Identification of glycoinositol phospholipids in rat liver by reductive ...

Vol. 267. No. 26, Issue of September 15,pp. 18581-18588.1992 Printed in U.S.A.

THEJOURNAL OF B~OLOGICAL CHEMISTRY 0 1992 by The American Societyfor Biochemistry and Molecular Biology, Inc.

Identification of Glycoinositol Phospholipids Rat in Liver by Reductive Radiomethylation of Amines but Notin H4IIE Hepatoma Cells or Isolated Hepatocytes by Biosynthetic Labeling with Glucosamine* (Received for publication, January 30, 1992)

Mark A. Deeg, NicoleR. Murray, and TerroneL. RosenberryS From the Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland,Ohio 44106

The identification of free glycoinositolphospholipids (GPIs) following biosynthetic labeling with [3H]glucosamine in culturedcells has been reported by several laboratories. We applied this procedure to two of the cell types used in these studies, H4IIE hepatoma cells and isolated hepatocytes, but were unable to detect a [3H]glucosamine-containing lipid that met any of the criteria for GPIs, including sensitivity to phosphatidylinositol-specific phospholipase C (PIPLC) or GPIspecific phospholipase D. Part of the difficulty in radiolabeling a GPI by this procedure was the rapid metabolic conversion of [3H]glucosamineto galactosamine and neutral or anionic derivatives. A PIPLCsensitive radiolabeled lipid was detected only after 16 h of labeling. The water-soluble fragments released from thislipid by PIPLC corresponded largely to myoinositol 1,2-cyclic phosphate and myo-inositol l-phosphate, products expected from PIPLC cleavage of phosphatidylinositol or lyso-phosphatidylinositol. In an alternative approach that we introduce here, free GPIs in lipid extracts from rat liver plasma membranes were labeled by reductive radiomethylation. This procedure, which radiomethylates primary and secondary amines, has been shown to label a glucosamine residue adjacent to inositol in all GPIs characterized to date. The labeled extracts were fractionated by a two-dimensional thin-layerchromatography,and cluster of polar labeled lipids were assigned as GPIs based upon the following observations. 1)They were cleaved by PIPLC, 2) after hydrolysis in 6 N HCl, both radiomethylated glucosamine and a glucosamine-inositol conjugate were identified by cation exchange chromatography, and 3) hydrolysis in 4 M trifluoroacetic acid generated a fragment consistent with glucosamine-inositol-phosphate. These results illustrate new criteria for the identification of GPIs. The labeled GPIs also contained radiomethylated ethanolamine, another component found in GPI anchors of proteins and in mature lipid precursors of GPI anchors, suggesting that theliver plasma membrane GPIs retained considerable structural homology to GPI anchors.

* This work was supported by Grant DK38181 (to T. L. R.) and National Research Service Award DK 08441 (to M. A. D.) 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 be hereby marked “oduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed.

Glycoinositol phospholipids (GPIs)’ serve as membrane anchors for a number of extracellularly oriented proteins in a wide range of organisms. A fewof these GPI anchors have been structurally characterized, including those of the trypanosome variant surface glycoproteins, rat brain Thy-1, scrapie prion protein, Leishmania major promastigote surface protease, and human erythrocyte acetylcholinesterase (reviewed in Cross, 1990).As described in the accompanyingpaper (Deeg et al., 1992), the core glycan sequence has been conserved among these diverse proteins and consists of 3 Man residues linked through nonacetylated GlcN to an inositol phospholipid. Heterogeneity among the GPI glycans derives from various groups that branch from the core glycan, including galactose oligomers in some variant surface glycoproteins and additional phosphoethanolamine residues in Thy-1 anderythrocyte acetylcholinesterase (Ferguson and Williams, 1988; Deeg et al., 1992). GPIs also have received considerable attention as free glycolipids. Most of the free GPIs whose structures have been established appear to be intracellular and involved in the biosynthesis of GPI anchors. The biosynthetic pathway has been described in Trypansoma brucei (reviewed in Doering et al. (1990)) and recently investigated in mammalian cells (Sugiyama et al., 1991; Hirose et al., 1991, 1992a, 1992b;Puoti et al., 1991). Free GPIs also have been proposed to play a role in insulin signal transduction (Saltiel andCuatrecasas, 1988). Inthis proposal, insulin binding toits receptor leads to phospholipase C cleavage of a GPI and generation of two intracellular signals, diacylglycerol and an inositol glycan. Extracts containing thisglycan appear to mimic the action of insulin in a number of intact cell and in vitro assays (reviewed in Romero (1991)). Reports of GPIs in partially purified lipid extracts from a number of insulin-sensitive cells, including H35 hepatoma cells (Mato et al., 1987a), BCaH1myocytes (Suzuki et al., 1991), adipocytes (Macaulay and Larkins, 1990), and activated T- and B-lymphocytes (Gaulton et al., 1988; Eardley and Koshland, 1990) have been based on biosynthetic labeling with several GPI anchor components, including [3H]GlcN and on cleavage of the labeled lipids with bacterial phosphatidylinositol-specific phospholipase C (PIPLC) or nitrous acid. However, thestructure of these glycolipids has not been definitively elucidated. Preliminary The abbreviations used are: GPI, glycoinositol phospholipid; PI, phosphatidylinositol; PIP2, phosphatidylinositol 4,5-bisphosphate; PIPLC, phosphatidylinositol-specific phospholipase C; GPI-PLD, glycoinositolphospholipid-specific phospholipase D; HPLC, high performance liquid chromatography; EthNMe, N-methylethanolamine; EthN(Me),, N,N-dimethylethanolamine;GlcNMe, N-methylglucosamine; GlcN(MeIz, N,N-dimethylglucosamine; GlcN(Me)2-Inos, N,N-dimethylglucosamine-inositol; GlcN(Me)2-Inos-P, N,N-dimethylglucosamine-inositol-phosphate;DMEM, Dulbecco’smodified Eagle’s medium.

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Identification of Glycoinositol Phospholipids

inLiver Rat

composition analyses of the glycolipids isolated from hepato- extracted twice with 30 volumes of ethanol/water/ethyl ether/pyricytes (Mato et al., 1987b) and BC3H1 cells (Suzuki et al., dine/NH40H (15:15:5:1:0.018, v/v) at 60 "C for 15 min, and the 1991) suggested 3-4 Gal residues per residue of GlcN and combined extracts were taken for analysis. Method 4 also was used toextractGPIs from trypanosomes inositol. Larner et al. (1988) separated two putative inositol (Menon et al., 1988). Pelleted cells (400 pl) were lysedby adding 2 ml glycan fractions from rat liver extracts, one containing GlcN of water at 0 "C, and the lysate was lyophilized and washed three that inhibited CAMP-dependentprotein kinase and adenylyl- times with 1 ml of chloroform, methanol, 6 N HC1 (100:500.1, v/v) cyclase and the other containing GalN that inhibited pyruvate and thenfive times with 3 ml of chloroform/methanol(2:1,v/v). The washed insoluble material was dried under NP andextracted with 5 dehydrogenase. To investigate structural similarities between the GPIs in ml of methanol/pyridine/water (2:1:1, v/v) overnight at 4 "C and then with 3 ml for 4 h at room temperature. The combined methanol/ protein anchors and insulin-sensitive glycolipids, we pursued pyridine/water extracts were dried in the SpeedVac, and the residue the isolation of free GPIs from liver-derived sources. We was partitioned with 1-butanol as in Method 2. report here that we are unable to detect GPIs labeled biosynMethod 5 has been applied to extract gangliosides from bovine thetically with [3H]GlcNin H4IIE hepatoma cells or isolated brain (Svennerholm and Fredman, 1980) and GPIs from L. major hepatocytes. However, by extending reductive radiomethyla- (McConville and Bacic, 1989). Pelleted hepatocytes (400 pl) were twice with 20 volumes of chloroform/methanol/water(4:83, tion procedures previously applied to GPIs in protein anchors,extracted v/v) for 30 min at room temperature. Water was added tothe we were able to label a number of glycolipids in lipid extracts combined extracts to give a final ratio of 485.6. The phases were from rat liver that fulfill the criteria for GPIs. separated by centrifugation. The lower phase was washed with 0.8 EXPERIMENTALPROCEDURES

volumes of methanol, 0.01 M KC1 (32, v/v) and centrifuged, and the two upper phases were combined, dried, and partitioned with 1butanol as described for Method 2. Radiomethylation of Lipids from Liver Plasma Membranes-Livers from fed, male Sprague-Dawley rats (175-250 g) were removed, and plasma membranes were prepared through step 11 as previously described (Neville, 1968) and lyophilized. Lyophilisate (200 mg from about one liver) was extracted by Method 4 above except that 4-ml rather than1-ml aliquots of the first wash solvent were applied. Total extracts from the chloroform, methanol, 6 N HCl wash, the chloroform/methanol wash, and the final 1-butanol phase were dried individually and resuspended by sonication in 100 p1 of water. Free amino groups in these lipid extracts were radiomethylated (Haas andRosenberry, 1985) with 10 mM ['4C]HCH0 (ICN, 56 mCi/mmol) and 63 mM NaCNBH3 at 37 "C for 60 min. After extensive dialysis against 10 mM sodium phosphate (pH 7.0), the radiolabeled samples were partitioned with 1-butanol, and the dried organic phases were stored at -20 'C. In typical radiomethylations with 1 pmol of ['4C]HCH0 (100 X lo6 cpm), about 9, 2, and 7% of the radioactivity was incorporated into the stored products from the first, second, and third lipid extracts, respectively. Enzymatic Cleavage of Lipids-Dried lipid samples resuspended by sonication in 50pl of 20 mM sodium phosphate (pH 7.0)were incubated with purified Bacillus thuringiensis PIPLC (Deeg et al., 1992) a t a final concentration of 2 pg/ml a t 37 "C for the time indicated. To assess lipid cleavage by GPI-PLD partially purified' from bovine serum (Davitz et al., 1989),lipids were dried, resuspended by sonication in 100 pl of 0.01% Nonidet P-40, 10 mM Tris-C1, 2.5 mM CaC12,10 mM NaCl (pH 7.8), and incubated overnight with GPIPLD (10 pl of a 1:20 dilution of stock) a t 37 "C. Cleavageof [3H]PI or the Proteinase K fragment of human erythrocyte acetylcholinesterase (Deeg et al., 1992)providedpositive controls for both enzymatic reactions. Thin Layer Chromatography-For one-dimensional TLC, dried samples were resuspended in water-saturated 1-butanol and loaded onto a Silica Gel60 plate. The plates were precoated with 1% potassium oxalate and preactivated by heating at 110 "C for 30-60 min. The samples were applied as 1-cm streaks and developed with chloroform, methanol, 4 M NH40H (453510,v/v). The dried plates were sprayed with EN3HANCE (Du Pont), dried overnight, and exposed to preflashed Kodak XAR filmfor 3-14 days at -70 "C. Twodimensional TLC was performed as described previously (McConville and Bacic, 1989). Briefly, lipid samples were applied to preactivated Silica Gel 60 plates and developed in the first dimension with chloroform, methanol, 0.2% KC1 (w/v) in water (10103, v/v). The dried plates were then developed a second time in the first dimension with 1-butanol, pyridine, 0.2%KC1(9:6:4, v/v). The plates were dried overnight, rotated go", developed in the second dimension with chloroform, methanol, 4 N NH,OH (45:35:10, v/v), and fluorographed as described above. Column Chromatography of Lipids-Lipid extracts (5-6 ml) were depleted of divalent cations by application to a 0.5 X 2-cm column of Chelex 100 (sodium form) that was prewashed with 5 ml of the lipid

Materiokr-Iatrobeads 6RS-8060 and Chelex 100 were purchased from Iatron Laboratories (Tokyo, Japan) and Bio-Rad, respectively. Silica Gel60 TLC plates were from EM Separations (Gibbstown, NJ), DMEM and DMEMIF-12 culture media and fetal calf serum were from GIBCO, and tunicamycin was from Sigma. H4IIE hepatoma cells were kindly provided by Dr. R. Hanson, Case Western Reserve University. Rats (Sprague-Dawley)were obtained from ZivicMiller (Pittsburgh, PA). All reagents were of analytical grade. Biosynthetic Labeling-H4IIE hepatoma cells were grown to confluence at 37"C on 100 X 15-mm platesin DMEMIF-12 media containing 22 mM glucose and supplemented with 10% fetal calf serum. Except where noted, each plate of cells was labeled for 16-20 h in 4 ml of DMEM containing 5.5 mM glucose, 10 pCi of [6-3H]GlcN (33 Ci/mmol; Du Pont), and 10% heat-inactivated fetal calf serum. Isolated hepatocytes were prepared from fed, male rats (250-350 g) as described previously (Brass et al., 1984). Hepatocytes were suspended to a final density of 1 X lo' cells/ml in 117 mM NaCI, 4.7 mM KC1, 1.2 mM CaC12,1.2 mM KHzP04, 2.4 mM MgS04, 2.5 mM NaHC03, and 0.1 mM D-glucose (pH 7.4). [3H]GlcN was added (20 pCi/ml) and the cells were incubated a t 37 "C for 2 h with an atmosphere of 95% 02,5% COz. When present, tunicamycin was added from 100 X stock 30 min before the 13H]GlcN.At the end of the labeling period, the cells were centrifuged for 2 min a t 1000 X g, the media was removed, and thelipids were extracted. Lipid Extraction-Five different methods were used based on published procedures. All lipid extracts were stored a t -20 "C until further analysis. Method 1 was applied to theextraction of putative insulin-sensitive GPIs from H35 hepatoma cells (Mato et al., 1987a), hepatocytes (Merida et al., 1988), and T-lymphocytes (Gaulton etal., 1988). Briefly, incubations were terminated by removing the media and adding 2 ml of 5% trichloroacetic acid at 0 "C to the plate of cells. Cells were collected by scraping and centrifugation a t 1500 X g for 5 min. The cell pellet (estimated a t 100 pl/plate) was mixed with 30 volumes of chloroform/methanol/HCl (1:2:0.013, v/v) for 15 min a t room temperature, and theinsoluble residue was further extracted with one-half the volume of the same solvent. The combined extracts were separated into organic- and aqueous-rich phases by adding 15 volumes each of 0.1 M KC1 and chloroform. The upper aqueous phase was washed with 15 volumes of chloroform and thecombined organic phases were washed with 15 volumes of 0.1 M KC1 in 50% methanol and dried in a Savant SpeedVac. Method 2 was used to extract GPIs from trypanosomes (Mayor et al., 1990). Plates of hepatoma cells were washed with phosphatebuffered saline, scraped, and centrifuged at 500 X g for 2 min. The cell pellet (100 pl) was mixed with 10 volumes of chloroform/methanol (2:1, v/v) and centrifuged again, and theinsoluble residue was further extracted with 30 volumes of chloroform/methanol/water (10103, v/v). The second extract was dried, resuspended in 0.5 ml of watersaturated 1-butanol and partitioned with an equal volume of water. The water phase was washed with an equal volume of water-saturated 1-butanol, and thecombined organic phases were taken for analysis. Method 3 was originally developed for the extraction of polar GPI-PLD activity per milligram of protein was increased about glycosphingolipids from yeast (Hanson and Lester, 1980) and was 10-fold by chromatography of dialyzed bovine serum on phenylused to extract GPIs from L. major (Turco et al., 1989). Incubations Sepharose. (D. Sevlever and T. L. Rosenberry, unpublished observawere terminated with trichloroacetic acid as in Method 1,cells were tions.)

Identification of Glycoinositol Phospholipids in Rat Liver extraction solvent. The column was washed with 3 ml of the solvent and the combined eluants were dried. Recovery of counts/min was typically >go%. The sample was resuspended in solvent A (chloroform/methanol/NH,OH, 65:296, v/v) and applied to an Iatrobead column (1 X 60 cm). Gradient elution with solvents A and B (chloroform/methanol/water/NH.OH, 42:40:12:6, v/v) was controlled by a Beckman 406 analog interface to two Beckman 112 HPLC pumps set at 1 ml/min according to the following schedule: 0-180 min, % B = 20(t/180)”‘ (Barr and Lester, 1984); 180-225 min, linear increase in % B to 30%;225-345 min, 30% B. Fractions of 2 ml werecollected. HPLC Analysis of Inositol Phosphates-Lipid samples were incubated with PIPLC and partitioned with 1-butanol, and the aqueous phase was analyzed by anion exchange Dionex CarboPac PA-1 HPLC (Deeg et al., 1992) with isocratic elution by 100 mM NaOH and 140 mM sodium acetate. Elution of myo-inositol cyclic 1,2-phosphate and myo-inositol 1-phosphate added as internal standards was monitored by pulsed amperometric detection (Deeg et al., 1992), and fractions (0.5 min) were scintillation counted for radioactivity. Cation Exchange Chromatography of Labeled Amines-bdiolabeled lipid samples corresponding to a dried organic extract, column fraction, or silica scraped from a TLC plate were hydrolyzed in 6 N HC1 a t 115 “C for the time indicated. The hydrolysate was subjected t o cation exchange chromatography on a Beckman 119CL amino acid analyzer (Haas and Rosenberry, 1985; Haas et al., 1986; Deeg et al., 1992). GlcN orGalN added as aninternal standard was detected with ninhydrin. Trifluoroacetic Acid Hydrolysis of Glycolipids-Radiolabeled lipid samples corresponding to a dried organic extract or to silica scraped from a TLC plate were hydrolyzed with 200 p1 of 4 M trifluoroacetic acid at 100 “C for 4 h. The dried hydrolysates were resuspended in 200 pl of water, clarified by centrifugation, and analyzed by anion exchange Dionex CarboPac PA-1 HPLC for the presence of GlcNcontaining fragments consistent with GPIs (see Deeg et al., 1992).

RESULTS

Survey of Hepatoma Lipids Labeled with PHJGlcN-We first investigated the metabolic labeling of hepatoma lipids with [3H]GlcNby a procedure reported to label GPIs in H35 hepatoma cells (Mato et al., 1987a). Following extraction of labeled cells and two-step TLC purification of lipids, two predominant lipid bands were observed with RFvalues of 0.42 and 0.45 that were comparable with those of the reported GPIs in H35 cells. However, these labeled lipids were not cleaved by PIPLC (Fig. lA). To determine if the labeled lipids corresponded to GPIs resistant to PIPLC cleavage, the lipid bands were hydrolyzed with trifluoroacetic acid. Trifluoroacetic acid hydrolysis of GPIs generates a characteristic fragment, GlcN-inositol-phosphate (Deeg et al., 1992), that can be identified by anion exchange chromatography. The trifluoroacetic acid hydrolysates of the hepatoma lipids gave two radioactive peaks resolved by anion exchange chromatography (Fig. lB), with hexosamines (GlcN and GalN)’ accounting for 65% of the radioactivity and anunidentified peakthat eluted between hexoses and hexose monophosphates with a retention time of 25 min accounting for 7%. No radioactivity corresponding to theGlcN-inositol-phosphate elution timeof 31 min was observed. In examining these protocols, we identified three concerns that may have accounted for these negative results: low cellular uptake of [3H]GlcN, inefficient extraction of [3H]GlcNcontaining lipids, and significant metabolic conversion of the [‘HIGlcN taken upby the cells. Under the labeling conditions in Fig. 1, only 9 f 4% (mean k S.E., n = 7) of the radioactivity in the medium was taken up after 20 h. By decreasing the incubation volume from 10 to4 ml and theGlc concentration from 22.5 to 5.5 mM, radiolabel uptake was increased nearly &fold to 46 f 10% (n = 2). This increase in cellular uptake was associated with an increase inthe percentage of radiolabel extracted aslipid from 0.5%f 0.2 ( n= 3) to 1.3% k 0.4 ( n = 2) of the initial [3H]GlcN present in themedium. After 16 h of labeling with thesemodifications, nearly 200,000 cpm/plate

E

18583

1000

0 PI

L e

500

0 0 0PIPLC

-

+

20

40

60

minutes

FIG.1. PIPLC resistance and trifluoroacetic acid hydrolysis of lipids labeled by incubation of hepatoma cells with [‘HI GlcN and isolated by two-step TLC. H4IIE cells were labeled for 20 h in DMEM containing 22 mM glucose, 10% fetal calf serum, and 2.5 mCi/ml of [3H]GlcN(10 ml/plate; Mato et al. (1987a)),and lipids were extracted by Method 1 as described under “Experimental Procedures.” The lipids were partially purified by a two-step TLC procedure (Mato et al., 1987a). Briefly, the initial lipid extract was spotted on Silica Gel 60 plates and developed twice in chloroform/ acetone/methanol/glacial acetic acid/water (50:2010105, v/v). Silica from the area 0.5 cm below to 1 cm above the origin was scraped and washed with methanol, and the concentrated washings were spotted on the second TLC plate and developed with chloroform, methanol, 4 N NH,OH (45:35:10, v/v). In a typical preparation, 9-10% of the applied radioactivity was recovered near the origin of the first plate. Panel A, lipids washed from the first TLC plate were dried, resuspended, treated with or without PIPLC as indicated, and spotted and developed on the second TLC plate. Quantitation of the radioactivity in each lane by radioscanning revealed no difference between the control and the PIPLC-treated samples. TLC markers indicate the solvent front ( F ) ,PI, and the origin (0).Panel B, silica containing the two major radiolabeled lipid bands scraped from the second TLC plate was hydrolyzed with trifluoroacetic acid and analyzed by anion exchange chromatography as described under “Experimental Procedures.” The elution times of internal standards (indicated by the triangles) correspond to the following order: GalN, GlcN, Glc, Man, inositol 2-phosphate, and Man 6-phosphate.

was extracted by the procedure used by Mato et al. (1987a) (Method 1 in Table I). The radiolabel in this extract was analyzed by cation exchange chromatography after hydrolysis in 6 N HCL and shown to consist of0.1% GlcN and 3.8% GalN. The remainder corresponded to anionic or neutral derivatives or metabolites of [3H]GlcN.Extraction with more polar solvents improved the yield of lipids containing hexosamine. Method 2, employing sequential extraction with chloroform/methanol (29, v/v) and chloroform/methanol/water (10:10:3, v/v), yielded 20% more total radiolabeled lipid than Method 1 but increased the recovery of labeled GlcN and GalN more than 10-fold (Table I). The basic polar solvent in Method 3 extracted 65% more radiolabel than in Method 1 with 32% corresponding to [3H]GlcN (Table I). Lipids extracted by Methods 1-3 gave qualitatively similar TLC patterns of six to seven major bands by TLC, butMethods 2 and 3 were more efficient in extractingthe more polar lipid bands with lower mobilities (Figs. 2 and 5B). The lipid extraction procedure in Method 3 was used for the remainder of the hepatoma studies. In optimizing the uptake of [3H]GlcN into hepatoma cells, it was observed that the presence of serum increased the amount of extracted radiolabeled lipid by 20-30% (data not shown). The increase in extracted radiolabel was associated with an increase in the proportion of radiolabel in the three most polar TLC bands(Fig. 3). This effect did not arise from

Identification of Glycoinositol Phospholipids

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TABLE I

inLiver Rat F -

Comparison of extraction procedures of pH]GlcN-labeled lipids in hepatoma cells H4IIE hepatoma cells were labeled with [3H]GlcN and extracted by Methods 1, 2, or 3 as outlined under “Experimental Procedures.” Aliquots of extracts were hydrolyzedwith 6 N HCl for 4 h and analyzed for hexosamine content by cation exchange chromatomaDhy. ~~~~~~~i~~ method

% of cpm in lipid extract in

% total cpm in

lipid extract

GalN GlcN

Neutral/anionic”

PC

-

PI

-

0.9* 0.9‘

4 80 0.1 10 23 54 0.4d 29 2 47 3 1.5 32 20 31 “Three peaks of radioactivity from the cation exchange column accounted for 75-80% of the recovered radioactivity in all hydrolysates. These included GlcN, GalN (see Footnote 3), and neutral or anionic components that were not retained on the column. Three unidentified minor peaks a t 17, 39, and 90 min, none of which exceeded 7% of the recovered radioactivity, accounted for most of the remaining counts/min. Recovery of the hexosamines was corrected for the 5%degradation of hexosamine standards during hydrolysis. A 3-fold increase in the relative volume of the extraction solvent did not increase the total counts/min extracted. First extract in chloroform/methanol (2:l). Second extract in chloroform/methanol/water(10103).

1 2

-F

El

1500 21 I

0 I

counts

r I

0 PIPLC

+

- +

-serum

-

+serum

FIG.3. Effect of serum on the radiolabeling of hepatoma lipids with [3H]GlcN. Hepatoma cells were radiolabeled for 20 h with or without 10% fetal calf serum as described under “Experimental Procedures.” Lipids were extracted by Method 3, and a portion of the extract (13,000 cpm) from each labeling condition was incubated with or without PIPLC as indicated. The radiolabeled lipids were analyzed by one-dimensional TLC. 0, origin; PC, phosphatidylcholine; F, solvent front.

metabolic conversion were examined. Total radiolabel in the culture medium disappeared linearly at about 4%/h for 8 h -PI and thendecreased slowly until 45-50% of the initial amount E3 remained at 24 h (data not shown). In contrast, the amount 1500 of [’HIGlcN in the medium decreased linearly, whereas the amount of labeled lipid increased linearly throughout the 24 counts h of labeling (Fig. 4A). The appearance of lipid radiolabel that no longer corresponded to GlcN was rapid. After 1 h of labeling, the percentage of lipid label corresponding to [3H] 0 0 4 a 12 16 GlcN was only 44%, and the amountdecreased to 20% after -0 24 h (Fig. 4B). Lipid label corresponding to GalN varied from El E3 crn 15 to20% throughout the 24 h of labeling, whereas radiolabel FIG. 2. Comparison of two lipid extraction procedures. Hep- in neutral or anionic components increased from 25% after 1 atoma cells were radiolabeled for 16 h, and aliquots of lipids were h to 35% after 24 h of incubation. extracted by Method 1 ( E l , 10,000 cpm) or Method 3 (E3, 13,000 Individual hepatoma lipid bands labeled after a 20-h incucpm)and analyzed by one-dimensional TLCas described under “Experimental Procedures.” The TLC plate lanes were both fluoro- bation with [3H]GlcNwere partially purified by column chrographed for 8 days (left) and scanned for radioactivity with a Bioscan matography on silica (Fig. 5A), and peaks of radioactivity Imaging Scanner System 200 for 60 min/lane (right). The detector corresponded to themajor bands seen by TLC (Fig. 5B). The response, represented as total counts, is 98%. Panel B (right),fractions corresponding to peaks of radioactivity were pooled, dried under vacuum, and analyzed by one-dimensional TLC. Lanes 1-6 in panel B correspond to peaks 1-6 in Panel A . Panel B (left),lipids from identically labeled hepatoma cells were extracted by Method 2, and portions (15,000 cpm) were treated with or without PIPLC as indicated. 0, origin; F, solvent front. B

A

F -

F -

PIPI

-

?? ”

0” PIPLC

0 -

+

E4

+

-

Tunicamycin

0 0.1 0.5 1

5 ug/mL

E5

FIG.6. [‘HIGlcN labeling of hepatocytes. Isolated hepatocytes were incubated with [‘HIGlcN, and lipids were extracted by Methods 3-5 as outlined under “Experimental Procedures.” Panel A , extract aliquots from Method 4 (E4, 27,000 cpm) or Method 5 ( E 5 , 23,000 cpm) were treated with or without PIPLC for 1.5 h. Panel B, cells preincubated 1 h with the indicated concentration of tunicamycin prior to addition of [3H]GlcN were extracted by Method 3, and 5% of each extract (4,000 cpm for the control without tunicamycin)was analyzed by one-dimensional TLC. 0, origin; F, solvent front. tocytes with [3H]GlcNsuggested that rates of GPI synthesis in these cells are low. Exogenous radiolabeling of lipids is an alternative approach in which GPI detection limits depend only on steady-state levels and not on synthesis rates. In particular, reductive radiomethylation is attractive because

Identification of Glycoinositol Phospholipids Ratin

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labeling is limited to primary and secondary amine groups, including those in the GlcN and ethanolamine components that are characteristic of GPIs. This procedure has been used to label these aminegroups in the GPIsof several mammalian GPI-anchored proteins (Rosenberryet al., 1989), and a report of putative GPIs in extracts of hepatic plasma membranes (Saltiel and Cuatrecasas, 1986) prompted us to apply the procedure to this tissue. Plasma membranes were prepared from rat liver and lipids were sequentiallyextractedwith increasingly polar solvents (Method 4). Amines were radiomethylated and analyzed by two-dimensional TLC (Fig. 7). In the first (Fig. 7, A and B ) and second (data not shown) extracts, no spotswere identified that were cleaved by PIPLC. However, in the final lipid extract, 8-10 clustered spots with low TLC mobilities characteristic of polar lipids were cleaved by PIPLC (Fig. 7, C and D).Treatment of the lipid extracts with GPI-PLD prior to radiomethylation removed only this cluster of spots from the two-dimensional TLC profiles (data not shown), supporting their assignment as GPIs. Spots in

A

FirstExtract

B 1

Liver

this cluster were analyzed either together or after division into groups a-c as indicated in the legend to Fig. 7. 6 N HC1 hydrolysis and cation exchange chromatographic analysis of the labeled amine groups revealed that the entire cluster of spots, as well as each of the groups a-c contained radiomethylated GlcN and ethanolamine (Fig. 8 and data not shown). The hydrolysates contained boththe mono- and dimethylated derivatives GlcNMe, G ~ C N ( M ~ EthNMe, )~, and EthN(MeIn, a characteristic hydrolysis degradation product of radiomethylated GlcN, and a peakof radioactivity that corresponded to radiomethylated GlcN-inositol (Deeg et al.,1992). To confirm the presence of Gl~N(Me)~-1nos-P in these radiomethylated lipids, trifluoroacetic acid hydrolysates of the PIPLC-sensitive spots andof the GPI anchor of radiomethylated human erythrocyte acetylcholinesterase were analyzed by anion exchange chromatography. Lipids a (Fig. 9B), b, and c (data not shown), and the acetylcholinesterase GPI (Fig. 9A) gave identical profiles with three peaks of radioactivity. The nonretained 2.5-min peak corresponded exclusively to EthNMe and EthN(Me),when analyzed by cation exchange chromatography, and the31-min peak had the same retention time as Gl~N(Me)~-1nos-P (Deeg et al., 1992). Further hydrolysis of this peak with 6 N HC1 followed by cation exchange chromatography confirmed the presence of GlcNMe, G ~ C N ( M and ~ ) ~their , hydrolysis products. 6 N HCl hydrolysates of the 43-min peak also contained only GlcNMe, G ~ C N ( M and ~ ) ~ their , hydrolysis products. DISCUSSION

C

ThirdExtract

Prior to this report, the identification of free GPIs has depended upon their biosynthetic labeling with GPI compo-0 nent precursors in cells or cell lysates. This approach led to elucidation of a GPI biosynthetic pathway in trypanosomes

D 1500

7 EthNIMe), \

1000

n

I

Ib control

+

o

120

B

EthNMe

PIPLC

FIG. 7. Two-dimensional TLC analysis of radiomethylated lipid extracts from rat liver plasma membranes. Plasma membranes were prepared and partitioned in three extracts of increasing polarity (Method 4), and extractswere radiomethylated and analyzed by two-dimensional TLC as outlined under “Experimental Procedures.” Samples (20 pl) of the first(chloroform/methanol/HCI, 0.4 X lo6 cpm) and third (methanol/pyridine/water,0.9 X lo6 cpm) radiolabeled extracts following dialysis were treated overnightwith or without PIPLC(2 pg/ml) and partitionedwith 1-butanol.The organic phases from the first (panels A and B ) and third (panels C and D ) extracts, containing >97% of the initial extract radioactivity, were subjected to two-dimensional TLC and fluorographed for 2.5 days (panels A and B ) or 5 days (panels C and D).The PIPLC-sensitive spots (arrow) were subdivided into three groups: a, the four spots most mobile in the first dimension; c, the three spots leastmobile in both dimensions; b, the remaining spots of intermediate mobility. Radioscanning of the TLC plate panel in C indicated that the PIPLCsensitive spots corresponded to 1.2% of the total radioactivity detected. The intersection of the solvent fronts in both dimensions is indicated by r. Phospholipid standards had the following R p values in the first andsecond dimensions, respectively: PI, 0.74, 0.39; phosphatidylcholine, 0.47, 0.44; PIPz, 0, 0.

GlcNMe

4 80 0

120

40

minutes

FIG.8. Identification of radiolabeled amine components in PIPLC-sensitive lipids from liver plasma membranes. Silica spots in groups a-c containing the PIPLC-sensitive lipids identified in Fig. 7 were scraped and hydrolyzed with 6 N HCl for 8 h at 115 “C. The labeled amine components were identified by cation exchange chromatography (panel B ) by reference to previously identified radiomethylated component standards (panel A ) from 6 N HCI hydrolysates of the GPI anchor from human erythrocyte acetylcholinesterase (Haas et al. (1986), Deeg et al. (1992); see “Experimental Procedures”). GlcNfMe), Product denotesacharacteristic hydrolysis degradation product of radiomethylated GlcN (Haas et al., 1986). Further 16-h 6 N HCl hydrolysis of the fractions in panel B corresponding to GlcN(Me),-Inos converted more than one-half of the radioactivity toGlcN(Me)zandits hydrolysis product (datanot shown).

Identification of Glycoinositol Phospholipids in Rat Liver 900 -

600 -

cpm 300 .

0 -: 200 -

cpm 100

-

0 -:

0

20

40

60

minuter

FIG. 9. Trifluoroacetic acid hydrolysis of PIPLC-sensitive lipids from liver plasma membranes. Silica spots in group a identified in Fig. 7 were scraped and treated with 4 M trifluoroacetic acid for 4 h at 100 "C. The labeled amine components were identified by anion exchange chromatography (panel B ) by reference to previously identified radiomethylated component standards (panel A ) from 4 M trifluoroacetic acid hydrolysates of the GPI anchor from human erythrocyte acetylcholinesterase (Deeg et al. (1992); see "Experimental Procedures").The peak at 43 min represents incomplete hydrolysis of species larger than GlcN(Me)2-Inos-P(Deeg et al., 1992).

(Doering et al., 1990) and to confirmation that thecore glycan structure of the products of this pathway corresponded to that of the GPI anchors of trypanosome proteins (Mayor et al., 1990; Menon et al.,1990; Field et al.,1991). A similar approach recently has identified a number of GPIs in mammalian lymphoma and HeLacells that also may be precursors of GPI anchors on proteins (Sugiyama et al., 1991; Hirose et al., 1991, 1992a, 1992b; Puoti et al., 1991). The mammalian GPIs could be labeled by incubation of the intact cells with t3H]Man in the presence of tunicamycin or with [3H]ethanolamine, anda number of criteria supported theirclassification as GPIs. These included 1) their cleavage by GPI-PLD and by HN02 and 2) the correspondence of their dephosphorylated HNOz fragments to trypanosome GPI fragment standards by both Bio-Gel P4 andDionex anion exchange chromatography. These mammalian GPIs were cleaved by PIPLC only after pretreatment with base, indicating acylation of an inositol hydroxyl group (Roberts et al., 1988). The biosynthetic labeling of GPIs in a number of mammalian cells including H35 hepatomas had been reported earlier (Saltieland Cuatrecasas, 1986; Mato et al., 1987a), and these GPIs were proposed to be cleaved by an endogenous phospholipase C in response to treatment of the cells with insulin. The glycolipids labeled inthese procedures differ significantly from the [3H]Man-and[3H]ethanolamine-labeled GPIs noted above. The insulin-sensitive glycolipids were labeled by 24-h incubation with tritiated GlcN, Gal, myoinositol, glycerol, or myristic acid, but not with tritiated Man or ethanolamine (Mato et al.,1987a; Gaulton et al.,1988; Suzuki et al., 1991).They were cleaved not only by HNOz but also by direct treatment with PIPLC without pretreatment with base (Gaulton, 1991).However, to our knowledge, neither their susceptibility to GPI-PLD nor the correspondence of their labeled glycan fragments to trypanosome GPI fragment standards hasbeen reported. The insulin-sensitive glycolipids may represent GPIs with a core glycan structure that diverges significantly from GPI anchors on proteins. In Leishmania, a family of free GPIs has been identified with core glycans

18587

similar to that of an extracellular lipophosphoglycan (McConville et al., 1990) and thusmay serve as lipophosphoglycan anchor precursors. This core glycan differs from that of the GPI protein anchors in retaining only the first Manadjacent to GlcN and replacing other residues of the core with sugars that include Gal (Turco et al., 1989). Alternatively, labeling of the insulin-sensitive glycolipids may reflect considerable metabolic conversion of the tritiated precursors. Our results in hepatoma cells indicated rapid metabolic conversion of [3H]GlcN to other components in extracted lipids and identified the radiolabel in the only PIPLC-sensitive labeled lipid as inositol. This PIPLC-sensitive lipid may berelated to a lipid labeled with CDP-glyceride and [3H]myo-inositolin human liver microsomes that exhibited TLC mobility similar to that reported for the insulinsensitive glycolipids (Thakkar et al., 1990). Further analysis of this labeled microsomal lipid revealed a mixture of lysophospholipids, including lyso-PI but no evidence of a GPI. The primary pathways for GlcN metabolism in hepatocytes include 5"epimerase conversion of UDP-GlcNAc to UDPGalNAc (Maley and Maley, 1959). In addition, GlcN is converted to sialic acid via mannosamine (Harms et al., 1973). All three of these hexosamine5 may be present in our [3H] GlcN-labeled hepatoma lipid extract^.^ Although sialic acid was not explicitly identified in our studies, this component would not be retained on a cation exchange column and may account for some of the radioactivity identified in peaks 1-4 in Fig. 5. GPI-anchored proteins have been reported in hepatocytes (Perelman and Brandan, 1989; Misumi et al., 1990) and presumably are present inhepatoma cells; thus, free GPI precursors of these anchors should be present. Our inability to identify a [3H]GlcN-labeledlipid that satisfied the criteria for a GPI suggests that the amounts of these lipids are small and thatthey aredifficult to detect by [3H]GlcNbiosynthetic labeling because of the rapid metabolic conversion of this precursor. In a new approach to identifying free GPIs, we employed a reductive radiomethylation procedure that has been used to label GlcN and ethanolamine components in the GPI anchors of human erythrocyte acetylcholinesterase (Haas et al., 1986) and decay-accelerating factor (Medof et al., 1986), rat brain Thy-1 (Fatemi et al., 1987), and rat kidney folate-binding protein (Lee et al., 1992). A group of a t least eight lipids from rat liver plasma membranes were identified that met several criteria for GPIs. These included 1)cleavage by PIPLC and GPI-PLD, 2) generation of radiomethylated GlcN, ethanolamine, and the GlcN-inositol conjugate by hydrolysis in 6 N HCl, and 3) identification of radiomethylated GlcN-inositolphosphate following hydrolysis in 4 M trifluoroacetic acid. Standards for the GlcN-inositol and GlcN-inositol-phosphate conjugates were established through analysis of the GPI anchor of human erythrocyte acetylcholinesterase as outlined in the accompanying paper (Deeg et al., 1992).It is unlikely that the radiomethylated GPIs represent anchors cleaved by proteolysis of an attached protein, since acid hydrolysis did not reveal the N"-radiomethylated amino acid that wouldbe expected from a residual peptide. However, the relationship of the free GPIs labeled by radiomethylation to thet3H]Manand [3H]ethanolamine-labeledGPIs identified in the biosynthetic studies above is unclear. Both classes of GPIs contain ethanolamine, a component found in all mammalian GPIAlthough the anion and cation exchange chromatography systems employed in this study clearly resolved GlcN and GalN, mannosamine could not be sufficiently resolved from GalN for unequivocal identification. Thus, components denoted GalN could be in part or whole mannosamine.

18588

Identification of Glycoinositol Phospholipids in Liver Rat

anchored proteins that have been characterized. In contrast to thebiosynthetically labeled GPIs, however, the radiomethylated GPIs were cleaved directly by PIPLC. Furthermore, the radiomethylated GPIs were obtained from a particulate fraction enriched in plasma membranes, a pool distinct from the GPIs biosynthetically labeled in microsomal fractions. Thus, theradiomethylated GPIs represent aclass of free GPIs not previously detected in mammalian cells. These GPIsmay derive from precursors that can be biosynthetically labeled and are deacylated during transit to the cell plasma membrane. Cell surface GPIs have been detected in L. major (McConville and Bacic, 1990). There are some limitations on the use of radiomethylation to identify GPIs. The low abundance of GPIs in mammalian tissues demands fractionation procedures with high resolution to distinguish radiomethylated GPIs from more abundant phospholipids and proteins that are radiomethylated. While two-dimensional TLC was used in this report, other methods, including selective lipid extraction and/orHPLC may be needed to identify all the GPIs, especially the less polar ones, in a given tissue. Furthermore, the application of cleavage procedures diagnostic for GPIs is limited by radiomethylation. Although PIPLC cleavage still proceeds with methylated GPIs, the methylation of GlcN renders GPIs resistant both to HNOz and to GPI-PLD.' It thus becomes difficult to find radiomethylated GPIs that are resistant to PIPLC because of inositol acylation (Roberts et al., 1988). To identify these GPIs, lipid extracts must be pretreated with GPI-PLD or nitrous acid prior to radiomethylation, and treated and untreated samples must be labeled and analyzed in parallel. However, radiomethylation offers some advantages over biosynthetic labeling as a procedure for identifying GPIs. Since it can be applied to tissue extracts, its sensitivity is limited only by thequantity of tissueextracted and the resolution of the lipid fractionation procedure, In our rat liver membrane extracts, the radiomethylated GPIs accounted for about 1%of the counts/min in the thirdlipid extract or about 0.5% of the totalcounts/min incorporated into all of the lipid extracts. If phosphatidylethanolamine and phosphatidylserine are the major lipids labeled by this procedure and account for 15%of the plasma membrane lipids (Ray et al., 1969), we can roughly estimate that thelabeled GPIs comprise 0.1% of the total lipids or about the same amount as PIPz in liver plasma membranes (Seyfred and Wells, 1984). Another advantage is that radiomethylation is targeted to the GlcN residue in GPIs regardless of the remainder of the glycan composition. Divergence of the core glycan from the Man3GlcN-inositol-phosphate sequence in GPI-anchored proteins, as has been confirmed for free GPIs in Leishmania (MCConville et al., 1990) and suggested for the insulin-sensitive glycolipids (Mato et al., 1987b), would not influence this labeling. Based on current evidence, the GlcN-inositol-phosphate sequence may be considered as thedefinitive feature of a GPI, and is it fortunate that this conjugate resists hydrolysis in 4 M trifluoroacetic acid. Radiomethylation of the GlcN permits both this conjugate to be identified by anion exchange chromatography andthe 6 N HC1-resistant GlcN-inositol species to be detected by cation exchange chromatography. Identification of these conjugates now provides important new criteria in establishing aGPI structure.

* D. Sevlever

and T. L. Rosenberry, unpublished observations.

Acknowkdgments-We thank Dr. Eric Brass, Case W e s t e r n ReserveUniversity, for preparation of the isolated hepatocytes and Dawn R. H u m p h r e y for excellent technical support. REFERENCES Barr, K., and Lester, R. L. (1984) Biochemistry 23,5581-5588 Bradford, M. M. (1976) Anal. Biochem. 72,248-254 Brass, E. P., Garrity, M. J., and Robertson, R. P. (1984) FEBS Lett. 169,2939OC

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""_

R116-Rl91 """

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