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.
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