A leukemia virus-related protein in the murine pancreas

Vol. 124, No. 2, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

October 30, 1984

Pages 344-349

A LEUKEMIA VIRUS-RELATED Gillian

M. Beattie,

John F. Reece,

Cancer Received

September

PROTEIN IN THE MURINE PANCREAS

Center,

Javier

F. Villela

University of California, La Jolla, California 92~093

and Nathan

0. Kaplan

San Diego

5, 1984

A protein that has been detected in the granules of islet cells in the murine pancreas is similar but not identical to the endogenous murine leukemia The pancreatic protein was detected by several virus envelope protein gp70. immunological methods using both polyclonal and monoclonal anti-murine gp70. On purification by affinity chromatography, it was shown to be different from murine gp70 in its subcellular location and its molecular size and the size of its precursor and by the effect of various reagents on its immunological activity as determined by the ELISA assay. 0 1984 Academic Press, Inc.

In our model the site this

of lymphomagenesis

of synthesis

system

(1).

of a protein

of the

During

in the islet

MuLV gp70 yet

is

somewhat

the

in athymic

endogenous course

cells

of our

studies,

with

lymphoma

we have detected

which

in some of its

MATERIALS

we have been investigating

MuLV* gp70 associated

of the pancreas

different

mice,

in many ways

high is

in levels

similar

to

properties.

AND METHODS

Mice: Conventional (Simonson Labs. Inc., Gilroy, CA) and athymic (Athymic Mouse Facility, UCSD) mice of the BALB/c background were used in this study. Histological studies: Immunoperoxidase staining of pancreatic tissues was used as previously described (2). Antibodies used for gp70 localization were: goat anti Rauscher gp70 (Becton Dickinson Labs., N. Carolina), rabbit anti goat IgG and goat peroxidase-anti peroxidase (Dako Corp., Santa Barbara, CA) and for insulin localization as previously described (2). Controls were run with normal serum replacing the anti gp70 and anti insulin sera. Subcellular fractionation of pancreas homogenates was carried out as previously described (3). Protein purification: MuLV gp7D from the virus infected xenograft T24 (4) and the pancreatic protein were purified by solubilising the appropriate subcellular fraction in 0.1% octyl glucoside and affinity chromatography using RI7-34.15, a rat monoclonal anti-Friend gp70, bound to sepharose, and subsequent elution with 6M guanidine. The monoclonal antibody was a gift from Dr. Jane Lesley, the Salk Institute for Biological Studies, La Jolla, CA. Metabolic labelling: Pulse labelling and immune precipitation of the virus infected xenograft cell line T24 and primary islet cell cultures using goat anti Rauscher gp70 to precipitate the proteins were carried out according to the method

*MuLV:

Murine

0006-291X/84 Copyright All rights

0

leukemia

virus

$1.50

1984 by Academic Press, of reproduction in any form

Inc.

reserved.

344

Vol. 124, No. 2, 1984 of Famulari mice by the Stability presence of Protein gp70 activity

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Primary islet cell cultures were --et al. (5). method of Leiter --et al. (6). and binding properties of the two proteins various reagents at OOC. was determined by the Lowry assay (7) or the by the ELISA assay as previously described

prepared

from

was compared Bradford (4).

10 day old in the

assay

(8)

and

RESULTS AND DISCUSSION In searching

for

with

lymphomagenesis

high

levels

mice,

both

(Table

(l-3

pancreas particles

model

(10).

and Like

technique

islet

has located

insulin

stains

with

either

only

(Figure

have

very

molecule

in

surprised

of

and pre lymphome

state

have previously

shown by immunoepithelium

of conventional

microscopy

by streptozotocin

of our mice using

this

molecule

like"

conventional

anti-insulin

of the islets,

mice.

has shown that

anti-gp70

the

diabetes

immuno-

in the cytosol

and athymic

or anti-gp70

C type

in a murine

of the pancreati "gp70

to find

the pancreati

shown by electron

damaged

of both

the $ cells

we were

the normal

--et al.

MuLV gp70 associated

of MuLV gp70 in the

--et al. cells

like"

in both

Lerner

study

of the pancreas

sections

islets

of a "gp70

the presence

histochemical

serial

the

(9)

of endogenous

mouse model,

and athymic,

in the

Further

cells

athymic

Ug/mg protein)

techniques

virus

islet

of synthesis

In the literature,

1).

peroxidase

in our

conventional

fluorescence murine

the site

stains

all

of the

Staining

of

while

anti-

the

cells

1).

TABLE I.

gp70 VALUES OF HOMOGENATESOF CONVENTIONAL MOUSE TISSUES

TiSSWS

nglml

Protein mglml

gp70 wlmg

Spleen Lymphnode Liver Intestine Epididymis Pancreas Gall bladder

200 < 2.5 ~25 < 25 410 10,000 ~25

3.8 1.6 26.4 5.4 3.1 10.5 0.24

53 0 0 0 132 952 0

gp70

Table I. Location of activity was determined by assaying homogenates of each tissue for gp70 activity in the ELISA assay using a sandwich of goat anti murine gp70 IgG and goat anti murine gp70 IgG conjugated to horseradish peroxidase. Protein was determined by the Lowry or Bradford assay. These values were for conventional mice. Values were similar for control and prelymphoma athymic mice. 345

of

Vol. 124, No. 2, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Figure 1. Serial histological sections from athymic mouse the immunoperoxidase technique specific for a) gp70 and b) using normal serum in place of antiserum. Note that while stain equally in a), the a cells mainly around the periphery unstained in b) which is specific for insulin, present only Magnification: x560. Staining of sections from conventional was similar.

Subcellular gp7Q ELISA (Table

fractionation

activity

II).

membrane

(see

III

Table

of the pancreas granules

(data

to release anti

copurified

In contrast,

and plasma

murine

of homogenates with

fraction

and Ref.

not shown). (data

fraction located murine

Immunohistochemical

have determined

activity

is usually

of MuLV infected

11).

of the pancreas

the granule

MuLV gp70

that

This not

the

data

shown).

gp7O has shown no cross

"gp70

like"

The ELISA

stained with c) is control of the islet islet are B cells. pancreas

has shown

in the endoplasmic tissues

microscopy

activity

assay

is

using

rabbit

reticulum

studies

inside

the Q$

polyclonal pancreatic

granules goat

granules

TABLE II. gp70 ELISA ASSAY VALUES OF SUBCELLULAR FRACTIONS FROM PANCREATI FROM CONVENTIONAL MOUSE, RAT AND RABBIT

wdmg

Rat wdmg

Rabbit ndmg

2,400 700 800

441 400 333

0 0 0

1,500 0 0 6,300

n.d. 0 0 333

n.d. 0 0 0

Mouse

Subcellular

Fractions

Homogenate Nuclei Mitochondria Microsomes (Endoplasmic reticulum, membrane and granules) Endoplasmic Reticulum Plasma Membrane Granules

plasma

Table II. Location of activity was determined by assaying each subcellular fraction for gp70 activity in the ELISA assay using a sandwich of goat anti murine gp70 IgG and goad anti murine gp70 IgG conjugated to horseradish peroxidase. Protein was determined by the Lowry or the Bradford assay. 346

the

and xenografts

by freeze/thawing

with

that

of the microsomes

electron

was confirmed

reactivity

pancreas insulin; all cells of the in the mouse

Vol. 124, No. 2, 1984

BIOCHEMICAL

TABLE III.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

COMPARISONOF MOLECULAR SIZE AND LOCATION OF MuLV gp70 AND PANCREATIC PROTEIN MuLV gp70

Gel analysis of bound material. from RI7-34.15 column

Mr Mr Mr

80,000

Gel analysis of immune recipitated material from 3 H pulse labelled cultures

Mr Mr

83,000

Subcellular location ELISA assay activity

of gp70

Pancreatic Mr Mr

68,000 70,000

Protein

70,000 30,000

Mr 182,000 Mr 166,000

72,000

plasma membrane and endoplasmic reticulum fractions

granule fraction

Table III. Solubilized proteins from the endoplasmic reticulum fraction of the virus infected xenograft T24 and the granule fraction of the pancreas were bound to RI7-34.15fsepharose. Sound material was eluted with 6M guanidine and analyzed by SDS-polyacrylamide gel electrophoresis in 12.5% acrylsmide slab gels. Protein was visualized by staining with Coomassie brilliant blue. Immune precipitated proteins from3H pulse labelled cultures of the MuLV infected T24 xenograft cell line and the primary pancreatic islet cell culture were analyzed by SDS-polyacrylamide gel electrophoresis in 7.5% acrylamide slab gels. Labelled protein was visualized by autoradiography.

and 5% reactivity that

the

quite

with

rat

immunological

binding

purification

of murine

of MuLV gp70

MuLV and of the murine these

were

pancreatic

two proteins.

eluted

with

of the bound

(Table

pancreatic

Both

bound

6M guanidine.

material

from

II).

These

granules

doublet

of Mr 68,000

and Mr 70,000,

with

analysis

have identified

data

indicate

to anti-gp70

polyclonal

also acids goat

molecule

was

with

Mr 70,000

indicated

important

and subsequent anti-murine

reticulum, comprises

from

cell

line

of the

analysis,

in molecular

size

precipitated

a precursor

of Mr 83,000

and gp70 of Mr 70,000 347

gel

a

gp70 and p15E) (12).

and a

In

granules,

we

III).

and primary

murine

two to four

hour

of labelled

proteins

we have again

identified

material. from

analysis

we identified

(Table

With

immunoprecipitation

differences

and both

MuLV gp70

and Mr 30,000

endogenous

differences

the pancreatic

differences.

gp70 and gel

some important

seen with

of the T24 xenograft

with

SDS polyacrylamide

that

typically

infected

RI7-34.15jsepharose

endoplasmic

of the bound material

labelling

cultures

of 3H amino

precursor

two proteins

Metabolic

has shown

subsequent

the xenograft

(the

a xenograft

to monoclonal

With

of Mr 80,000

contrast,

from

protein

protein

cell

granules

specific. Affinity

in

pancreatic

We have

the T24 xenograft

islet pulses with

identified cell

line,

Vol. 124, No. 2, 1984 which

is

a pattern

simllat

from primary

cultures

two proteins

of Mr

Incubation conditions

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS to xenotropic

of islet 182,000

gp70.

cells,

some differences

and Mr 166,000

determination

in the binding

and stability

after

methosulfate,

EDTA or monoclonal

gp70 and the pancreatic potassium

thiocyanate,

protein.

This

gp70 ELISA activity with

ovomucoid

the MuLV gp70 but latter

observation

(Table

IV).

TABLE

of conformation urea

both

pattern:

under

a variety

of

gp70 ELISA activity

on

OF STABILITY

with

guanidine

was seen only

with

the pancreatic

protein

or

is more

the MuLV gp70. serum albumin

A increased of

OF MuLV

led

to an increase

protein.

the pancreatic

gp70

Relative

The

PROTEIN

Increase

Pancreatic Protein

1.0 1.4 1.7 0.6 0.8 0.7 1.3 0.8 1.1 0.7 2.6 1.1 7-15

Table IV. Incubation was carried out at O°C for 2 hours 5 pg pancreatic protein followed by the ELISA assay for Control: incubation with no additives.

of

two proteins

AND PANCREATIC

MuLV gp70

activity

protein.

of the

in

However,

the gp70 ELISA

in glycosolation

348

MuLV

with

than

Control - no additive Bovine serum albumin 10 mg/ml Ovomucoid 1 mg/ml Dicyclohexyldiimide 1 mM Phenazine methosulfate 1 mM Ethanol 2% Guanidine 6M EDTA 10 mM Glycerol 20%. RI7-34.15 150 pg/ml UREA 2l4 Potassium thiocyanate 0.l.M Concanavalin A 25-250 pgfml

both

incubation

the pancreatic

Additive

A decrease phenazine

was observed

after

the activity

a difference

has shown

two proteins.

MuLV gp70 and the pancreatic

had no effect

COMPARISON

different

protein

RI7-34.15

or bovine

or concanavalin

suggests

IV.

that

a very

dicyclohexyldiimide,

in activity

indicates

glycerol, with

with

material

III).

of these

However,

a decrease

to alteration with

incubation

protein.

observation

Incubation

incubation

(Table

of the

in gp70 ELISA activity ethanol,

the precipitated

we have observed

of MuLV gp70 and the pancreatic

and subsequent

susceptible

With

1.0 1.3 1.0 0.1 0.6 0.6 0.3 0.9 1.8 0.7 6.0 0.3 1.0 using 5 pg MuLV gp70 activity.

gp70

or

Vol. 124, No. 2, 1984 Although related

protein

interesting,

exercised parameters

results

in

(13-15).

It

pancreatic

be used protein

should

we find

of the

cells

islet

findings

also

molecules

out

by immunological with

now under

the presence

of a virus

of the murine

of the relationship

be pointed

in conjunction is

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

preliminary,

of the recent

in identifying must

are

the granules

in view

and viruses

of this

these

BIOCHEMICAL

that

between

caution

methods

immunological

pancreas'most

alone. evidence.

should

diabetes be

Other The nature

investigation.

ACKNOWLEDGMENTS The research was supported by National Institutes of Health Grants and CA-23052 to the Athymic Mice Facility of the University of California, San Diego.

CA-11683

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

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