©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Uptake of Exogenous sn-1-Acyl-2- lyso-phosphatidylinositol into HeLa S3 Cells
REACYLATION ON THE CELL SURFACE AND METABOLISM TO GLUCOSAMINYL(ACYL)PHOSPHATIDYLINOSITOL (*)

Adisak Wongkajornsilp (1) (2), Terrone L. Rosenberry (1)(§)

From the (1) Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106 and the (2) Department of Pharmacology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 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.


INTRODUCTION

Phosphatidylinositol (PI)() 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.

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 10molecules/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.


EXPERIMENTAL PROCEDURES

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 NHOAc,and [H]lyso-PI (1 Ci/mmol) was eluted at 35% n-propyl alcohol. The P-labeled lipids were resolved prior to PLAtreatment by TLC in CHCl/CHOH/CHCOCH/CHCOOH/HO (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 10cells). 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/CHOH (1:1, v/v) to a final mixture of CHCl/CHOH/HO (10:10:3, v/v). The remaining cell pellet was re-extracted twice with 1 ml of CHCl/CHOH/HO (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 HO-saturated n-butyl alcohol and 200 µl of n-butyl alcohol-saturated water. The aqueous phase was removed and extracted with 200 µl of HO-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/CHOH/HO (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/CHOH/HO (10:10:3, v/v), partitioned with n-butyl alcohol and water, and analyzed by TLC as described above.


RESULTS

[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.

To conclude that exogenous [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.

The medium was also depleted of [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% COat 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 PIPbased 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.




DISCUSSION

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 [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 PIPfrom 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.

In contrast to the final distribution of cell-associated labeled inositol phospholipids, the distribution of the labeled species in the medium differed for exogenous [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.

It is noteworthy that we observed substantial sn-2 acylation of exogenous [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).

Almost all the label remaining in the medium after cell incubation with [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

HeLa S3 cells were cultured as outlined under ``Experimental Procedures'' except that 25 m M HEPES (pH 7.4) was added. Mixtures of [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.



FOOTNOTES

*
This work was supported by Grant DK38181 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

The abbreviations used are: PI, phosphatidylinositol; lyso-PI, sn-1-acyl-2- lyso-phosphatidylinositol; PIP, phosphatidylinositol 4-phosphate; PIP, 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.

D. Sevlever, D. R. Humphrey, and T. L. Rosenberry, submitted for publication.

D. Sevlever, D. Schiemann, and T. L. Rosenberry, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Dr. Daniel Sevlever for generous gifts of the HeLa S3 subline and [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.


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