

membrane gives

species,

molecular

by maltosyl

erythrocyte

because

dalton

of the 100,000

a considerable

erythrocyte

upon proteolysis

It

inactivation

into

in Triton

transporter.

as cytochalasin

experiments

exposed

lower

glucose

incorrect.

irreversible

anion

This

probably

fortuitously

weight

to a 55,000

isolated

such

COMMUNICATIONS

of the membrane

proteases.

of substances,

that

has a molecular

is

observed

component

RESEARCH

converted

to the

hypothesis

and Langdon

BIOPHYSICAL

Upon dissolution

transporter.

component

by the

AND

of label transporter,

is a major

proteins.

Moreover,

rise

to fragments

(20)

and added

in the (21)

proteases.

ACKNOWLEDGEMENTS We are logical

deeply

methods.

Institutes

indebted Supported

to Dr.

Stanley

by grants

Froehner

GM 22996

for

his

and AM 25336

advice from

on immunothe National

of Health.

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

8. 9. 10. 11. 12. 13. 14. 15.

Kasahara, M., and Hinkle, P.C. (1977) J. Biol. Chem. 252, 7384-7390. Baldwin, S.A., Baldwin, J.M., Gorga, F.R., and Lienhard, G.E. (1979) Biochim. Biophys. Acta 552, 183-188. Kahlenberg, A., and Zala, C.A. (1977) J. Supramol. Struct. 7, 287-300. Sogin, D.C., and Hinkle, P.C. (1978) J. Supramol. Struct. 8, 447-453, Gorga, F.R., Baldwin, S.A., and Lienhard, G.E. (1979) Biochem. Biophys. Res. Commun. 91, 955-961. Batt, E.R., Abbot, R.E., and Schachter, D. (1976) J. Biol. Chem. 251, 7184-7190. Lienhard, G.E., Gorga, F.R., Orasky, J.E., Jr., and Zoccoli, M.A. (1977) Biochemistry 16, 4921-4926. Mullins, R.E., and Langdon, R.G. (1980) Biochemistry 19, 1199-1205. Mullins, R.E., and Langdon, R.G. (1980) Biochemistry 19, 1205-1212. Goding, J.W. (1978) J. Immunol. Methods 20, 241-253. Steck, T.L., and Kant, J.A. (1974) Methods Enzymol. 31, 172-180. Bennett, V., and Stenbuck, P.J. (1980) J. Biol. Chem. 255, 2540-2548. Matsudaira, P.T., and Burgess, D.R. (1978) Anal. Biochem. 87, 386-396. Steck, T.L., and Yu, J. (1973) J. Supramol. Struct. 1, 220-232. Burridge, K. (1978) Methods Enzymol. 50, 54-64.

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Adair, W.S., Jurivich, D., and Goodenough, U.W. (1978) J. Cell Biol. 79, 281-285. Raney, J.P., and McLennan, B.D. (1979) J. Immunol. Methods 29, 65-70. Peterson, G.L. (1977) Anal. Biochem. 83, 346-356. A. (1976) J. Immunol. 116, Tung, A.S., Ju, S.-T., Sato, S., and Nisonoff, 676-681. Tarone, G., Hamaski, N., Fukuda, M., and Marchesi, V.T. (1979) J. Membr. Biol. 48, 1-12. Steck, T.L., Ramos, B., and Strapazon, E. (1976) Biochemistry 15, 11541161. Steck, T.L. (1974) J. Cell Biol. 62, l-19. Zoccoli, M.A., Baldwin, S.A., and Lienhard, G.E. (1978) .I. Biol. Chem. 253, 6923-6930.

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