Vol.

174,

January

No.

2, 1991

BIOCHEMICAL

BIOPHYSICAL

AND

RESEARCH

COMMUNICATIONS Pages

31, 1991

YEAST

PROTEINS

G.A.

of

School

Received

REACTIVITY TO ANTIBODIES MAMMALIAN APOLIPOPROTEINS

Keesler,'

'Department 2

WITH

S. Moore,' Microbiology,

of Life

D.C.

Usher,2

ELICITED

and

L.W.

North Carolina Raleigh, N.C.

and Health

631-637

Parks'* University,

State

Science University Newark, DE.

AGAINST

of

DelaiJare,

December 13, 1990

rabbit apolipoprotein Hyperimmunized anti-rat antibodies showed reactivity to proteins in Saccharomvces cerevisiae. Antiapolipoproteins Al, B and E reacted with proteins in both a crude extract and a lipid enriched fraction. Protein reactivity was dependent on antisera dilution. Furthermore, the different antiapolipoproteins tested reacted with individually distinct yeast proteins, possibly suggesting the presence of a variety of yeast apolipoproteins with distinct cellular functions as is the case with mammalian apolipoproteins. The specificity of the antibody was directed toward the yeast protein and not a lipid moiety. e' 1491 Rcadc2"lcPress, 1°C. A previous

report

interconversion

of

from

sterol

form in Saccharomvces culture cycle dependent is

used

principally

is

sequestered

form these mediate It

the

to

us

that

are

That proteins observed in many and

functions

evolution.

biogenesis lipid

occur

with

all

in

the

yeast,

that

the

lipid

correspondence

proteins example

of

mammalian

storage

B,

apo

631

esterified In

must

order

exist

and steryl the

E and

suggests

lipids Apo

that

in

corn

Al

the

been conserved conservation

bodies

should

the

for which

esters. may

be by

have

been

apolipoproteins.

have this

of

this to be a of ergosterol

vesicles.

of sterols

similar to apo other vertebrates, these

shown form

while

mobilization to

reversible

to an esterified

vehicles

trafficking

similar

the

a free

We have the free

membrane

One extreme

associated

* To whom

of

described from

to protein-coated

intracellular

occurred

proteins

to

laboratory

cerevisiae (1). event and that for

processes

our

(ergosterol)

is

seeds

structures throughout a protein (2).

The

be addressed.

All

Copright 0 IY9/ r-ighrs of’ reprodwrim

0006-291X/91 $1.50 b! Academic Press. 1~. irl atq form rexn,ed.

Vol.

174,

No.

2, 1991

BIOCHEMICAL

carboxyterminal with

amino

a conserved

mammalian results binding

acids

of the

repeating

In mammalian flotation

which

that might

domain

(4). the

density

of

low

Apolipoprotein to the apo

E, B,E

like

lipoproteins

to

clearance

metabolism

of

LDL

Al by

apoB, also receptor

In this Al, that these

B

and exist proteins

paper E cross in

in

regulation

in

we show react

that

of

at the Therefore, lipid

mammalian

associated

antisera

against

MATERIAL

with the

of

binding

which

mediates

circulation

(5). of

binding these

internalization

and

cholesterol, level there

this

in

of 3-hydroxyare distinct

mobilization,

cerevisiae. with

proportions

transport,

systems.

differentially

Saccharomyces are

to

release

apolipoproteins

medium,

high affinity The binding

leads

subsequent

by their salts

a receptor from

mediates (6,7).

receptor

lipid

is an essential cofactor lecithin cholesterol acyl

lipoprotein

functions

and

and

LDL receptor

cholesterol biosynthesis reductase (8).

for

size

several These

with

high

B 100 possesses

density

in

apo Al.

characterized in

their

turn regulates 3-methylglutaryl-CoA metabolism

found

associated are

lipoprotein

(LDL) the LDL and

sequence

ultracentrifugation

Apolipoprotein

COMMUNICATIONS

a 40% homology

apo E and

lipoproteins

via

low

the

for

share

structures conserved.

density heterogeneity, and and lipids (3). Apolipoprotein the esterification of cholesterol

transferase

L3 protein acid

their proteins in

RESEARCH

include

certain be highly

systems,

properties

BIOPHYSICAL

11 amino

apolipoproteins suggest proteins

AND

rat

a number

A significant lipid

apolipoprotein of

proteins number

of

fraction.

and METHODS

StrainThe strain of Saccharomvces cerevisiae used in the experiments was haploid X2180-1A (Mata SUC2 mal gal2 CUPl), obtained from the Yeast Culture Collection (Berkeley, CA.). Antisera preparationNew Zealand White rabbits were initially immunized intramuscularly with 0.5mg of lipoprotein or apolipoprotein emulsified in complete Freund's adjuvant. This was followed by a series of 3 intramuscular and subcultaneous injections spaced three weeks apart with 0.5mg/ml of immunogen emulsified in incomplete Freund's adjuvant and 2 intravenous injections spaced three weeks apart on immunogen (O.lmg) in saline. The rabbits were then rested for six weeks before being given a final intravenous injection. After an additional 7 days, serum was collected. Three different immunogens were used. Rat apolipoproteins Al and E were gifts from Julian Marsh, Medical College of Pennsylvania. Rat LDL was prepared from rat plasma by sequential ultra-centrifugation with a Beckman Ti60 rotor according to Have1 (9). After adjusting the density of the plasma with NaBr, --et al. a 1.019lOO, 11=35). d- yeast proteins reacting to apo E antiserum (1=71.5). FIGURE

635

Vol.

174,

No.

2, 1991

BIOCHEMICAL

AND

2

1

BIOPHYSICAL

3

4

RESEARCH

5

COMMUNICATIONS

6

FIGURE 3. Titration of apo B antiserum with delipidated human apolipoprotein B showing specificity of the antibody to an apo B antigen. Each lane was loaded with 20 bg crude extract. Primary antibody dilution was 1:500. Lanes: l- control, 2- 0.5 pg/ml apo apo B, 4- 10 pg/ml apo B, 5- 100 pg/ml apo B, 6B, 3- 1 pg/ml normal rabbit serum (NRS).

as

a competitive

inhibitor

delipidated the

human

84.5

kDA

reactivity

apo

yeast

to

delipidated

(figure B was

protein

the

to

was

of

inhibit

virtually

proteins

apo

A concentration

sufficient while

yeast

human

3).

10 pg/ml

reactivity

complete

achieved

with

inhibition

at

100

of

,ug/ml

B. DISCUSSION

We have yeast a

shown One

(12).

that of

quantitatively

the the

on

excess

the

steryl

ester

to

Therefore,

appropriate

partitioning

and have

reactivity

to

distinct Only

proteins with

protein exist

as

part

of

antibodies

elicited

form

B's

did

we

(B

particles

for

extra-cellular

molecular

weight

domains

saturated,

ester.

When

hydrolase

the

the

yeast

(1).

for

yeast

normal

observe

48)

in and

sterol

two

with

a yeast

transport;

B

B 100.

It

additional

B 26

(13,

14). of

therefore, to

be

novel

trafficking.

reactivity

stabilization

be expected 636

these

Apolipoprotein

and

show

functionally that

termed

B 74 the

which

three for

protein

involves would

in

is

interconverts

suggest

size. kDa

designated

function

ester

against

molecular

products

sterol

functions

549

acyl readily

This

distinct

a large

fatty

in

amount

Once

cell

in

apolipoproteins. AI

whose

sterol.

exist

sterol represents

sterols.

proteins

a truncated apo

must of

identified

as

degradative

to via

for

function

sterol

free

the

vehicles

similar

predominately

of

sterol

apolipoprotein

"bulk"

free

reached,

free

may have

of

of

converted

is

trafficking

mammalian

yeast

is

sterol

functions

the

supply

sterol

free

multiple

pool

available

free

for

We

are

functions,

variable

dependent demand

there

these

conserved.

also

--in vitro A large

large only

exist

can

lipid small We show

a

Vol.

174,

No.

several

distinct

Perhaps

these

However,

we

yeast

protein

apolipoprotein apolipoproteins documented

BIOCHEMICAL

2, 1991

yeast are

AND

proteins

reactive

degradative

can

not

rule

is

not

due to

BIOPHYSICAL

to anti

products

out

that

a highly

antibody tested. across species lines

RESEARCH

the

of

in

apo B antibodies. a

larger

reactivity

conserved The higher

COMMUNICATIONS

molecule.

seen region conservability eucaryotes

with

within

the each

is

of well

(15,16,17). Acknowledgments

This research was supported in part by the National Science Foundation (DCB-8814387), the North Carolina Agricultural Research Service, State of Delaware Grant (DRP-88-17), and a Terumo Medical Corporation Grant.

REFERENCES

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

Taylor, F.R. and Parks, L.W. (1978) J. Bacterial. 136: 531537. Vance, V-B., and Huang, A.H.C. (1987) J. Biol. Chem. 262: 11275-11279. Schen, B.W., Scanu, A.M., and Kezdy, F.J. (1977) Proc. Natl. Acad. Sci. USA 74: 837-841. Fielding, C.J., Shore, V.G. and Fielding, P.E. (1972) Biochem. Biophys. Res. Commun. 46: 1493-1498. Goldstein, G.L. and Brown, M.S. (1977) Ann. Rev. Biochem. 46: 897-930. Mahley, R.W. (1988) Science 240: 622-630. Brown, M.S. and Goldstein, J.L. (1986) Science 232: 34-47. Assmann, G., Brown, B.G., and Mahley, R.W. (1975) Biochemistry 14: 3996-4002. Havel, R-J., Eder, H.A., and Bragdon, J.H. (1955) J. Clin. Invest. 34: 1345-1353. Bottema, C.D.K., Mclean-Bowen, C.A., and Parks, L.W. (1983) Biochim. Biophys. Acta 734: 235-248. Lammelli, U.K. (1970) Nature 227: 680-685. Rodriquez, R-J., Low, C., Bottema, C.D.K., and Parks, L.W. (1985) Biochim. Biopys. Acta 837: 336-343. Myrseth, L., Hagve, T., and Prydz, H. (1989) Analyt. Biochem. 181: 86-89. Gustafson, A., Kane, J.P., and Havel, R.J. (1988) Eur. J. Clin. Invest. 18: 75-80. Burton, P.M. and Chiou, Y.M. (1989) Comp. Biochem. Physiol. 92B: 667-673. Law, A. and Scott, J. (1990) J. Lipid Res. 31: 1109-1120. Luo, C-C., Li, W-H., and Chan, L. (1989) J. Lipid Res. 30: 1735-1746.

637

Yeast proteins with reactivity to antibodies elicited against mammalian apolipoproteins.

Hyperimmunized rabbit anti-rat apolipoprotein antibodies showed reactivity to proteins in Saccharomyces cerevisiae. Antiapolipoproteins A1, B and E re...
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