From the
A HeLa S3 subline is unusual in accumulating relatively large
amounts of glucosaminyl(acyl)phosphatidylinositol (GlcN(acyl)PI), a
derivative of phosphatidylinositol (PI) in which both GlcN and a fatty
acid are linked to inositol hydroxyl groups (D. Sevlever, D. Humphrey,
and T. L. Rosenberry, submitted for publication). This lipid is a
proposed intermediate on the biosynthetic pathway for glycosyl-PI (GPI)
anchors of membrane proteins. In this study we demonstrate for the
first time that exogenous inositol phospholipids can enter this
biosynthetic pathway and be metabolized to GlcN(acyl)PI. When HeLa S3
cells were incubated for 24 h with exogenous PI or
sn-1-acyl-2- lyso-phosphatidylinositol (lyso-PI)
labeled with
Phosphatidylinositol (PI)
A HeLa S3 subline was chosen as an initial model for
uptake studies since these cells contain a significant amount of
GlcN(acyl)PI (about 10
[
To conclude that exogenous
[
The medium was also depleted of
[
When HeLa S3 cells were incubated with exogenous labeled PI
or lyso-PI for periods approaching 24 h, the cellular lipid metabolic
products included PI, lyso-PI, PIP, and GlcN(acyl)PI. The final
distribution of metabolites was similar to that observed for endogenous
inositol phospholipids produced by labeling with
[
In contrast to the final distribution of
cell-associated labeled inositol phospholipids, the distribution of the
labeled species in the medium differed for exogenous
[
It is noteworthy that we observed substantial
sn-2 acylation of exogenous [
Almost all the label remaining in the
medium after cell incubation with [
HeLa S3 cells were
cultured as outlined under ``Experimental Procedures'' except
that 25 m
M HEPES (pH 7.4) was added. Mixtures of
[
We thank Dr. Daniel Sevlever for generous gifts of the
HeLa S3 subline and [
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
H in the inositol group, 25-30% of the
label was recovered in cell-associated lipids and most of the remaining
70-75% in hydrophilic metabolites in the medium. The predominant
labeled lipid was PI, with smaller amounts of lyso-PI,
phosphatidylinositol 4-phosphate (PIP), and GlcN(acyl)PI. Both
exogenous lipid precursors gave the same distribution of labeled
lipids, and a similar distribution was observed for endogenous inositol
phospholipids metabolically labeled with
[
H]inositol. Addition of excess inositol had no
effect on the conversion of [
H]lyso-PI to
[
H]GlcN(acyl)PI, indicating that the conversion
did not result from breakdown to [
H]inositol
followed by resynthesis. The cellular orientation of incorporated PI
and lyso-PI was determined by incubating cells at 4 °C with
PI-specific phospholipase C (PI-PLC). This enzyme cleaves only PI and
lyso-PI on the outer leaflet of the cell membrane. After 24-h
incubation with either precursor, only about 15% of cell-associated
[
H]PI or [
H]lyso-PI was on
the outer leaflet. However, more than 60% of the
[
H]PI was on the outer leaflet after 1-h
incubation with either precursor, suggesting that substantial
sn-2 acylation of exogenous [
H]lyso-PI
occurred in the outer leaflet. This suggestion was confirmed by
examining labeled lipids in cells after uptake of
[
H]lyso-PI at 4 °C. No transmembrane
translocation of lyso-PI, PI phosphorylation, or PI glycosylation
occurred at this temperature, but some sn-2 acylation was
apparent and more than 90% of the [
H]PI formed
was on the outer leaflet. These data indicate that sn-2
acylation can occur in the outer leaflet of the cell membrane, perhaps
by transacylation from other cell surface phospholipids.
(
)
is an
important precursor to many inositol phospholipids including
glycoinositol phospholipid (GPI) anchors. In the affected cells of
patients with paroxysmal nocturnal hemoglobinuria (PNH), a somatic
mutation blocks the first step in the GPI biosynthetic pathway, the
linkage of GlcNAc to PI (Armstrong et al., 1992; Takahashi
et al., 1993; Hidaka et al., 1993). As a consequence
of the mutation, affected PNH cells fail to express GPI-anchored
proteins on the cell surface, several of which are important in the
regulation of complement activation (Yamashina et al., 1990).
One possible therapeutic strategy for PNH is to provide exogenous
GlcNAc-PI or other early precursors in the GPI biosynthetic pathway
that could bypass the genetic block. As a prelude to investigating cell
uptake of these PI derivatives, we have initiated studies on the uptake
of exogenous PI and
sn-1-acyl-2- lyso-phosphatidylinositol (lyso-PI). The
spontaneous transfer of phospholipids like phosphatidylcholine (PC),
phosphatidylethanolamine, and phosphatidylglycerol from donor vesicles
to cells is consistent with a monomer transfer model in which lipid
monomers dissociate from the donor vesicles, diffuse through the
aqueous phase, and associate with the cell membrane (Ferrell et
al., 1985; Gardam et al., 1989; Silvius and Zuckermann,
1993). After cell association, the exogenous lipids can be transferred
inside the cell by transbilayer translocation (flipping) or
endocytosis. The incorporated lipids will be susceptible to further
metabolism in compartments where the appropriate metabolic enzymes are
available.
molecules/cell), a proposed
intermediate on the GPI biosynthetic pathway.
(
)
The goal of this study was to characterize the generation
of radiolabeled lipids from exogenous PI or lyso-PI that were
H-labeled in the inositol group. The steady-state levels of
these labeled lipids were compared with those of endogenous inositol
phospholipids labeled by incubation with
[
H]inositol. After the addition of either
[
H]PI or [
H]lyso-PI to the
cells, we observed interconversion of PI and lyso-PI and a
time-dependent appearance of phosphatidylinositol 4-phosphate (PIP) and
GlcN(acyl)PI. This is the first report of the metabolic labeling of a
free GPI with exogenous inositol phospholipid precursors. The
transbilayer distribution of PI and lyso-PI was also studied with the
use of PI-PLC to cleave PI and lyso-PI in the outer leaflet of the
plasma membrane bilayer.
Radiolabeled PI and
Lyso-PI
[H]PI was taken directly from a
commercial stock (DuPont NEN, 13 Ci/mmol) in ethanol.
[
H]Lyso-PI and [
P]lyso-PI
were prepared by phospholipase A
(PLA
, bee
venom, Sigma) treatment either of commercial
[
H]PI or of endogenous lipids produced by
incubation of [
H]inositol (American Radiolabeled
Chemicals, Inc., 20 Ci/mmol) or
[
P]orthophosphate (American Radiolabeled
Chemicals, Inc.) with HeLa S3 cells. The
H-labeled
endogenous lipids were fractionated by chromatography on
octyl-Sepharose with a linear gradient of 5-60% n-propyl
alcohol in 100 m
M NH
OAc,
and
[
H]lyso-PI (1 Ci/mmol) was eluted at 35%
n-propyl alcohol. The
P-labeled lipids were
resolved prior to PLA
treatment by TLC in
CHCl
/CH
OH/CH
COCH
/CH
COOH/H
O
(30: 10: 40: 10: 5, v/v). The [
P]PI band was
washed from the scraped silica with n-butyl alcohol and
incubated with bee venom PLA
(0.1 mg/ml) in 5 m
M
Tris-Cl, 1 m
M NaCl, 0.6 m
M CaCl
(pH 8) at
25 °C for 16 h, and the product [
P]lyso-PI
was isolated by extraction as described (Darnell et al.,
1991). TLC mobilities of the metabolically labeled inositol
phospholipids were calibrated by comparison to standard
[
H]PI and [
H]lyso-PI
derived from the commercial [
H]PI. Susceptibility
of the [
H]lyso-PI and
[
P]lyso-PI to PI-PLC, but not to
PLA
, also was confirmed. Aliquots of radiolabeled inositol
phospholipids in ethanol were added to serum-free medium (
1%
ethanol), and the mixtures (1-20 n
M labeled lipid) were
bath-sonicated for 5-10 min prior to incubation with cells. The
distribution of metabolic products obtained from cells incubated with
radiolabeled lyso-PI was the same when this precursor was derived from
either commercial [
H]PI or endogenous labeling
with [
H]inositol or
[
P]orthophosphate. Furthermore, addition of
exogenous PC or PI (10 µ
M) to the sonication mixture also
did not affect the distribution of metabolic products from
[
H]lyso-PI incorporation.
Cells and Culture Conditions
HeLa S3 cells were
maintained on a 60-mm diameter dish in 3 ml of Dulbecco's
modified Eagle's medium (DMEM; Life Technologies, Inc. or Celox),
10% horse serum or 10% fetal bovine serum, 50 units/ml penicillin, 50
µg/ml streptomycin, and 5% COat 37 °C until nearly
confluent (4-8
10
cells). The HeLa S3 line (a
cloned HeLa cell line provided to the American Type Culture Collection
after about 400 passages) was shown previously to produce mannosylated
GPIs (Hirose et al., 1992; Ueda et al., 1993). During
passaging leading up to the current studies, these cells were collected
by scraping from culture flasks rather than by trypsinization. The
passaging and culture procedures reproducibly generated cells with a
more rounded morphology and a much higher level (3-4-fold) of
GlcN(acyl)PI than the parental S3 cells.
(
)
Karyotype analysis showed the same modal chromosome count
(65, 66, 67) and the same copy numbers of HeLa
markers (one of M1, one of M2, two of M3, one of M4) reported for HeLa
S3.
Metabolism of Inositol Phospholipids in HeLa S3
Cells
For metabolic labeling with
[H]inositol, cells were incubated with
inositol-free DMEM supplemented with 10% dialyzed newborn calf serum
for 60 h. Cells (1.5
10
) were metabolically labeled
with [
P]orthophosphate (200 µCi) in
phosphate-free DMEM supplemented with 10% fetal bovine serum. For lipid
uptake and metabolism studies, cells were washed to remove serum
protein before addition of serum-free DMEM containing 0.02-0.3
µCi of radiolabeled lipids and returned to the incubator for
indicated times (0.5-24 h). The cell viability was determined to
be over 95% in the presence of serum-free medium for 24 h by trypan
blue exclusion. At each time point the conditioned medium was removed
from one plate of cells, and the labeled cells were rapidly scraped
from the dish in 3 ml of prechilled phosphate buffer saline (140 m
M NaCl, 10 m
M sodium phosphate, pH 7). Pelleted cells were
washed twice with phosphate buffer saline and lysed by addition of 1 ml
of CHCl
/CH
OH (1:1, v/v) to a final mixture of
CHCl
/CH
OH/H
O (10:10:3, v/v). The
remaining cell pellet was re-extracted twice with 1 ml of
CHCl
/CH
OH/H
O (10:10:3, v/v), and
the combined extract supernatants were dried in a SpeedVac
concentrator. The dried supernatants were partitioned by resuspending
in 400 µl of H
O-saturated n-butyl alcohol and
200 µl of n-butyl alcohol-saturated water. The aqueous
phase was removed and extracted with 200 µl of
H
O-saturated n-butyl alcohol, and the combined
n-butyl alcohol phases were re-extracted with 200 µl of
n-butyl alcohol-saturated water. The resultant aqueous phases
also were combined. One ml of conditioned medium removed from the cells
at each time point also was dried and partitioned with n-butyl
alcohol. The n-butyl alcohol phases from both the cell lysate
and the conditioned medium were dried and resuspended in 15 µl of
n-butyl alcohol to spot on TLC (Silica Gel 60). The TLC plate
was developed in a solvent system of
CHCl
/CH
OH/H
O (10:10:3, v/v) and
scanned to determine the mobility and the amount of each labeled band
with a BioScan System 200 Imaging Scanner and Nscan software. Bands in
silica from extracts of cells labeled with a mixture of
[
H]- and [
P]lyso-PI were
scraped into Formula 963 (DuPont) for 2 days prior to scintillation
counting with channel settings that gave >97% resolution of
H and
P. Label in the dried aqueous phase from
conditioned medium was resuspended in 6 ml of water and analyzed by
chromatography on a Dowex 1-X8 column (2 ml) (from Baker, Phillipsburg,
NC; Berridge et al., 1983; Ross and Majerus, 1986). Standards
were eluted as follows: inositol and
sn-glycero-3-phospho-
D1- myo-inositol were
not retained; inositol cyclic 1,2-phosphate was eluted with 20 and 50
m
M ammonium formate and inositol mono- and polyphosphates with
150 and 400 m
M ammonium formate (also see Volwerk et
al., 1992).
PI-PLC Treatment of Intact Cells
PI-PLC was
prepared from the medium of Bacillus subtilis which
overexpresses PI-PLC of Bacillus thuringiensis (Henner et
al., 1988; Deeg et al., 1992). The medium was
concentrated by tangential filtration (Millipore Pellicon 0.45-µm
filter), and the enzyme was purified by column chromatography on
octyl-Sepharose and DEAE-cellulose. Cells were incubated with
radiolabeled lipid, and at the designated time the conditioned medium
with residual radiolabel was removed and the cells were washed once
with prechilled phosphate buffer saline. Fresh serum-free medium
containing 0.15-0.25 µg/ml PI-PLC was prechilled to 4 °C
and added to the cells. The incubation was continued in an environment
of 5% COat 4 or 37 °C for an additional hour before
cell harvesting. This concentration of PI-PLC was sufficient to cleave
all the PI and lyso-PI on the outer leaflet of the cell membrane
( e.g. see text below). The medium containing PI-PLC was
removed and the PI-PLC-treated cells were washed three times before
scraping to ensure a complete removal of PI-PLC. The harvested cells
and the medium were extracted with
CHCl
/CH
OH/H
O (10:10:3, v/v),
partitioned with n-butyl alcohol and water, and analyzed by
TLC as described above.
H]PI Incorporation-In an initial
set of experiments, [
H]PI was prepared as a
liposomal suspension in serum-free medium and added to HeLa S3 cells as
they approached a confluent state of cell growth. Samples of cells and
medium were then taken at various times up to 24 h and partitioned
between n-butyl alcohol and water, and the lipids in the
n-butyl alcohol phase were fractionated by TLC. Label
associated with the cells approached a steady state of about
30-35% of the total added label with a t of less than 30
min, and this level of cell-associated label was maintained up to 24 h.
TLC analysis revealed that about 20% of the total label corresponded to
cell-associated [
H]PI after 1 h of incubation,
and only a slight further increase in this percentage was observed over
the next 24 h (Fig. 1 A). At least four labeled metabolic
products were detected by phase partitioning and TLC analysis. The two
more abundant products corresponded to [
H]lyso-PI
and to a class of derivatives appearing in the aqueous phase that we
denoted ``hydrophilic metabolites.'' The appearance of these
products with time also is shown in Fig. 1 A. Two less abundant
products, [
H]PIP and
GlcN(acyl)[
H]PI, also were identified (see
below). The level of cellular [
H]lyso-PI showed a
biphasic pattern in Fig. 1 A, rising to 8% of the total
label at 1 h and gradually declining to 3% by 24 h. The levels of
hydrophilic metabolites and the minor lipid species increased
continuously up to 24 h.
Figure 1:
[H]PI uptake by
HeLa S3 cells. Cells were incubated with 0.02 µCi of commercial
[
H]PI at 37 °C for the indicated time and
extracted, and samples of the extract and the medium were partitioned
between n-butyl alcohol and water and analyzed by TLC as
described under ``Experimental Procedure.'' A, the
quantities of cell-associated label migrating as
[
H]PI (
) or
[
H]lyso-PI (
) or partitioning into the
aqueous phase (
) were calculated as a percentage of the total
label applied to the tissue culture dish. The label in the aqueous
phase is denoted as hydrophilic metabolites. B, the quantities
of label in the medium were calculated as in A. Data points
were obtained from seven experiments.
The total recovery of H label
in the experiments in Fig. 1was greater than 95%. In addition to
the 30-35% of label associated with the cells, 65% of the total
label was recovered in the medium. More than one-half of the medium
label corresponded to [
H]PI throughout most of
the labeling period (Fig. 1 B). The basis of this incomplete
uptake of [
H]PI was unclear and is considered
further under ``Discussion.'' Most of the remaining medium
label was in hydrophilic metabolites, with only a trace amount of
[
H]lyso-PI. The hydrophilic metabolites in the
medium reached a plateau of 20% of total label by 1 h and increased
slowly up to 24 h. [
H]Lyso-PI Incorporation-A number of
fibroblast cell lines have been shown to take up
[
H]lyso-PI rapidly (Volwerk et al.,
1992). When HeLa S3 cells were incubated with
[
H]lyso-PI under the same conditions as in
Fig. 1
, the initial cellular uptake of label was greater than
that with [
H]PI (Fig. 2 A). About 60% of
the total label became cell associated within 1 h. However, the
cell-associated label did not remain as
[
H]lyso-PI. Cell-associated
[
H]lyso-PI declined rapidly from 30% of the total
label at 1 h to 11% by 5 h and 3% at 24 h. The loss in cellular
[
H]lyso-PI was accompanied by a rise in cellular
[
H]PI, which reached a maximal level of 40% by
3-5 h. The pronounced uptake and conversion of
[
H]lyso-PI into [
H]PI
indicated extensive acylation of exogenous
[
H]lyso-PI at the sn-2-hydroxyl group of
glycerol. As a result of this conversion, the distribution of labeled
species inside the cell following [
H]lyso-PI
labeling progressively approached that obtained with
[
H]PI labeling, and after a 24-h labeling period
the distribution and levels of labeled species obtained from both
precursors were virtually identical.
H]lyso-PI or [
H]PI is
metabolically converted directly to other radiolabeled lipid products,
it was important to document that exogenous phospholipid was not
degraded to [
H]inositol and in turn utilized in
de novo synthesis of
H-labeled products. This
possibility seemed unlikely, as addition of a large excess of inositol
(10 m
M) to the medium during 24-h labeling with
[
H]lyso-PI as in Figs. 2, A and
B, did not change the distribution of label in
[
H]PI, [
H]PIP, and
[
H]GlcN(acyl)PI (data not shown). In a more
rigorous test, lyso-PI mixtures radiolabeled with
H in
inositol and
P in phosphate were presented to cells, and
the ratios of
P to
H in the lipid products
were compared with the ratio in the initial mixture. PI is synthesized
from CDP-diacylglycerol and inositol (see Catt and Balla, (1989)). If
lyso-PI were degraded to [
H]inositol and
[
P]phosphate prior to resynthesis of labeled PI,
PIP, and GlcN(acyl)PI, the
P/
H ratios in these
lipid products should differ dramatically from that in the lyso-PI
precursor pool. As shown in , however, these ratios
remained essentially unchanged, indicating that the lipid products are
derived directly from lyso-PI.
H]lyso-PI rather rapidly
(Fig. 2 B). Only 7% of the total label corresponded to
[
H]lyso-PI in the medium at 2.5 h, and just a
trace amount of organic phase label ([
H]lyso-PI
plus [
H]PI) was in the medium after 5 h. Most of
the medium label was in hydrophilic metabolites after 2.5 h. The
virtual complete removal of [
H]lyso-PI from the
medium contrasted with the incomplete uptake of
[
H]PI noted above, although the percentage of
[
H]lyso-PI degraded to labeled hydrophilic
metabolites in the medium was only slightly larger than that observed
with [
H]PI in Fig. 1. The composition of
these hydrophilic metabolites was investigated by a conventional Dowex
1-X8 chromatography procedure in which inositol and inositol phosphates
are resolved by stepwise elution with ammonium formate (Berridge et
al., 1983; Ross and Majerus, 1986). After 1 h of incubation with
[
H]lyso-PI, approximately 33% of the hydrophilic
metabolite pool in the medium corresponded to inositol and/or
sn-glycero-3-phospho-
D1- myo-inositol, 19% to
inositol cyclic 1,2-phosphate, and 48% to inositol phosphates. After
2.5 h of incubation, the percentages were 52, 19, and 29%,
respectively. The nature and the location of the enzymes involved in
the degradation to hydrophilic metabolites was studied by adding
[
H]PI or [
H]lyso-PI to
conditioned medium that had been incubated with unlabeled cells for
6-15 h. After the incubation, [
H]PI or
[
H]lyso-PI was recovered without detectable
metabolic breakdown, indicating that the hydrophilic metabolites had to
be produced by cell-associated enzymes that presumably were on the cell
surface.
Figure 2:
[H]Lyso-PI uptake by
HeLa S3 cells. Cells were incubated with 0.02 µCi of
[
H]lyso-PI at 37 °C for the indicated time,
and quantities of [
H]PI (
),
[
H]lyso-PI (
), or hydrophilic metabolites
(
) associated with the cells ( A) or in the medium
( B) were calculated as in Fig. 1. Data points were obtained
from four experiments.
Lipids on the Outer Leaflet of the Plasma Membrane
To
clarify the processes by which the metabolic products derived from
exogenous [H]PI or
[
H]lyso-PI were produced, the transbilayer
orientations of the cellular [
H]lyso-PI and its
lipid metabolic products were studied. PI and lyso-PI on the external
leaflet of the plasma membrane bilayer can be determined quite
selectively by treatment of intact cells with purified bacterial
PI-PLC, since this enzyme hydrolyzes only inositol phospholipids
located on this outer leaflet without penetrating the membrane (Higgins
et al., 1989). With this technique, the transbilayer
distribution of PI in the plasma membrane of red blood cells from
human, ox, sheep, and pig was shown to be asymmetric, with 80-95%
of the PI in the cytosolic or inner leaflet of the plasma membrane (Low
and Finean, 1977; Butikofer et al., 1990). HeLa S3 cells were
incubated with [
H]lyso-PI for various times, and
the washed cells were subjected to PI-PLC treatment at 4 °C. This
low temperature treatment should inhibit endocytosis nonspecifically
(Al-Awaqati, 1989), and it also impeded exogenous lipid translocation
and metabolism in experiments described below. After 1 h of labeling,
PI-PLC removed more than 80% of the cellular
[
H]lyso-PI (Fig. 3), but this percentage
decreased to 15% at 24 h. These data indicate that
[
H]lyso-PI initially was in the outer leaflet
where it was susceptible to PI-PLC and progressively was translocated
(flipped) to the inner leaflet where it was resistant to PI-PLC. It is
noteworthy that the initial [
H]PI formed after
brief incubation with [
H]lyso-PI for less than 1
h was up to 60% susceptible to PI-PLC, whereas less than 15% of the
[
H]PI was cleaved after 24-h labeling
(Fig. 3). This observation suggests that at least some acylation
of cell-associated [
H]lyso-PI occurred on the
outer leaflet of the plasma membrane, a possibility that is addressed
further below.
Figure 3:
Labeled lipids on the cell surface are
susceptible to cleavage by PI-PLC. HeLa S3 cells were labeled with 0.02
µCi [H]lyso-PI as in Fig. 1. At the indicated
time point, the medium was removed and cells were incubated with or
without PI-PLC at 4 °C for 1 h as outlined under
``Experimental Procedures.'' Cell extracts were analyzed as
in Fig. 1. Data indicate the percentage of cell-associated
[
H]PI (
) or [
H]lyso-PI
(
) removed by PI-PLC treatment. Data points were obtained from
four experiments.
When cells with incorporated
[H]lyso-PI are treated with PI-PLC at 37 °C
instead of at 4 °C, not only the initial
[
H]PI and [
H]lyso-PI
available on the cell surface will be susceptible to cleavage, but
labeled lipids that translocate from the inner to the outer leaflet
(flop) during the exposure of cells to PI-PLC will also be cleaved. To
assess the extent to which flopping occurs in HeLa S3 cells, cells were
incubated with [
H]lyso-PI for 3 h, washed, and
then exposed to PI-PLC for 1 h of continued incubation at 37 °C.
About 25% of the [
H]PI formed after the 3-h
incubation was cleaved at 37 °C (data not shown), an amount
comparable with that removed by 4 °C cleavage in Fig. 3.
Since no more labeled PI was cleaved by the 1-h PI-PLC treatment at 37
°C than at 4 °C, the flopping of PI over this period at 37
°C was not significantly faster than at 4 °C. We show below
that flipping of lyso-PI and PI becomes negligible at 4 °C, so we
would expect that flopping also would not occur at this temperature. It
thus appears that flopping of PI in HeLa S3 cells is not significant
over 1 h at 37 °C. In human erythrocytes, the t for PI to
reach a steady-state flip-flop distribution was reported to be 3 h
(Butikofer et al., 1990).
Label Incorporation at 4 °C
The observation
that a higher percentage of [H]PI was cleaved by
4 °C PI-PLC treatment after shorter incubations with
[
H]lyso-PI (Fig. 3) implied that at least
some acylation of [
H]lyso-PI into
[
H]PI occurred on the outer leaflet, although it
remained possible that some [
H]PI initially
generated on the inner leaflet flopped back to the outer leaflet. To
resolve this question, HeLa S3 cells were incubated with
[
H]lyso-PI in 5% CO
at 4 °C
(instead of 37 °C as in Fig. 2) for various times before
lipid extraction (Fig. 4 A). Incorporation of the label
was slightly retarded at 4 °C, but the cell-associated label still
reached nearly 60% of the total at 5 h. Cellular
[
H]PI (Fig. 4 A) and hydrophilic
metabolites in the medium (Fig. 4 B) were markedly
reduced relative to the amounts observed during 37 °C incubation in
Fig. 2
, and no [
H]PIP or
GlcN(acyl)[
H]PI was detected under these
conditions. To determine the quantity of
[
H]lyso-PI and [
H]PI in the
outer leaflet of plasma membrane, the labeled cells were cleaved with
PI-PLC for an additional hour at 4 °C as in Fig. 3.
Cell-associated [
H]lyso-PI after incubation for
up to 16 h was completely cleaved by PI-PLC (>96%, data not shown).
Furthermore, the small amount of cell-associated
[
H]PI that was generated (8% by 15.5 h) also was
more than 90% susceptible to PI-PLC (data not shown). Since almost all
of the cell-associated labeled lipid was readily cleaved by PI-PLC,
flipping of both [
H]PI and
[
H]lyso-PI were markedly decreased by lowering
the temperature to 4 °C. Furthermore, although lipid metabolism
also was minimized at 4 °C, acylation of
[
H]lyso-PI into [
H]PI could
still occur on the outer leaflet of the cell membrane. In contrast,
phosphorylation and glycosylation, which require the presence of the
labeled lipid precursors inside the cells either by flipping or
endocytosis, were temperature-sensitive and not detectable at 4 °C.
Effects of Raising the Temperature on [
H]Lyso-PI
Incorporated at 4 °C-Since cell incubation at 4 °C was
shown to allow cellular association but not flipping of
[
H]lyso-PI, a rapid shift from 4 °C to 37
°C should initiate a competition between three pathways for the
externally oriented [
H]lyso-PI: 1) flipping to
the internal leaflet, 2) acylation to PI, or 3) degradation to
hydrophilic metabolites. To examine this competition, HeLa S3 cells
were incubated with [
H]lyso-PI at 4 °C for 5
h as in Fig. 4. The medium was then replaced by fresh medium at
37 °C, and the incubation was continued at 37 °C for an
additional hour. The distribution of label at the end of the 37 °C
treatment was compared with that obtained by direct incubation of cells
with [
H]lyso-PI for 1 h at 37 °C as shown in
Fig. 5. The levels of cell-associated [
H]lyso-PI
and medium hydrophilic metabolites obtained following the temperature
shift were not significantly different from those obtained after uptake
at 37 °C. The level of cell-associated [
H]PI
was about 38% in the temperature-shift experiment, whereas it reached
only 25% in the direct 37 °C incubation, but this difference
included about 5% formation of [
H]PI during the
incubation at 4 °C prior to the temperature shift. The same
susceptibility of cell-associated [
H]lyso-PI and
[
H]PI to PI-PLC (80 and 50%, respectively) also
was observed after both treatments (data not shown). These data
indicate that preadsorption of [
H]lyso-PI at 4
°C had little effect on the relative rates through the three
pathways available to this lipid at 37 °C.
Figure 4:
[H]Lyso-PI uptake by
HeLa S3 cells at 4 °C. Cells were incubated with 0.02 µCi of
[
H]lyso-PI at 4 °C for the indicated time,
and quantities of [
H]PI (
),
[
H]lyso-PI (
), or hydrophilic metabolites
(
) associated with the cells ( A) or in the medium
( B) were calculated as in Fig. 1. Data points were obtained
from two experiments.
Label Incorporation into PIP and GlcN(acyl)PI
We
noted above that when HeLa S3 cells were incubated with
[H]PI, two minor lipid products were observed
along with [
H]PI and
[
H]lyso-PI. The distribution of these labeled
lipids on TLC after a 24-h labeling period is shown in Fig.
6 A. The most polar of these lipids was assigned as PIP
based on its mobility, its resistance to PI-PLC and to a
GPI-specific phospholipase D (GPI-PLD), and its conversion to PI with
alkaline phosphatase. PIP was detected in the cell extract as early as
1 h. The least polar lipid corresponded in mobility to GlcN(acyl)PI,
whose structure was assigned previously after labeling with
[
H]inositol (as in Fig. 6 C)
according to the following criteria.
It was the only
labeled lipid susceptible to cleavage by GPI-PLD, and treatment of the
labeled cleavage product with methanolic KOH released the acyl group
from inositol and produced GlcN-inositol, which was identified by mass
spectrometry and by cation exchange chromatography. The peak of
identical relative mobility in Fig. 6, A and B,
also produced GlcN-inositol on 6
N HCl hydrolysis, confirming
its assignment as GlcN(acyl)PI. GlcN(acyl)PI was detectable only after
3 h, and levels of both PIP and GlcN(acyl)PI increased gradually during
incubation with [
H]PI for up to 24 h. A nearly
identical distribution of labeled lipids was observed when cells were
labeled with [
H]lyso-PI for 24 h (Fig.
6 B) or with [
H]inositol for 60 h (Fig.
6 C), except that less cell-associated
[
H]lyso-PI was obtained with
[
H]inositol.
Figure 6:
Exogenous [H]lyso-PI
is metabolically converted to [
H]GlcN(acyl)PI.
The TLC profiles show cell-associated labeled lipids resulting from
incubation of HeLa S3 cells for 24 h with 0.02 µCi of
[
H]PI ( A) or
[
H]lyso-PI ( B) or for 60 h with 1
µCi of [
H]inositol ( C). The
counts/min in 0.78-mm intervals in each lane are expressed as
percentages of the total counts/min recovered. While the absolute
mobilities of the four labeled lipids varied slightly from plate to
plate, their relative mobilities were very consistent. Each labeled
species was characterized in reference to standard
[
H]PI and [
H]lyso-PI
( arrows) run in parallel on the same TLC plate. Peaks
corresponding to PIP and GlcN(acyl)PI were assigned as indicated in the
text.
H]inositol. Furthermore, the percentages of
[
H]PI and [
H]lyso-PI in the
outer leaflet of the cell membrane were similar following the
incubation with either [
H]lyso-PI or
[
H]inositol (data not shown). PI and its
polyphosphorylated derivatives PIP and phosphatidylinositol
4,5-bisphosphate (PIP
) typically account for 5-10% of
the total membrane phospholipid (reviewed in Ganong (1991)). The
synthesis of PIP and PIP
from exogenous
[
H]PI was demonstrated previously in Friend
erythroleukemic cells (Hohengasser et al., 1986) and in human
red blood cells (Butikofer et al., 1990). Although it is
currently accepted that GPI biosynthesis begins with the transfer of
GlcNAc to PI, the location and structure of the precursor pool of PI
has remained elusive. To our knowledge, there are no previous reports
in which exogenous [
H]PI or
[
H]lyso-PI has been incorporated into a GPI
structure. Furthermore, the glycerolipid groups in GlcNAc-PI and
GlcN(acyl)PI are much more resistant to base hydrolysis than those of
PIs extracted from the same cells (Stevens and Raetz, 1991).
These observations have suggested that a subset of cellular PI
may be selected for GPI biosynthesis either by its compartmentalization
in the endoplasmic reticulum or by the specificity of the GlcNAc
transferase (Englund, 1993). Our data indicate that exogenous
[
H]PI and [
H]lyso-PI can be
taken up and translocated to the site of GPI biosynthesis, at least in
this HeLa S3 subline.
H]PI or [
H]lyso-PI
precursors. Exogenous lyso-PI was rapidly and completely removed from
the medium, whereas up to 30-35% of exogenous PI still remained
in the medium after 24 h. Although PI became the major cell-associated
labeled lipid when either [
H]PI or
[
H]lyso-PI was the precursor, the lyso-PI
precursor gave greater initial cellular incorporation than PI. Loss of
the sn-2 fatty acid renders lyso-PI more hydrophilic and thus
can improve its transfer from the medium to an acceptor membrane.
[
H]PI formed stable bilayer vesicles in these
experiments because its concentration in the medium (1-2
n
M) should be well above its critical micelle concentration.
Lyso-PI was added at a similar concentration, but its higher critical
micelle concentration is in the micromolar range (7 µ
M for
C16-thio-PI, Hendrickson et al., 1992; 7 µ
M for
lyso-PC, Ferrell et al., 1985). Therefore lyso-PI remained in
a monomeric form and readily integrated into cells. Two possible
explanations were considered for the observation that about
30-35% of the [
H]PI did not associate with
the cells, but both were rejected. The first investigated whether this
percentage reflected a dynamic equilibrium involving uptake of
[
H]PI from the labeling medium balanced by
reverse transfer from the cell membrane to medium liposomes. This
possibility was ruled out by measuring the extent to which labeled
lipids dissociated from HeLa S3 cells in the presence of unlabeled PI
liposomes. Cellular PI was labeled with
[
H]inositol and incubated for 24 h in fresh
medium with or without sonicated 2 µ
M PI. Only about 1% of
the total label was released from the cells, primarily as
[
H]PI, into either medium, indicating that the
presence of unlabeled PI liposomes did not help to stabilize or
accumulate [
H]PI in the medium. A second possible
explanation was that [
H]PI in the outer leaflet
of the liposome membrane bilayer was transferred to cells much faster
than that in the inner leaflet. To test this point,
[
H]PI liposomes remaining in the medium after
incubation with cells for 0.5-24 h were treated with PI-PLC at 4
°C. A constant 50-60% of the [
H]PI in
these liposomes was susceptible to PI-PLC cleavage throughout the
incubation period, presumably in the outer leaflet of the bilayer,
indicating no change in the transbilayer distribution of
[
H]PI in the donor liposomes as a function of
time. The factor(s) involved in stabilizing medium
[
H]PI liposomes and preventing complete transfer
of [
H]PI to HeLa S3 cells remain to be
established.
H]lyso-PI.
The topology of this acylation was studied by using PI-PLC at 4 °C
to cleave lyso-PI and the PI product on the outer leaflet of the cell
membrane. The levels of labeled lyso-PI and PI that remained cell
associated after PI-PLC treatment indicated intracellular lipids. The
labeled PI produced after brief incubation with
[
H]lyso-PI at 37 °C was distributed in the
outer leaflet to a greater extent than that produced after longer
incubations (Fig. 3), implying that at least some acylation
occurred in the outer leaflet. This was confirmed by examining labeled
lipids in cells after uptake of [
H]lyso-PI at 4
°C. No flipping of lyso-PI, PI phosphorylation, or PI glycosylation
occurred at this temperature, but some sn-2 acylation was
apparent (Fig. 4), and the [
H]PI formed was
almost entirely on the outer leaflet. Preliminary data on uptake of
[
H]lyso-PI at 15 °C indicate that over 40% of
the total label was converted to [
H]PI after 2.5
h, more than 85% of which remained on the outer leaflet (data not
shown). Although we expect that lyso-PI can be acylated to PI by fatty
acyl CoA-dependent enzymes in the cell interior, these data indicate
that acylation also can occur in the outer leaflet of the cell
membrane, perhaps by transacylation from other cell surface
phospholipids. The extent to which other lyso phospholipids
can be acylated in the outer leaflet of the cell membrane is unclear.
Acylation of exogenous lyso-PC was demonstrated in the plasma membrane
fraction of Vero cells, but no information on the topology of this
reaction was presented (Besterman and Domanico, 1992). The acylation
observed here was enzymatic, as [
H]lyso-PI
sonicated with 10 µ
M PI or PC showed no conversion to
[
H]PI in the 37 °C incubator for 24 h.
Furthermore, the enzyme(s) and other factors that catalyze the
acylation of lyso-PI observed here are not secreted by the cells. When
[
H]lyso-PI incorporated into PI or PC liposomes
was incubated with the cells, no acylation to
[
H]PI in the medium was observed over a 1-h
period (data not shown).
H]lyso-PI at
37 °C for 3 h corresponded to hydrophilic metabolites. Since these
metabolites were not formed when [
H]lyso-PI was
incubated with conditioned medium taken from the cells, they must
result from cell-associated phospholipase(s). Initial association of
lyso-PI with the cell surface may involve a ``nonspecific''
interaction in which the lyso-PI is more susceptible to cell surface
phospholipase(s). Rapid degradation of this nonspecifically associated
lyso-PI could account for the initial burst of hydrophilic metabolites
in the medium at the earliest time points in Figs. 2 B and
4 B. Subsequent integration of lyso-PI in a more
``specific'' way with the outer leaflet could decrease the
rate of degradation as indicated by later time points.
[
H]PI also may associate with the cells initially
in a similar nonspecific fashion that renders it highly susceptible to
degradative phospholipase(s). Both cell-associated lyso-PI and
hydrophilic metabolites in the medium are generated rapidly within the
first hour in Fig. 1and subsequently are produced at much slower
rates (Fig. 1). An attempt to bypass the proposed stage of
nonspecific interaction by preincubating cells with
[
H]lyso-PI at 4 °C before shifting the
temperature to 37 °C was unsuccessful. The same amounts of
hydrophilic metabolites were produced with or without preadsorption of
[
H]lyso-PI at 4 °C, and sn-2
acylation was only slightly enhanced by 4 °C preadsorption
(Fig. 5). Several murine cell lines, primarily fibroblasts in a
quiescent state of proliferation, have been shown to express a PI-PLC
activity on the cell surface (Ting and Pagano, 1990; Volwerk et
al., 1992). Exogenous fluorescent PI or
[
H]lyso-PI was taken up from the medium and
cleaved by this enzyme at 4 °C. Typically 5-15% of the total
[
H]lyso-PI was cleaved within the first few
minutes of addition (Volwerk et al., 1992), an observation
remarkably similar to that in Fig. 4 B. However, the
products of this cleavage released to the medium were exclusively
inositol phosphates and inositol cyclic 1,2-phosphate, in contrast to
the wider distribution of hydrophilic products in the medium, including
inositol and/or
sn-glycero-3-phospho-
D1- myo-inositol, that
we see after uptake of [
H]PI or
[
H]lyso-PI at 37 °C.
Figure 5:
Distribution of label following
[H]lyso-PI incubation with a temperature shift.
HeLa S3 cells were divided into two pools. One pool was incubated with
0.02 µCi of [
H]lyso-PI for 5 h at 4 °C,
washed with fresh DMEM, and then incubated for 1 additional h at 37
°C (
). Alternatively, the second pool was incubated
directly with 0.02 µCi of [
H]lyso-PI for 1 h
at 37 °C as in Fig. 2 (
). The levels of cellular lipids
(lyso-PI and PI) and medium hydrophilic metabolites were compared by
defining total label as the cellular [
H]lyso-PI
after 4 °C incubation (
) or the
[
H]lyso-PI added to the medium (
). Data
were from one experiment with duplicate plates of
cells.
GlcN(acyl)PI is an
important intermediate for GPI biosynthesis and is downstream from the
defect found in PNH. The ability to produce GlcN(acyl)PI in the
appropriate biosynthetic compartment of the stem cells affected by the
PNH mutation may enable the maturation of GPI-anchored proteins and
protect the cells from complement-mediated cell lysis. The accumulation
of labeled GlcN(acyl)PI from exogenous lipid precursors or from
incubation with [H]inositol in HeLa S3 cells is
unusual. It was not observed following [
H]PI or
[
H]lyso-PI incorporation into Madin-Darby canine
kidney cells or into primary cultures of rat hepatocytes or
Drosophila Schneider (S
) cells (data not shown).
Only PIP, lyso-PI, and PI could be observed after the addition of PI to
the two mammalian cell types, but no lipid modification was observed in
the insect cells. The accumulation of GlcN(acyl)PI in HeLa S3 cells
relative to other inositol phospholipids may prove to be a useful
indicator of the efficiency with which structurally defined PI
precursors enter the GPI biosynthetic pathway. For example, incubation
of Friend erythroleukemia cells with a labeled PI fraction enriched in
saturated fatty acids and lacking arachidonate resulted in negligible
metabolic conversion by phosphorylation or hydrolysis (Hohengasser
et al., 1986). Mammalian GPI anchors are known to be enriched
in sn-1-alkyl-2-acyl-glycerolipids (Roberts et al.,
1988; Walter et al., 1990; Singh et al., 1994), and
about 15% of the GlcN(acyl)PI in HeLa S3 cells appears to contain
alkylglycerol, in contrast to only a 1% alkylglycerol content in the PI
of these cells.
It is important to determine whether the
sn-1-alkyl-2- lyso analog of PI can be taken up and
converted to a GlcN(acyl)PI more efficiently than the lyso-PI studied
in this report.
Table:
Retention of H and
P in
inositol phospholipids metabolized from lyso-PI
H]lyso-PI (300,000 cpm) and
[
P]lyso-PI (40,000 cpm) were incubated with the
cells for 8 or 24 h with or without addition of 20 m
M sodium
phosphate. TLC chromatography of
H- and
P-labeled lipids extracted from the cells showed
distributions similar to that in Fig. 6 A for both isotopes.
Ratios of
P counts/min to
H counts/min for the
indicated TLC bands were determined as outlined under
``Experimental Procedures.'' Ratios in each band were similar
under the four labeling conditions, and means and standard errors are
shown.
, phosphatidylinositol
4,5-bisphosphate; GlcN(acyl)PI, GlcN-PI with a fatty acid acyl group on
inositol; PLA
, phospholipase A
; PI-PLC,
PI-specific phospholipase C; GPI, glycoinositol phospholipid; GPI-PLD,
GPI-specific phospholipase D; PC, phosphatidylcholine; lyso-PC,
sn-1-acyl-2- lyso-phosphatidylcholine; PNH, paroxysmal
nocturnal hemoglobinuria; DMEM, Dulbecco's modifed Eagle's
medium; TLC, thin layer chromatography.
H]lyso-PI and for helpful
discussions. We also thank Dr. Stuart Schwartz, Director of the
Cytogenetics Laboratory at Case Western Reserve University, for
karyotype analysis.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.