Solubilization of membrane H-2 isoantigens: Chromatographic separation of· specificities determined by a single H-2 genotype

Vol. 29, No. 6, 1967

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

SOLUBICIZATION

OF MEMBRANE -H-2 ISOANTIGENS:

OF SPECIFICITIES Akira

CHROMATOGRAPHIC SEPARATION

DETERMINED BY A SINGLE -H-2 GENOTYPE

Shimada

and Stanley

G. Nathenson

Department of Microbiology and Iaxaunology Albert Einstein College of Medicine, Bronx, N.Y.

Received

November

Membrane (H-2)

locus

located

isoantigens

and at least

rejection five

been detected

further

(Nathenson

moreover,

separated

from

by the

MATERIALS

AND METHODS.

Dr.

Frank

BALB/C, cells

latter

were

(H-2d);

H-2H,

were

maintained

other

found

to have

1967).

of 50,000

evidence

that

differs

specificities

in a

the general

in the range

specificity

one

in

determined

by

can be physically

on Sephadex

determined

have

isoantigens

by the H-2d allele

by chromatography

by the H-2b

G-150.

Products

locus

can also

carbe

technique. Mice:

Bar Harbor,

Lilly,

is complex,

We now present

antigenic

specificities

The -H-2 locus

the H-2

weights

1967).

several

the ~-2.31

separated

Labs,

molecular

are

involved

1967 and Shreffler,

releases

they

specificities

specificities

Snell,

(cf.

determined

from

the others

these

by papain

and Shimada,

that

two antigenic

controlling

with

properties

transplants.

purified

(H-2.31)

by the Histocompatibility-2

of antigenic

studies

of glycoproteins

solubilization

Jackson

regions

membranes

and when

of the specificities

rying

of tissue

ordered

of cell

form,

composition

-,H-2d*

an array

by recombination

Digestion

to 70,000

determined

in the mouse carry

in immunological

soluble

15, 1967

The following

Maine,

used: (H-2h);

inbred

or obtained

B.lO.A

(H-2a);

ARR, BlO.BR

in BALE/C mice

from

&.

828

purchased

the breeding

C57BL/10,

and E.L.

mice.

mice,

c57BL/6,

colonies

of

(H-2b);

DBA/2,

Meth A (H-2d) 4 (H-2b)

tumor

from

cells

ascites

tumor

in C57BLf6

Vol. 29, No. 6,1967

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Isoantisera described of

and test

previously

(Nathenson

immune cytolysis

determined H-2.31:

system:

Isoantigenic and Davies,

(Sanderson,

1964,

by use of the following BIO.A

H-2.3,8:

anti-Meth

C57BL/lO

H-2.4,10,13: H-2.5:

BlO.D2

H-2.2:

(BALB/C

H-2.6,14,27,28,29: Membrane

A serum

anti-Meth

anti-E.L.4

tested

were

on BlO.BR

0

I TIME

as described

2

cells;

tested

cells;

on Bl0.A

target

cells;

cells;

on H-2H target

serum tested

were

cells.

target

target

as

of inhibition

The specificities

target

on BlO.BR

serum tested

prepared

by the method

and test

A serum

anti-C5-7BL/lO

was detected

1965).

on BlO.D2

anti-Meth

x C3H)F1 anti-E.L.4

fractions

Wigzell,

tested

serum tested

BlO.BR

1966)

isoantisera

A serum

(ARR x C57BL/6)F1

activity

on BlO.D2 (Nathenson,

cells. target

cells.

1967).

3

(HRSJ

Fipure 1. Time course of papain solubilization of H-2d specificities. Crude membranes from DBA/2 spleen cells (upper fig.) and Meth A tumor cells (lower fig.) were suspended at 10 mg/ml in Tris-Cl O.OSM, pH 8.4, with papain at 0.5 mgfml and cysteine 0.06 mg/ml and incubated at 37O for times indicated. The reaction was stopped with neutralized iodoacetic acid (final concentration O.OlM) and membranes and supernatant fluids separated by centrifugation at 144,000 x g for one hour and then assayed. For both DBA/2 and Meth A preparations, membrane isoantigen activity is plotted in the set of curves starting at 100% and supernatant activity is plotted in the set of curves H-2.3,8 o----O ; H-2.4,10,13 ; and starting at 0%. Specificities H-2.31.----* were measured. The results are average determinations from two different membrane preparations of each source and three separate time course studies. 829

Vol. 29, No. 6, 1967

RESULTS.

Figure

specificities tumor

the sets

1 presents

from DBA/2

membranes

of either

BIOCHEMICAL

type

(lower

spleen

half

H-2.31

is

DBAf2 membrane three

sets

from

solution

course

membranes

of Fig.

of release (upper

1).

part

The data

very

H-2.3,8

higher

Ammonium sulfate

time

solubilized

of specificities,

in considerably

(Fig.

the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

slowly

of different of Fig.

show that and in

and H-2.4,10,13,

antigenic

1) and Meth A from membranes

low amount,

are

released

whereas

rapidly

and

yield. fractionation

preparations

of specificities

showed

that

was soluble

at 75% saturation.

2) of the redissolved,

of the solubilized

95% of the activity at 50% saturation,

Chromatography dialyzed

TUBE

-H-2 isoantigens for but

on a Sephadex

50 to 75% ammonium sulfate

from

each of the precipitated G-150

column

precipitate

NO.

Figure 2. Chromatographic separation of specificities of H& isoantigens. A sample of H-2d isoantigen was solubilized by incubating USA/2 cell membranes from 1000 sp=s at 20 mg/ml, with crude papain (Sigma Chemical Co.) 6 mg/ml, and cysteine, 0.3 mg/ml in Tris buffer O.O5M, pH 8.4 at 3i'O for 2 hours. Iodoacetic acid at a final concentration O.OlM was added to stop the reaction, and supernatant recovered by centrifugation at 144,000 x g for one hour. The 50-75% saturated ammonium sulfate fraction, after dialysis against O.OlM Tris-Cl, pH 8.4, was placed on a Sephadex G-150 column (95 cm x 4 cm) equilibrated and eluted with O.OlM Tris-Cl, 0.15M NaCl buffer pH 8.4 at 4'C, and 22.5 ml samples collected. V, was 382 ml (tube 17). Absorbance at 280 millimicrons ; and the quantity of H-2 specificities H-2.3,8 W and H-2.31 O- ----• were determined on each tube.

830

Vol. 29, No. 6, 1967

showed

that

the isoantigen

and H-2.4,10,13 at tubes H-2.31,

BIOCHEMICAL

(not

however, of units

103% for

H-2.4,10,13

ity

~-2.5

membranes G-150

showed

pattern

for

of C57/BL6 column

a single

(Fig. sharp

were

spleen 3).

other

95% for

at tube

findings

membranes.

isoantigenic

(H-2b)

was chromatographed

cells

The activity

profile

28, whereas

32.

H-2.3.8;

chromatographic cell

a peak

carrying

fractionated

peak at tube

Fipure 3. Chromatographic separation A sample of H-2b isoantigen prepared H-2d preparation of Figure 2, except Absorbance280 ; H-2.5 -

with

species

27 and the

from Meth A tumor

TUBE

' The set of specificities specificities determined and 29.

the molecular

Identical

ammonium sulfate

H-2.3,8

to each other

to the column

H-2.31.

preparations

of specificities

identical

one at tube

applied

and 93% for with

the sets

almost

two peaks,

solubilized,

same Sephadex

were

of activity

have been obtained

from cell

for

The elution

revealed

Recovery

Papain

profiles

plotted)

28 and 29.i

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

for

material on the

the specific-

specificity

H-2.2

NO.

of specificities of H-2b isoantigens. and chromatographed as described for the membranes from 2000 spleens were used. ; H-2.2 O-----m .

H-2.6,14,27,28,29, by H-2, d likewise 831

as a measure of the showed a single peak

remaining at tube 28

Vol. 29, No. 6, 1967

peaked

at tube

H-2.31.

27 and 32 to give

Specificity

eluted

were

BIOCHEMICAL

in a pattern

mice homozygous

with

solubilized

arates

molecules

molecules Since

and H-2.4,10,13 conclude

(H_zd)

without

specificities expressed

uniquely

molecule.

This

techniques,

were

homozygous

further protein, molecular further order

weight chemical

to precisely

-H-2 specificity single

It

specificity The present

others

coexist

bf hybrid

sepfrom

the

both

respect

the

(Shimada

studies

will

make this

findings

bear

H-2.5

sugar

with

those

which from

nature

and H-2.3,8

he

mice

have been

composed

and 2-3% sialic

of about acid,

in preparation).

of the purified

products

of the antigenic

The separation task

are

on another

findings

preparations

and Nathenson,

on the glycoprotein.

these

in solubilization

to be glycoproteins

the chemical

carrying

to the -H-2 allele.

5% amino

and structural

for

H-2.3,8

2) we cannot

-H-2 specificities

specificities

found

(Fig.

together

in our

soluble

e.g.,

some specificities

differences

disagreement

that

carbohydrate,

define

that

with

apparent

with

possible

while

on the same molecule,

of 66,000

evidence

of an -H-2 genotype,

is quite

together

were

spec-

the H-2b allele)

(from

the molecules

and both

other

x &)F,

whether

containing

8% neutral

of an (H-2b

fractionation

for

molecules

chromatography

on Sephadex

and heterozygous

purified

Sephadex

from

the H-2d allele).

who concluded

The molecules

carrying

together

possibility,

found

that

specificity

on one molecule,

(1967),

show that

We have further

of specificities

separate.

plotted)

preparations

chromatographed

may account

of Davies tested

(from

further are

-H-2 allele.

the H-2.5

some of the sets

isoantigen H-2 alleles

preparations

H-2.3,8

of

(neither

from molecules

in preparation)

carrying

carrying

solubilized

two different

same

isoantigen

to that

to H-2.5.

to

by the

similar

H-2.6,14,27,28,29

can be separated

and Nathenson,

papain

remarkably

set,

papain

respect

determined

(Shimada

and the

with

one specificity

ificities

a profile

similar

Our results

DISCUSSION.

carrying

H-2.33

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of molecules

with

832

way on the question

a

Of course, are site

needed

in

of each showing

easier.

in a general

85%

of the

a

Vol. 29, No. 6, 1967

molecular

relationships

the membrane. molecules from

may be derived

solubilization precedent

properties

for

bilization

has split sizeable

one specificity.

site

or sites

be localized

is

a very

for

be supported

by the finding

and Wallach

on plasma possible,

(1967)

however,

membrane

it

on the

l),

intermixed

that

the proteolytic into

ceron

solu-

smaller, uniquely

seem that

but con-

the antigenic

membrane

macromolecule

with

sites

the

have

and others

some of which

larger

of different

who showed

macromolecule

would

must

of other

specificities. ACKNOWLEDGEMENT.

The authors

Fabian

for

expert

technical

Osborn

for

helpful

ed in part a career

Mrs.

assistance,

suggestions

by U.S.P;H.S. development

thank

grant

awardee

with

Margareta and Dr.

Tuckman E. J.

the manuscript.

AI-07289,

N.S.F.

of the U.S.P.H.S.

grant

and Mrs.

Hehre This

and Dr.

research

GB-5399.

Dagmar M. J. support-

S.G.N.

is

(AI-11569).

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.

Davies, D. A. L., Transp. 5, 31 (1967). for publication (1967). Nathenson, S. G., submitted Proc. Natl. Acad. Sci. (U.S.). Nathenson, S. G. and D. A. L. Davies, 56, 476 (1966). in "Viral Tumor and Transplantation Nathenson, S. G. and Akira Shimada, Antigen Isolation Symposium", Oak Ridge, Tennessee, October 1967, Transp., in press. Ozer, J. H. and D. F. H. Wallach, Transp. 2, 652 (1967). Sanderson, A. R., Brit. J. Exp. Path. 45, 398 (1964). 1, 163 (1967). Shreffler, D. C., Ann. Rev. Genetics Snell, G. D., In Earl L. Green (ed.), Biology of the Laboratory Mouse. McGraw Hill, New York, 1966. Wigzell, H., Transp. 2, 423 (1965)

a33

in

or in fact,

and would

fragments

that

subunits)

and not

(Fig.

membrane

case also,

such a specificity region

H-2.31

--in situ

on separate

would

(perhaps

In that

to a single

we found

on the membrane,

complex

glypoproteins

tain

which molecules

of Ozer

It

may exist

separate

expressed

reticulum.

as it

the specificity

in the experiments

endoplasmic

still

from This

are

product

specificities

membranes.

specificities

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of the -H-2 cellular

The solubilized

separate

tain

BIOCHEMICAL