THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol ,268 , No. 15, Issue of May 25, pp. 11394-114OO, 1993 Printed in U.S.A.

P-selectin Is Acylated with Palmitic Acid and Stearic Acid at Cysteine 766 through a Thioester Linkage*

(Received for publication, January 25, 1993, and in revised form, March 5, 1993)

Tetsuro FujimotoS, Eric Stroudj, Ralph E. Whatleyj, Stephen M. Prescottj, Laszlo Muszbekll, Michael Laposatall, and Rodger P. McEverS** From the 4 W. K. Warren Medical Research Institute, Departments of Medicine and Biochemistry, University of Oklahoma Health Sciences Center and Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, the §Nora Eccles Harrison Cardiovascular Research and Training Institute, Departments of Medicine and Biochemistry, University of Utah School of Medicine and the Veterans Affairs Medical Center, Salt Lake City, Utah 84112, the IIDepartment of Clinical Chemistry, University School of Medicine, Debrecen, H-4012, Hungary, and the I( Massachusetts General Hospital, Department of Pathology, Harvard Medical School, Boston, Massachusetts 02114

We report that the adhesion receptor P-selectin can be metabolically labeled with ['Hlpalmitic acid in hu- man platelets. Analysis of alkaline methanolysis prod- ucts from labeled protein demonstrated that the radio- activity associated with P-selectin was covalently bound palmitic acid. ['HIPalmitic acid was cleaved by hydroxylamine treatment at neutral pH and by reduc- ing agents, indicating that acylation occurred through a thioester linkage. Both stearic acid and palmitic acid were detected by gas chromatography-mass spectrom- etry analysis of alkaline hydrolysates of purified P- selectin. Deletion or mutation of Cys7" eliminated ['HI palmitic acid labeling of P-selectin in transfected COS- 7 cells. We conclude that the cytoplasmic domain of P- selectin is acylated at Cys7" through a thioester bond. Fatty acid acylation may regulate intracellular traf- ficking or other functions of P-selectin.

P-selectin (CD62, GMP-140, PADGEM) is an adhesion receptor for myeloid cells and lymphocyte subsets that is expressed on thrombin-activated platelets and endothelial cells (reviewed in Refs. 1 and 2 ) . Like the related proteins, E- selectin (expressed on cytokine-activated endothelium) and L-selectin (expressed on leukocytes), P-selectin contains an N-terminal lectin domain, followed by an epidermal growth factor-like motif, a series of consensus repeats, a transmem- brane domain, and a short cytoplasmic tail (3). The selectins mediate the initial contacts of leukocytes with activated plate- lets or endothelium through Ca2+-dependent interactions of the lectin domain with carbohydrate ligands on target cells (1, 2, 4). Thus, the selectins play pivotal roles in the early phases of inflammation and hemostasis, leading to intense interest in the mechanisms that regulate both their expression and their recognition of cell surface ligands.

P-selectin is constitutively synthesized by megakaryocytes and venular endothelial cells where it is sorted to membranes

* This work was supported by National Institutes of Health Grants HL 34363, HL 45510, HL 34127, HL 43689, and DK 43159, and by the Department of Veterans Affairs Medical Research Program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** To whom correspondence should be addressed: W. K. Warren Medical Research Inst., University of Oklahoma Health Sciences Center, 825 N.E. 13th St., Oklahoma City, OK 73104. Fax: 405-271- 3137.

of secretory granules: the LY granules of platelets (5, 6) and the Weibel-Palade bodies of endothelial cells (7-9). Activation of these cells with thrombin or histamine induces rapid redis- tribution of P-selectin to the cell surface following fusion of granule membranes with the plasma membrane. The surface expression of P-selectin on activated endothelial cells is nor- mally transient because the protein reenters the cell by en- docytosis within 30-60 min (9, 10). When P-selectin cDNA is transfected into heterologous AtT20 pituitary cells, the ex- pressed protein is also sorted into secretory granules, indicat- ing that the sorting signal is conserved across cell types and species (11, 12). The signal has been localized to the cyto- plasmic domain where it may interact with sorting compo- nents in the cytosol (11). The cytoplasmic tail is also required for endocytosis of P-selectin, presumably at least in part through interactions with coated pits.' The murine and hu- man sequences of the 35-residue cytoplasmic domain of P- selectin are very highly conserved, further supporting the functional importance of this region (3, 13, 14).

Posttranslation modifications of the cytoplasmic tail could potentially modulate sorting, endocytosis, or other functions of P-selectin. One common modification is fatty acid acyla- tion, usually with palmitic acid, by a thioester linkage to cysteine (15, 16). The cytoplasmic tails of both human and murine P-selectin contain a single cysteine residue that is located 12 residues from the transmembrane domain. In this paper, we demonstrate that human P-selectin is acylated with both palmitic acid and stearic acid, predominantly through a thioester linkage to the single cysteine of the cytoplasmic domain.

MATERIALS AND METHODS

Platelet Labeling with fH/Palmitic Acid-Preparation of [3H]pal- mitic acid-labeled platelets was performed as described previously (17, 18). Platelet-rich plasma was obtained from 50 ml of human blood in acid/citrate/dextrose solution and prostaglandin El and then centrifuged at 1300 X g for 15 min. Platelets were resuspended (3-6 X 10s/ml) in 140 mM NaCI, 2.5 mM KC], 0.1 mM MgClz, 10 mM NaHC03, 0.5 mM NaH2P04, 5.6 mM glucose, 10 mM Hepes pH 7.4, containing 3.6 mg/ml fatty acid-free bovine serum albumin (BSA)'

300 rCi/ml [9,10-3H]palmitic acid (60 Ci/mmol, Du Pont-New Eng- (Sigma), 1 unit/ml apyrase, 0.3 PM prostaglandin E1 (Sigma), and

H. Setiadi and R. P. McEver, unpublished observations. * The abbreviations used are: BSA, bovine serum albumin; @-ME,

(3-mercaptoethanol; HPLC, high performance liquid chromatography; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; GC, gas chromatography; MS, mass spectrometry; PCR, polymerase chain reaction; DMEM, Dulbecco's modified Eagle's medium; HBSS, Hanks' balanced salt solution.

11394

Acylation of P-selectin 11395

land Nuclear). Before use, [3H]palmitic acid was dried completely under nitrogen gas and then solubilized in the buffer. Platelets were incubated for 1 h at 37 "C, then pelleted by centrifugation, resus- pended in the same volume of above buffer without BSA and [3H] palmitic acid, and incubated at 37 "C for an additional 30 min.

Immunoprecipitation of P-selectin-Immunoprecipitations were carried out using biotinylated monoclonal antibody and streptavidin- conjugated beads (19). The labeled platelet suspension (1 X lo9 platelets) was centrifuged at 1300 X g for 15 min, and the platelet pellet was lysed in 0.5 ml of ice-cold solubilizing buffer (150 mM NaC1, 20 mM Tris-HC1, pH 7.4, containing 2 mM EGTA, 10 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 100 pg/ml leupep- tin, and 1% Triton X-100). After centrifugation at 10,000 X g for 15 min the supernatant was first incubated for 30 min at 37 "C with a 100-pl suspension of streptavidin beads (ImmunoPure immobilized streptavidin from Pierce Chemical Co.) to remove proteins that might absorb nonspecifically to the beads. Monoclonal antibody was biotin- ylated with NHS-LC-biotin (Pierce) in 0.1 M sodium bicarbonate buffer, pH 8.25, and then separated from unbound biotin on a PD-10 Sephadex G-25M column (Pharmacia LKB Biotechnology Inc.). Two monoclonal antibodies were used, S12 directed against human P- selectin (20) and, as a negative control, HPC4 directed against human protein C (21). Both antibodies are of the IgG, subclass. Twenty pg of biotinylated antibody was incubated with 100 p1 of streptavidin beads at 37 "C for 1 h and then washed twice with Tris-buffered saline (150 mM NaC1, 20 mM Tris-HC1, pH 7.4) to remove unbound antibodies. Platelet lysate was added to the pelleted streptavidin beads saturated with biotinylated antibody. After incubation at 37 "C for 1 h, the beads were washed 5 times with solubilizing buffer. Bound proteins were eluted by boiling in SDS-PAGE sample buffer (62.5 mM Tris-HC1, 2% SDS, 10% glycerol, 0.002% bromphenol blue, pH 6.8). For reduced samples, 6-mercaptoethanol @-ME) was added at a concentration of 5% (v/v) after boiling.

Alkaline Methanolysis and Identification of Released Fatty Acid by High Performance Liquid Chromatography (HPLC)-To identify the 3H-labeled compounds which bound to P-selectin, the P-selectin immunoprecipitates were subjected to delipidation and then alkaline methanolysis to disrupt ester linkages. BSA (1 mg/ml) was added as a carrier protein to 0.2 ml of immunoprecipitate in SDS-sample buffer, and then the sample was precipitated with 6 ml of methanol. The precipitated protein was extracted twice with 3 ml of chloro- form:methanol(2:1) and once with 3 ml of ch1oroform:methanol:water (kk0.3). In each extraction, the mixture was vortex-mixed for 1 min, incubated at room temperature for 5 min, and then centrifuged at 4,500 X g for 45 min to pellet the protein. To remove the noncova- lently associated lipids completely, the extraction was repeated until radioactivity was no longer detected in the organic solvent. Finally, the protein was extracted with acetone and dried under nitrogen gas. The delipidated protein was incubated with 2 ml of 0.2 M KOH in methanol at 37 "C for 30 min. After centrifugation, the supernatant was supplemented with 2 M HC1 and extracted 3 times with 1.5 ml of hexane. Fatty acids released by methanolic KOH and converted to fatty acid methyl esters and any nonderivatized fatty acid were recovered into the hexane phase, which was then dried, and the lipids were resuspended in 50 p1 of methanol. To identify the radioactive fatty acids and their methyl esters, 150 pg each of unlabeled myristic acid, palmitic acid, stearic acid, methyl myristate, methyl palmitate, and methyl stearate (all from Nu-Check Prep) were added as stand- ards, and the sample was analyzed by reverse-phase HPLC using a C18 column (Ultrasphere-ODS, 25 cm X 4.6 mm, ALTEX Scientific Inc.), eluted with 94% (v/v) acetonitrile:H20 at a flow rate of 1 ml/ min. One-ml fractions were collected and counted in 4 ml of Aquasol- 2 scintillation fluid (Du Pont-New England Nuclear). The retention time of the radioactive product was compared with those of the six standard lipids monitored at 205 nm.

Hydroxylamine Treatment and Deacylation with Reducing Agents- TO determine how [3H]palmitic acid was linked to P-selectin, P- selectin immunoprecipitates from t3H]palmitic acid-labeled platelets were treated with hydroxylamine as described previously (22). Each immunoprecipitate in SDS-sample buffer was separated into 4 equal aliquots. Two samples were dialyzed against 1 M hydroxylamine adjusted with NaOH to pH 7 or 11, respectively. As controls, two other samples were dialyzed against 1 M Tris-HCI, pH 7 or 11, respectively. After dialysis a t room temperature overnight, all samples were redialyzed against Tris-buffered saline and then analyzed by SDS-PAGE and fluorography.

TO determine whether the fatty acid linkage was sensitive to reducing agents, equal aliquots of immunoprecipitates were incubated

with dithiothreitol (DTT) (0.06 or 0.6 M) or &ME (0.4 or 4 M ) for 3 h at 37 "C under nitrogen. Control samples were incubated without reducing agents for 3 h at 37 "C. To ensure that mobilities did not vary because of different degrees of disulfide reductions, 0.6 M DTT or 4 M 6-ME was added to samples just before SDS-PAGE.

SDS-PAGE and Fluorography-SDS-PAGE was performed on 5- 20% polyacrylamide gradient gels (23), and gels were fixed and washed for several days as previously described (17, 18). Gels were then soaked in Amplify solution (Amersham Corp.) for 45 min, dried at 60 "C, and exposed to Kodak X-Omat AR film (Eastman Kodak CO.) at -70 "C for 2 weeks.

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis of the P-selectin Fatty Acid Modification-All glassware used in this proce- dure was new and was rinsed with ch1oroform:methanol (1:l) prior to use. 1 mg (approximately 7 nmol) of purified P-selectin was separated on a 12% SDS-polyacrylamide gel. The gel was stained to identify the band containing P-selectin and then destained. The band con- taining P-selectin was cut from the gel, diced into small pieces, and placed in a 15 X 75-mm screw-top tube. A control band of equal size was treated in identical fashion and placed in a separate tube. Each gel was extensively washed as follows: 2 x 10 ml of deionized water over 1 h; 3 X 10 ml of 90% methanol in water over 6 h; 1 X 10 ml of 50% methanol in water for 12 h. The final wash was then removed, and 4.75 ml of methano1:chloroform:water (2.5:1.25:1) was added to each of the tubes, which were then rocked at room temperature for 10 min. These extraction solutions were removed and transferred to a new tube. Addition of water (1 ml) and chloroform (1.5 ml) resulted in a biphasic solution (24); the chloroform phase was removed and transferred to a clean tube for later derivatization and analysis.

The gels were dried under nitrogen and then subjected, sequen- tially, to mild alkaline hydrolysis followed by strong acid hydrolysis to hydrolyze ester and amide linkages, respectively. Fatty acids in ester and thioester linkage were hydrolyzed using mild alkaline hy- drolysis; 0.70 ml of 1.5 N NaOH was added to the dried gels, and they were incubated at 30 "C for 3 h, with rocking. The tubes were then placed on ice, and 0.30 ml of 6 N HCI was added. To this was added 3.75 ml of methano1:chloroform (2:1), and the tubes were rocked at room temperature for 10 min. The extraction solutions were trans- ferred to a clean tube; each gel was then washed with 1.5 ml of chloroform, and this wash was added to the extraction solutions. One ml of water was added to each extraction; the chloroform phase of the resulting hiphasic solution was removed and transferred to a clean tube. The gels were dried under nitrogen and then subjected to strong acid to hydrolyze fatty acids in amide linkage; 1 ml of 6 N HC1 was added to each tube, which was incubated for 4 h a t 100 "C and then cooled. Methano1:chloroform (2:l) (3.75 ml) was added to each tube and incubated at room temperature for 10 min with rocking. The extraction solutions were transferred to a clean tube; each gel was then washed with 1.5 ml of chloroform, and this wash was added to the extraction solutions. To this was added 1 ml of water; the chloroform phase of the resulting hiphasic solution was removed and transferred to a clean tube.

Fatty acids in the chloroform phase of each extraction (metha- no1:chloroform wash, mild alkaline hydrolysis, acid hydrolysis) were then derivatized to their methyl esters. First, 2.5 ml of acidified methanol (100 mM acetic acid in methanol) was added to each chloroform phase and the entire sample dried under nitrogen. Then, 1 ml of petroleum ether and 1 ml of BF3:methanol (Supelco, Belle- fonte, PA) were added to each tube, which was capped and heated (100 "C for 10 min). The solutions were cooled, and 0.5 ml of water and 2 ml of petroleum ether were added. The tubes were shaken, and the resulting biphasic solution was generated by a brief centrifugation (400 X g for 5 rnin). The upper phase was transferred to a conical bottom screw-top tube and dried under nitrogen. The derivatized fatty acids were resuspended in hexane and analyzed by gas chro- matography-mass spectroscopy.

GC-MS was carried out with a Hewlett-Packard 5890 mass spec- trometer with a dedicated microcomputer for instrument control and data reduction. Gas chromatography was on a 15-meter DB-5 (J & W, Rancho Cordova, CA) capillary column of 0.25-mm internal diameter and 0.1-pm film thickness with helium as the carrier gas. Development was 125 "C for 10 min, increased 2 "C/min to 140 "C, where it was held for 3 min, then increased 10 "C/min to 250 "C, where it was held for 5 min. The injector temperature was 260 "C; the detector was at 290 "C. Fatty acid methyl esters were identified by comparison with authentic standards by analysis of the ion chro- matograms of candidate peaks, by comparison of those ion chromat- ograms to a library of standards, and by selective ion monitoring of

11396 Acylation of P-selectin the molecular ion of the fatty acid methyl ester (palmitate methyl ester: m/z = 270).

A series of control experiments were performed to ensure that the SDS-PAGE separation completely removed fatty acids that were not covalently associated with protein. Ovalbumin (Sigma) in water was incubated (1 h a t room temperature) with radiolabeled 13H]palmitate (28.5 Ci/mmol, Du Pont-New England Nuclear) a t a ratio of 2 mg of ovalbumin/O.l mCi of ['HHIpalmitate. The ovalbumin was subjected to SDS-PAGE, and the extraction and derivatization procedures were performed as detailed above. There was no [3H]palmitate in the ovalbumin band on the gel as measured by liquid scintillation spec- troscopy. In addition, GC-MS analysis of the hydrolysates of the gel containing the ovalbumin demonstrated no fatty acid methyl esters.

cDNA Constructs-The cDNA clone encoding full-length human P-selectin (3) was excised from the cloning vector pIBI2O (IBI) and cloned into pcDNAI (Invitrogen) at the XhoI site. pcDNAI is a plasmid expression vector that contains a cytomegalovirus promotor. Three P-selectin mutants (Fig. 7) were constructed by using the polymerase chain reaction (PCR) according to the strategy previously described (11). In the first construct (tail-less), a stop codon was introduced at the junction between exon 14 and exon 15, resulting in deletion of the cytoplasmic tail after Asp'62. This construct lacks Cys7=. In the second (Cys'= + Stop), a stop codon was created at base 2460 immediately after Cys'=. In both cases, PCR products were made, using P-selectin cDNA as a template, from the XbaI site a t base 2240 to the stop codon after base 2447 or after base 2459, followed by an additional XbaI site. In the third construct ( C Y S ~ ~ + Ala), Cys'= (TGC) was mutated to Ala (GCC). An 18-mer sense oligonucleotide (5'-GG AAA GCC CCC TTG AAT C-3') and its antisense oligonucleotide ( 5 ' 4 A T T CAA GGG GGC TTT CC-3') were prepared, and two separate PC& were performed the first from the XbaI site a t 2240 to the antisense primer and the second from the sense primer to base 2534. After gel purification the two PCR products were mixed, and a second PCR was performed using the two outside primers. In all cases, PCR fragments were digested by XbaI, isolated by agarose gel electrophoresis, and then used to replace the XbaI fragment of the wild type P-selectin cDNA extending from the XbaI site in the cDNA to the XbaI site in the multicloning site of pcDNAI. All constructs were verified by nucleotide sequencing of the region encoding the PCR products using a DNA sequencing system (Promega).

Transfection and rHJPalmitic Acid Labeling of COS-7 Cells-COS- 7 cells (American Type Culture Collection, ATCC no. CRL1651) were grown in Dulbecco's modified Eagle's medium (DMEM) with high glucose (GIBCO/BRL) supplemented with 10% bovine calf serum, 4 mM glutamine, 200 units/ml penicillin, and 200 pg/ml streptomycin under 5% C02 a t 37 "C. Cells were transfected with Transfectam (Promega) according to the manufacturer's instructions. Seventy percent confluent cells in 15-cm dishes were incubated with the Transfectam and plasmid mixture in DMEM without serum for 4 h a t 37 "C and then incubated in complete medium. After 36 h, fresh medium was added containing 6 mM sodium butyrate, which increases transcription by cytomegalovirus promotors (25). After 48 h, cells were rinsed three times with Hanks' balanced salt solution (HBSS, GIBCO/BRL) and then labeled with I3H]palrnitic acid (200 pCi/ml) in DMEM supplemented with 10% delipidated serum and 5 mM sodium pyruvate for 4 h a t 37 "C. Delipidated serum was made from dialyzed fetal bovine serum by acetone-ethanol precipitation as pre- viously described (26). After labeling, cells were rinsed twice with HBSS and lysed in 1 ml of ice-cold solubilizing buffer. The cell lysates were subjected to S12 immunoprecipitation, SDS-PAGE, and fluo- rography as described above. T o evaluate the expression levels, im- munoblotting analysis was performed using the same cell lysates. Equal aliquots of each lysate were fractionated by SDS-PAGE under nonreducing conditions, transferred to Immobilon-P membrane (Mil- lipore), and probed with S12 followed by a biotinylated anti-murine immunoglobulin reagent coupled to a peroxidase-streptavidin detec- tion system (Vectastain ABC kit, Vector Laboratories).

RESULTS

Incorporation of PHIPalmitic Acid into P-selectin-To de- termine whether P-selectin was acylated by long-chain fatty acids, the protein was immunoprecipitated from [3H]palmitic acid-labeled platelet lysates with the monoclonal antibody S12 and analyzed by SDS-PAGE and fluorography. Fig. 1 shows that S12 precipitated a single radiolabeled protein that

[3H]Polrnitic Acld-labeled Platelets

Antibody: 'HPC4 S12 HPCL S12'

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FIG. 1. Fluorogram of immunoprecipitates from ['Hlpalmi- tic acid-labeled platelets. Human platelets were labeled with ['HI palmitic acid, lysed, and subjected to immunoprecipitation with an anti-P-selectin monoclonal antibody (S12) or a control antibody to human protein C (HPC4). The immunoprecipitates were resolved by SDS-PAGE on 5-20% gradient gels under nonreducing (left) or reducing (right) conditions. lZ5I-Labeled purified P-selectin was elec- trophoresed in parallel lanes. Molecular weight standards are shown on the [eft.

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FIG. 2. HPLC analysis of alkaline methanolysis products released from P-selectin. P-selectin was immunoprecipitated from lysates of ['Hlpalmitic acid-labeled platelets. The immunoprecipi- tates were subjected to delipidation followed by alkaline methanolysis. Released 3H-labeled fatty acids were resolved by HPLC and moni- tored by liquid scintillation counting. Arrows indicate the elution positions (detected by absorbance a t 205 nm) of standard fatty acids: myristic acid (MA), palmitic acid (PA), stearic acid (SA) , methyl myristate ( M M ) , methyl palmitate ( M P ) , and methyl stearate (MS).

comigrated with purified '251-labeled P-selectin under both reducing and nonreducing conditions. The control antibody HPC4 (directed against protein C) did not precipitate any labeled proteins. Thus, P-selectin was selectively labeled with ['Hlpalmitic acid in washed platelets.

HPLC Analysis of Fatty Acids Released from PHIPalmitic Acid-labeled P-selectin-To determine if the radioactivity as- sociated with P-selectin was covalently bound fatty acid, a P- selectin immunoprecipitate was delipidated to remove non- covalently associated lipids. I t was then subjected to alkaline methanolysis, which disrupts 0-ester and thioester linkages but not amide linkages. After alkaline methanolysis more than 90% of radioactivity was recovered in the organic sol- vent. HPLC analysis of alkaline methanolysis products re- vealed a single radioactive peak with a retention time that corresponded to the methyl palmitate standard (Fig. 2). These

Acylation of P-selectin 11397

results demonstrated that the radioactivity associated with P- selectin was covalently bound ['Hlpalmitic acid in ester link- age when the platelets were incubated for 1 h with ['HI palmitic acid.

Deacylation of P-selectin by Hydroxylamine and Reducing Agents-To identify whether ['Hlpalmitic acid was linked through a thioester or 0-ester bond, P-selectin immunopre- cipitates were first treated with hydroxylamine, which dis- rupts both thioester and 0-ester linkages a t alkaline pH but only thioester linkages a t neutral pH (27). Fig. 3 demonstrates that 1 M hydroxylamine treatment a t both neutral and alka- line pH substantially reduced the radioactivity associated with P-selectin, whereas control treatments with 1 M Tris did not affect the degree of labeling. Immunoprecipitates were next treated with reducing agents. Thioester linkages are cleaved by these agents, but they are more resistant than intramolec- ular disulfide bonds, and the degree of cleavage is dependent on the concentration of reducing agent and the incubation time (28). We compared the effects of relatively high and low concentrations of DTT and @-ME. Fig. 4 shows that most of the radioactivity was released from P-selectin a t concentra- tions of 0.6 M DTT or 4 M @-ME. These results indicated that palmitic acid was coupled to P-selectin through a thioester linkage and therefore must be linked to a cysteine.

GC-MS Analysis-To confirm the results obtained with ['Hlpalmitate-labeled P-selectin, we used a complementary approach to determine whether fatty acid modifications were present on the unlabeled protein. This analysis reflected the fatty acid composition of the P-selectin-bound fatty acids in the steady state, rather than following a 1-h incubation with radiolabeled palmitate. P-selectin isolated from human plate- lets was further purified by preparative SDS-PAGE, ex- tracted, subjected to alkaline and acid hydrolysis, and the hydrolysates analyzed by GC-MS. To confirm that these procedures effectively removed all noncovalently associated fatty acids, ovalbumin that had been incubated with ['HI palmitate was subjected to SDS-PAGE and hydrolysis. There

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FIG. 3. Hydroxylamine treatment of [SH]palmitic acid-la- beled P-selectin. P-selectin immunoprecipitates from ['Hlpalmitic acid-labeled platelets were separated into four samples of identical volume. Two of them were treated with 1 M hydroxylamine a t pH 7 or 11, and the other two were treated with 1 M Tris at pH 7 or 11 as controls. The treated samples were then analyzed by SDS-PAGE and fluorography.

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with reducing agents. P-selectin immunoprecipitates were divided into equal aliquots and incubated with various molar concentrations of DTT (0.6, 0.06, or 0) or &ME (4, 0.4, or 0). After incubation, samples were analyzed by SDS-PAGE and fluorography.

was no palmitate in these hydrolysates as assessed by the absence of radiotracer.

GC-MS analysis of the alkaline hydrolysates of P-selectin demonstrated peaks identified as palmitic acid methyl ester (7.9 min) and stearate methyl ester (17.7 min) (Fig. 5A) . Two peaks at 7.9 and 17.7 min were also identified in ion-selective chromatograms for major molecular ions common to fatty acid methyl esters (m/z = 74, 87, and 143) (Fig. 5 B ) . The methyl ester of palmitate was identified by several criteria: 1) its retention time on gas-liquid chromatography was identical to a standard; 2) an ion chromatogram of this peak demon- strated the molecular ion ( m / z = 270) and other major ions characteristic of palmitic acid methyl ester (Fig. 6); and 3) an ion-selective chromatogram for the molecular ion (m/z = 270) demonstrated a peak at the retention time of the standard. The methyl ester of stearate was identified using the same criteria, including demonstration of the molecular ion (m/z = 298) (Fig. 6). Palmitate and stearate methyl esters were not seen in the ch1oroform:methanol wash or in the acid hydrol- ysate of the gel containing P-selectin. Neither palmitate nor stearate was present in any of the extracts of the control gel (containing no P-selectin) or in control gels containing oval- bumin that had been incubated with palmitate prior to SDS- PAGE. Although several other peaks had retention times similar to the palmitic acid methyl ester (Fig. 5 ) , their ion chromatograms were not characteristic of fatty acid methyl esters. Thus, in the steady state, stearate was bound to P- selectin in greater amounts than palmitate.

fH]Palmitoylation of Recombinant P-selectin and Its Mu- tants in COS-7 C e 1 l s - C ~ ~ ~ ~ was the most likely site for acylation of P-selectin since it is the only cysteine within the transmembrane and cytoplasmic regions (3). To confirm that

was acylated, we made three different cDNA constructs of P-selectin (Fig. 7). In the first (tail-less), most of the cytoplasmic tail was deleted, including the cysteine. In the second (Cys7= + Stop), a smaller deletion was made in which

was preserved as the C-terminal residue. In the third (Cys7= + Ala), the cysteine was mutated to alanine. These constructs as well as wild type P-selectin cDNA were trans- fected into COS-7 cells to determine whether the expressed proteins could be labeled with ['Hlpalmitic acid. Immunoblot analysis showed that all constructs were comparably ex- pressed in COS-7 cells (Fig. 8). However, immunoprecipita- tion of ['Hlpalmitic acid-labeled cells showed that only wild type P-selectin and the Cys7= + Stop mutant that retains the cysteine were palmitoylated (Fig. 8). No labeling with ['Hlpalmitic acid was detected in the tail-less mutant that lacked Cys7= or the C Y S ~ ~ + Ala mutant in which the cysteine

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FIG. 5. GC-MS analysis of alkaline hydrolysate from purified P-selectin. Purified P-selectin was separated by SDS-PAGE, and the band containing P-selectin was excised and subjected to mild alkaline hydrolysis as described under "Materials and Methods." Derivatized fatty acids from alkaline hydrolysates were subjected to GC-MS analysis. A, total chromatogram; B, ion-selective chromatograms for the major molecular ions common to the fatty acid methyl esters: m/z = 74 (upper), 87 (middle), and 143 (lower). Two peaks identical to methyl palmitate (7.9 min) and methyl stearate (17.7 min) were seen.

Acylation of P-selectin 11399

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40001 3000 I I FIG. 6. Ion chromatograms of the peaks of palmitate methyl ester and stearate methyl ester. The ion chromatograms of peaks

a t 7.9 min ( A ) and 17.7 min ( B ) are shown in the upperpanels. The ion chromatograms of standard fatty acid methyl esters are shown in the lower panels (methyl palmitate in A and methyl stearate in B ) .

TM ST c1 c2 Wild Type ~ R K R F R O K D DOK PLNPHS HLGTVGVF,NAAFDPSP 7

766

Tail-less 1

Cys7~6- , s top lJm=l I cl

Cys766->AIa I C I * A

FIG. 7. Schematics of the cytoplasmic domain of P-selectin cDNA constructs. The amino acid sequence (3) of the cytoplasmic domain of human P-selectin is shown at the top. Exon 14 encodes the transmembrane domain (TM) and the stop-transfer ( S T ) se- quence, which includes the first eight amino acids of the cytoplasmic tail. The remainder of the cytoplasmic tail is divided into two regions, C 1 and C2, which are encoded by exons 15 and 16, respectively (44). Below the wild type sequence are schematics of mutant constructs.

was changed to alanine. These results indicated that Cys'% was required for acylation with [3H]palmitic acid. Taken together with the results of Figs. 3 and 4, we conclude that palmitic acid is coupled to Cys7% of P-selectin by a thioester bond.

DISCUSSION

We used two complementary approaches to demonstrate that P-selectin was modified by fatty acids. In the first ap- proach, we found that P-selectin was labeled with [3H]pal- mitic acid in intact cells. The radiolabel was recovered as methyl palmitate after alkaline methanolysis of P-selectin immunoprecipitates exhaustively extracted with organic sol-

? Mr x l o "

-200-

- 116 - - 97 - - 66 - - L 5 - - 31 - - 21 - - 1L -

0"

- - Fluorography lmmunoblotting

FIG. 8. [SH]palmitoylation of recombinant wild type and mutant P-selectin in COS-7 cells. COS-7 cells were transfected with various P-selectin cDNA constructs as described under "Mate- rials and Methods." After labeling with [3H]palmitic acid, cell lysates were subjected to immunoprecipitation by S12 followed by SDS- PAGE and fluorography (left). Equal volumes of cell lysates were also subjected to immunoblotting with S12 (right).

vents, indicating that the palmitic acid was covalently linked to the protein. P-selectin was labeled in both platelets and in COS-7 cells transfected with P-selectin cDNA, suggesting that palmitoylation of P-selectin is a general event in different cell types. P-selectin can be added to the list of previously described membrane proteins in platelets that can be labeled

11400 Acylation of P-selectin

with [3H]palmitic acid (18). Although palmitoylation of pro- teins occurs posttranslationally soon after the synthesis of a protein (291, it is very likely that acylation-deacylation is an ongoing process. Thus, in cells such as platelets that synthe- size little or no protein the radiolabeled fatty acid is probably bound to proteins by exchange mechanisms. In the second approach, we directly measured fatty acids released by alka- line methanolysis from P-selectin by gas chromatography and mass spectroscopy. By this technique, we detected methyl stearate as well as methyl palmitate. The enzyme that couples fatty acids to cysteine through thioester Iinkages has not been isolated or characterized. Metabolic labeling experiments with [3H]palmitate led to the suggestion that palmitate was the primary fatty acid involved in this type of protein modifica- tion. However, some studies have indicated that saturated fatty acids other than palmitate also participate in this process (22, 30-32), and recently it has been shown that another straight chain fatty acid, myristate, also binds to protein via thioester linkage in platelets (33). The failure to detect [3H] methyl stearate after alkaline methanolysis of P-selectin from [3H]palmitic acid-labeled platelets is consistent with the lim- ited conversion of palmitic acid to stearic acid in these cells during short-term labeling experiments (34).

The ability of reducing agents and of hydroxylamine at neutral pH to remove [3H]palmitic acid from P-selectin indi- cated that palmitic acid was linked by a thioester bond. This conclusion was confirmed by demonstrating that mutants of P-selectin lacking the single cysteine at position 766 of the cytoplasmic domain were not labeled with [3H]palmitic acid in transfected COS cells. Although not directly determined, it is most likely that stearic acid is also coupled to Cys766 by a thioester linkage.

As in most previous reports concerning proteins modified by fatty acid, we were unable to accurately measure the stoichiometry of acylation. When immunoprecipitated in par- allel from the same [3H]palmitic acid-labeled platelet lysates, the intensity of labeled P-selectin on fluorograms was weaker than that of GPIb@ or GPIX, two membrane proteins previ- ously demonstrated to be palmitoylated (17) (data not shown). While this finding may reflect a lower degree of acylation for P-selectin than the other proteins, it may also indicate less efficient incorporation of labeled palmitic acid into P-selectin. Stoichiometry was also not measured by direct GC-MS analy- sis because of difficulties in determining the recovery of protein and lipids from the polyacrylamide gel.

The functional significance of acylation of P-selectin re- mains unclear. Diverse roles have been ascribed to palmitoy- lation of other membrane proteins, including fusion between different cellular compartments (35, 36), interactions of the cytoplasmic domain with G proteins (37), associations with the cytoskeleton (38), inhibition of endocytosis (39), and regulation of mobility in the membrane (40). Potentially some of these functions could be regulated by cycles of acylation and deacylation. Indeed, cellular agonists increase incorpo- ration of [3H]palmitic acid into certain membrane proteins (39, 41). Although we did not detect increased incorporation of [3H]palmitic acid in P-selectin immunoprecipitated from

lysates of thrombin-stimulated platelet^,^ it remains possible that acylation of P-selectin is regulated. Acylation does not appear to promote binding of P-selectin to the cytoskeleton, as the protein is not detectably associated with the Triton X- 100-insoluble cytoskeleton of resting or activated platelets (42).3 Acylation might modify the conformation of the cyto- plasmic domain of P-selectin to modulate sorting, endocyto- sis, or other trafficking functions. Regulation of lateral mo- bility might also affect the rate of contact of P-selectin with cell surface ligands on leukocytes under the shear forces found in the circulation (43). Further studies are required to deter- mine possible functions for the acylation of Cys7= in P- selectin.

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