Candidate Glycophospholipid Precursor for the

0 downloads 0 Views 2MB Size Report
variant surface glycoprotein polypeptide. We report the purification and chemical characterization of a glycolipid from T. brucei that has properties consistent.
THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 19% by The American Society for Biochemistry and Molecular Biology, Inc.

VOl. 263,No. 4, Issue of February 5,, pp. 1970-1977,1988 Printed in U.S.A.

Candidate Glycophospholipid Precursor for the Glycosylphosphatidylinositol Membrane Anchor of Trypanosoma brucei Variant Surface Glycoproteins* (Received for publication, September 4, 1987)

Anant K. Menon$, Satyajit Mayore, Michael A. J. FergusonT, Michael Duszenko11 ,and George A. M. Cross From T ~ Rockefeller Q Uniuersity, New York,New York 10021

Trypanosoma brucei variant surface glycoproteins attached to the protein by ethanolamine in an amide linkage are apparently synthesized with a hydrophobic car- to the carboxyl-terminal amino acid. In many cases, some boxyl-terminal peptide that is cleaved and replaced by fraction of these proteins could be released from membranes a complex glycosylphosphatidylinositol membrane an- by treatment with a phosphatidylinositol-specificphospholichor within 1 min of the completion of polypeptide pase C. Although compositional data are available for the synthesis. The rapidity of this carboxyl-terminal mod- glycolipid anchors of several proteins, including rat Thy-1 ification suggests the existence of a prefabricated core (Tse et al., 1985),humanerythrocyte acetylcholinesterase en bloc tothe glycolipid thatwouldbetransferred (Haas et al., 19861, and decay-accelerating factor (Medof et variant surface glycoprotein polypeptide. We report al., 1986), analysis of the variant surface glycoproteins (VSG)’ the purificationandchemicalcharacterization of a glycolipid fromT.brucei that has properties consistentof the parasitic protozoan Trypanosoma brucei has provided with a roleas a variant surface glycoprotein glycolipidthe most detailed structural information (Ferguson et al., 198513, 1987). donor. This candidate glycolipid precursor has been The surface of T. brucei is dominated by lo7VSG molecules, defined by thin-layer chromatography of extracts of which form a dense protective coat. Sequential expression of trypanosomes metabolically labeled with radioactive individual genes encoding immunologicallydistinct VSGs permyristicacid,ethanolamine,glucosamine,mannose, and phosphate and by enzymatic, chemical, and gas mits a trypanosome population to evade the immune response chromatographic-massspectrometric analysis. Mild of its mammalian host (Cross, 1984).cDNA sequence analyses alkali released 100%of the myristic acid, and reaction (Boothroyd et al., 1980) suggested that VSGs are synthesized with hydrophobic peptide sequences at the amino and carwith phospholipase Az released 50%. Nitrousacid deaminationgenerated dimyristylphosphatidylinosi- boxyl termini. The N-terminal extensionappears to be a tol, and periodate oxidation released phosphatidic acid. conventional signal sequence, which is cleaved upon transloTreatment of purified glycolipid with phosphatidyli- cation of the nascent polypeptide across the endoplasmic nositol-specific phospholipase C released dimyristyl- reticulum membrane (McConnell et al., 1981). The hydrophoglycerol and a water-soluble glycan that was sized on bic carboxyl-terminal tailhas never been detected, either Bio-Gel P-4 columns.Thecandidateprecursorconattached to theprotein or as a cleavage product. Instead, the tainedmannose,myristicacid,phosphate,and eth- carboxyl terminus of the mature protein ismodified by a anolamine with an unsubstituted amino group, but notglycosyl inositol phospholipid, which anchors the VSG to the galactose. membrane. Studies of VSG biosynthesis indicate that the glycolipid anchor is attached to theprotein immediately after translation of the nascent polypeptide (Bangs et al., 1985; Ferguson et al., 1986).The timing of this modification suggests Several eukaryotic externally disposed plasma membrane that itoccurs at 3r closeto the site of protein synthesis inthe glycoproteins are modified by complex glycosylinositol phos- endoplasmic reticulum. The rapidity of the process suggests pholipids, which appear to provide the sole means of mem- that the glycolipid is added en bloc and implies the existence brane attachment (Cross, 1987; Low, 1987). In all the chemi- of a precursor glycolipid, a molecule with the core structural cally characterized examples, the glycophospholipid anchor is characteristics of the VSG glycolipid anchor. Krakow et al. (1986) have indicated the existence of such a molecule by * This work wassupported inpart by National Institutesof Health analyses of [3H]myristi~acid-labeled lipid components exGrant A121531. Work performed at The Rockefeller University Ex- tracted from trypanosomes. Inthis paper, we report the tended Range Mass Spectrometric Research Resource was supported isolation and compositional analysis of a trypanosome glycoby National Institutes of Health Grant RR00862. The costs of publication of this article were defrayed in part by the payment of page lipid that is remarkably analogous to the VSG glycolipid charges. This article must therefore be hereby marked “aduertise- membrane anchor and which has properties consistent with ment” in accordance with 18 U.S.C. Section 1734 solely to indicate a role as a VSG glycolipid precursor. A second compositionally this fact. $ To whom correspondence should be addressed The Rockefeller University, 1230 York Ave., New York, NY 10021-6399. § Supported by the Lucille P. Markey Charitable Institute, Miami,

FL. 1 Present address: Dept. of Biochemistry, Oxford University, Oxford, OX1 3QU, United Kingdom. 11 Present address: Physiologisch Chemisches Institut der Universitiit mbingen, nbingenD-7400, Federal Republic of Germany.



The abbreviations used are: VSGs, variant surface glycoproteins; PI, phosphatidylinositol; mfVSG, membrane form of VSG sVSG, soluble form of VSG, GPI, glycosylphosphatidylinositol; HPTLC, high performance TLC; HPLC, high performance liquid chromatography; GC-MS, gas chromatography-mass spectrometry; HEPES, 4(2-hydroxyethyl)-l-piperazineethanesulfonicacid; dansyl, 5-dimethylaminonaphthalene-l-sulfonyl; dabsyl, 4-dimethylaminoazobenzene4’-sulfonyl.

1970

Glycolipid Precursor for Membrane Protein Glycolipid Anchor

1971

similar but otherwise disparate lipid has also been character- water (25:15:42)). Lipid standards were located by exposing the airdried TLC platesto iodine vapor. Radioactive lipids were detected by ized.

scraping the TLC plates in 0.5-1.0-cm-wide strips and measuring radioactivity by liquid scintillation counting. The butanol fraction (as described above) of radiolabeled trypanMaterials-[1-3H]Ethan-l-01-2-amine hydrochloride (12-30 Ci/ osomes was chromatographed on Silica Si 50000 HPTLC plates (10 mmol) was purchased from Amersham Cow. [9,10-3H]Myristicacid x 20 cm; Merck) using solvent system d (chloroform, methanol, 30% (22.4 Ci/mmol), ~-[6-~H]glucosamine hydrochloride (30 Ci/mmol), ammonia, 1 M ammonium acetate, water (18014098:23)) or on ~-[2-'H]mannose(23.5 Ci/mmol), D-[l-3H]galactose (11.5 Ci/mmol), cellulose sheets (20 x 20 cm; Kodak Chromagram Sheet 13256)using [9,10-3H]palmiticacid (17.3 Ci/mmol), and [32P]orthophosphoricacid solvent system e (l-butanol/acetic acid/water (5:2:3)). Radioactive were purchased from Du Pont-New England Nuclear. Fatty acid lipids were located by scraping and counting as described above or by radiolabels were coupled to defatted bovine serum albumin a t a molar spraying the dried plates with EN3HANCE (Du Pont-New England Nuclear), followed by exposure to Kodak X-Omat film at -70 'C. ratio of 1:l for biosynthetic labeling experiments. Calf intestinal All silica plates were activated a t 100 "C for 1-5 h before use and alkaline phosphatase, beevenom phospholipase A,, and Bacillus cereus phospholipase C (grade I) were from Boehringer Mannheim. cooled to room temperature before application of samples. All plates PI-specific phospholipase C from Staphylococcus aureus was a gift were developed in tanks lined with Whatman No. 3MM paper and from Dr. M. G. Low (The Rockefeller University) and GPI-specific pre-equilibrated with the developing solvent system. Enzyme Treatments-Radioactive lipids were dried down before phospholipase C was prepared from T. brucei by Dr. J. A. Fox (Columbia University) as described (Fox et al., 1986).Lipid and sugar being resuspended in 0.2-1.0 ml of detergent-containing buffer for standards were purchased from Sigma. All solvents were reagent- or enzyme reactions. PI-specific phospholipase C reactions were carried out in 0.1 M Tris-HC1, pH 7.4, with 0.1% deoxycholate using 1-3 pg HPLC-grade. Trypanosomes-Trypanosome clones of the Molten0 Institute Try- of enzyme. Phospholipase A, reactions were performed in the same panozoon antigenic types 1.4 and 1.5 (variant clones 117 and 118, buffer but with 1 mM CaCl, using 25 units of enzyme. GPI-specific respectively) of T. brucei strain 427 were purified from infected rat phospholipase C was used in 25 mM Tris-HC1, pH 8.0, with 0.5% Nonidet P-40. Samples were incubated for 30 min at 37 'C and then blood as previously described (Cross, 1975). Radiolnbelingof Trypanosomes-All labeling experiments were per- acidified with 1-2 pl of acetic acid and extracted with toluene or formed at 37 "C in a shaking water bath for 1-2 h. Trypanosomes water-saturated butanol. Aliquots of the toluene or butanol extracts were washed in the appropriate medium (see below), resuspended at were counted or analyzed by TLC. Periodate Clea~age-[~H]Myristic acid-labeled lipids were sus0.2-1 X 108/ml, and incubated a t 37 'C for 10-15 min before the addition of radiolabel (3H:25-60 pCi/ml; 3 2 P 500 pCi/ml). Fatty acid pended in 475 pl of sodium periodate (25 mg/ml) or 475 pl of water. and ethanolamine labeling was carried out in Eagle's minimum es- 1 drop of toluene was added, and thesuspensions were incubated for sential or RPMI 1640 (GIBCO); [3H]mannose, [3H]galactose, and 48 h a t 37 "C. A t the end of the incubation, the periodate samples [3H]glucosaminelabeling were performed in glucose-free RPMI 1640 were at pH 5. All samples were then acidified with 2 pl of 6 M HCl medium supplemented with 3 g/liter glycerol; and [32P]phosphate and extracted with 2 ml of chloroform/methanol (2:l). Half the CM labeling was performed in phosphate-free Eagle's minimum essential extract was dried and taken for liquid scintillation counting. The remainder of the extract was analyzed by TLC on Silica Gel 60using medium. All labeling media were buffered by 25 mM HEPES, pH7.4. Trypanosome viability (motility) was monitored throughout and was solvent systems b and c. Nitrous Acid Dearninatior~-[~H]Myristic acid-labeled lipids were usually greater than 95%. Extraction of Radiolabeled Lipids-At the end of an incubation, the suspended in 400 pl of 0.1 M sodium acetate, pH 4, followed by 400 trypanosomes were centrifuged, the supernatant was removed, and pl of 0.33 M sodium nitrite or 400 pl of water. Samples were left at the cells were lysed by briefly vortexing the cell pellet in ice-cold ambient temperature for 3 h before being acidified with 5 p1 of 6 M water a t a concentrationof about 10' cells/ml. The lysate was rapidly HCl and extracted first with 1.0 ml of diethyl ether and then 2.1 ml frozen in dry ice/ethanol and then lyophilized. The lyophilized ma- of chloroform/methanol (2:l). Aliquots of the ether and CM extracts terial was washed two to three times with 0.5-1 ml of chloroform, were dried and taken for liquid scintillation counting. The remaining methanol, 6 N HCl (10050:l) and then extracted four to five times CM extract was analyzed by TLC on Silica Gel60 using solvent with 0.5-1 ml of chloroform/methanol (2:l) (referred to as CM system c. Amine and Carbohydrate Analysis of Radiolabeled Lipids-In order extracts). Typically, the last extract contained less than 0.1% of the radioactivity in the first extract. The CM extracts were pooled and to determine the identity of the radioactivity in the butanol fraction retained for occasional analysis. The lyophilized material was then of radiolabeled trypanosomes or TLC-purified radioactive lipids, they were dried down in microdispenser tubes (100-200 pl, sealed at one dried in a stream of nitrogen to remove residual chloroform/methanol end). 20-75 pl of constant boiling HCl (6 M; Pierce Chemical Co.) before extraction with 0.5-1 ml of methanol/pyridine/water (2:l:l) (referred to as MPW extract). This extraction was carried out by was added, the tubes were sealed, and samples were incubated for 4incubating samples at room temperature for several hours or at 4 "C 8 h at 100 "C in an oven. The tubes were broken open, and the overnight, with occasional vortexing or sonication in a bath sonicator samples were dried down in a Speed-Vac, resuspended in 10-60 pl of (Heat Systems-Ultrasonics, Inc., Farmingdale NY). The MPW ex- water, and spotted onto TLC plates. The identity of [3H]mannose tract was removed and dried in a Speed-Vac evaporator (Savant and [3H]glucosaminewas established by TLC on Silica Gel G using Instruments, Inc.), and thedried residue was vortexed vigorously with l-propanol/water (7:l) (Gal, 1968). The identity of [3H]glucosamine a mixture of 0.5 ml each of water and water-saturated butanol. The was also confirmed by TLCon cellulose sheets using l-butanol, phases were separated by mild centrifugation, and theupper butanol- pyridine, 0.1 N IICl (5:3:2) (Got et al., 1967). The identity of [3H] rich phase was transferred to a fresh tube where it was "back- ethanolamine was confirmed by derivatizing the hydrolysate with 4extracted with 0.3 ml of water. The final material, described hence- dimethylaminoazobenzene-4'-sulfonyl chloride (dabsyl chloride; Pierce Chemical Co.) prior to analysis by HPLC using a Zorbax ODS forth as thebutanol fraction, was stored at 4 "C. HPLCAnaZysis-Initial analyses of MPW extractsof [3H]myristic column and anacetate buffer/acetonitrile system described by Chang acid-labeled trypanosomes were performed by HPLC using a Varian et al. (1981). In all cases, the identity of the radioactivity from 5500 ternary liquid chromatograph. 5-50 pl of extract was injected hydrolyzed lipid samples was established by comparing it to identiinto a Supelcosil LC-CN column (25 x 0.46 cm; Supelco, Inc., Belle- cally treated radioactive standards. Dansylation An~lysis-[~H]Ethanolamine and [3H]ethanolaminefonte, PA; preceded by a 2-cm guard column packed with the same material) and equilibrated in isopropyl alcohol/water (2080). The labeled, silica HPTLC-purified glycolipids weredried in microdispengradient elution program was linear, going from an isopropyl alcohol/ ser tubes in aSpeed-Vac evaporator. Samples were dansylated either water ratio of 2080 to 1000 in 30 min at 0.7 ml/min. Fractions were prior to hydrolysis (6 M HCI, 8-10 h, 110 "C) or after hydrolysis. For dansylation, the dried material was resuspended in 3 p1 of0.2 M collected every 0.5 min. Thin-layer Chromatography of Radiolabeled Lipids-Neutral lipids NaHC03, pH9, and 3 pl of 2.5 mg/ml5-dimethylaminonaphthalenewere chromatographed on Silica Gel G TLC plates (20 x 20 cm; l-sulfonyl chloride (dansyl chloride; Pierce Chemical Co.) in acetone Merck) using solvent system a (petroleum ether/diethyl ether/acetic wasadded. Tubes were sealed, covered with aluminum foil, and acid (70:30:2)). Phospholipids were separated on Silica Gel 60 TLC incubated at 37'C for 30 min. At the end of the incubation, the plates (20 x 20 cm; Merck) using either Solvent System b (chloroform, samples were dried, reconstituted in 5 pl of 50% aqueous pyridine, methanol, 30% ammonia (13:5:1)) or c (first development in acetone/ and analyzed by TLC using cellulose sheets developed in 1-butanol/ acetic acid/water (5:2:3). In this system, dansylethanolamine mipetroleum ether (1:3), followedby chloroform/methanol/acetic acid/ EXPERIMENTALPROCEDURES

1972

Glycolipid Precursor for Membrane Protein Glycolipid Anchor

grated almost at the solvent front (RF = 0.97), and underivatized ethanolamine had an R F of 0.55-0.65. Bio-Gel P-4 Gel Filtration-TLC-purified [3H]ethanolamine or [3H]glucosamine-labeledlipids or thebutanol fraction of radiolabeled trypanosomes was treated with PI-specific phospholipase C in 300 pl of buffer as described. The reaction mixtures were extracted with water-saturated butanol. In each case, the aqueous phase was dried down, resuspended in 300 p1 of water, loaded onto a Bio-Gel P-4 column (101 cm x 0.5 cm'), and eluted with a phosphate/citrate buffer, pH 2.6, with ionic strength of 0.5 M (3.1 g/liter Na,HP04, 17.1 g/liter citric acid, 34.2 g/liter KCl). Flow rate was measured before and after each run and was about 8 ml/h. Fractions were collected every 3.5 min, and the column was standardized with [3H]glucose oligomers obtained from Dr. Jacques Baenziger (Washington University School of Medicine, St. Louis, MO). Large-scale Preparations of Lipids-Larger quantities of lipids for GC-MS or amino acid analysis were prepared from 1-2 X 10" trypanosomes lysed in ice-cold water containing 50 pg/ml leupeptin and 0.1 mM Nu-p-tosyl-L-lysine chloromethyl ketone and centrifuged at 45,000 X g for 1 h. Supernatants were withdrawn and stored at -20 "C. Lysed cell pellets were frozen rapidly in dry ice/ethanol and lyophilized prior to extraction of lipids as described earlier. Glycolipids were purified by TLC on silica HPTLC plates as follows. The plates were "cleared"by development in methanol/diethyl ether ( 4 1 ) and then air-dried prior to a second "clearing" development in chloroform/methanol/pyridine/water (2:41:1) (Muthing etal., 1987). The butanol fraction was dried down and resuspended in less than 50 pl of water-saturated butanolfor TLC and streaked evenly over a 2-cmwide strip which was 2 cm from the bottom and at least 1 cm away from the edge of the plate. An adjacent, otherwise identical strip was used as a blank. The butanol fraction of [3H]myristic acid-labeled trypanosomes was used as a marker. The plates were developed in solvent system d; and then thesample and blank areas were covered with aluminum foil, whereas the marker lane was scraped a t 1.0-cm intervals and taken for liquid scintillation counting. The plate was never allowed to dry completely and, for this purpose, was kept in the TLC tank with solvent-moist paper lining after pouring off the bulk solvent. Peaks of radioactivity in the marker lane were identified, and corresponding areas of silica in thesample and blank laneswere scraped and extracted with 1.0 ml of methanol/pyridine/water ( 2 l : l ) . The MPW extracts were stored a t 4 "C until required for analysis (within 1 month). Ethanolamine and GlucosamineAnalysis-Analyseswere performed by R. Sherwood (Cornel1 University Department of Chemistry, Biotech Analytical Facility, Ithaca, NY), using Waters Pico-Tag system. Samples were hydrolyzed in 6 M HCl for 17 h at 110 "C under nitrogen, followed by conversion to the phenylthiocarbonyl derivatives. A modified Waters Pico-Tag procedure was used where the samples were not re-dried and theamount of derivatizing reagent was doubled to ensure that any residual HCl did not interfere with the Edman reaction. Separation was accomplished on a CIa Nova-Pak radial compression column in a heated RCM-100 unit (Waters) using an acetic acid/triet5ylamine/acetonitrile and acetonitrile/water gradient elution program. Ethanolamine and glucosamine were identified and quantified using one or separate elution gradients. Homoserine was used as aninternal standard. GC-MS-Capillary GC-MS was performed by Y. T. Ng and Dr. Aladar Benscath (The Rockefeller University Extended Range Mass Spectrometric Research Resource) using a Hewlett-Packard 5790A series gas-liquid chromatograph interfaced via a direct capillary coupling to a VG 70-250 two-sector, high resolution, computer-controlled magnetic sector mass spectrometer. A fused silica column (25 m x 0.3 mm) coated with a cross-linked 5% phenylmethyl silicone polymer was used. The gas-liquid chromatography temperature program was 120 "C for 2 min, followedby a linear gradient at 10 "C/min to 250 "C. The final temperature was maintained for 7 min. The interface temperature was 250 'C. Isobutane chemical ionization was used a t a source temperature of 250 "C and pressure of 5 X torr. Injection temperature was 250 'C, and the column pressure was 8 p.s.i. with a flow rate of 3 ml/min. Lipid and corresponding background samples were analyzed for neutral sugars and fatty acids. All samples including fatty acid and sugar standards were methanolyzed (75 pl, 0.5 M methanolic HCl (Supelco, Inc.), 110 "C, 8 h, in sealed microdispenser tubes) and then *.xtracted three times with 75 pl of hexane each time. The hexane extracts were pooled, dried down, and resuspended in 10 pl of hexane. This phase was used for analysis of the fattyacid methyl esters. The acid phase used for analysis of sugars was neutralized with a small

quantity of silver carbonate. The silver carbonate residue was separated by centrifugation, and the supernatant was transferred to a clean tube and dried in a Speed-Vac evaporator. 16 pl of bis(trimethylsily1)trifluoroacetamide (Supelco, Inc.) was added, and the tube was sealed and incubated at 70 "C for 30 min to convert neutral sugars to their trimethylsilyl derivatives. Trimethylsilyl sugars andfatty acid methyl esters were identified by their GC retention times (scan numbers) and characteristic fragmentation patterns by comparing them to identically treated standards. Greater than 95% of the mannose standard chromatographed as one peak, the major fragment ions being m/z 451 (lo%),361 (loo%), 319 (lo%),and 271 (10%).The galactose standard was primarily a doublet with the major fragment ions being: early peak, m/z 451 (5%),361 (55%), 319 (15%), and 271 (100%);and late peak: m/z 451 (5%),361 (loo%),319 (IO%), and 271 (20%) (see Fig. 5, BI-B3).

RESULTS

Extraction and Analysis of a [3H]MyristicAcid-labeled Lipid Precursor-The fatty acid composition of the diglyceride moiety of the membrane form of VSG (mfVSG) is exclusively myristic acid. (3H]Myristic acid is readily incorporated into mfVSG, and it was assumed that this would also be true for a lipid precursor of the mfVSG glycolipid membrane anchor. Trypanosomes were labeled with [3H]myristi~acid and then osmotically lysed and lyophilized. The dried residue was exhaustively extracted in chloroform/methanol (2:l) to reduce background radioactivity from excess radiolabel, neutral lipids, phospholipids, and less polar glycolipids. Analysis of these extracts by TLC (Silica Gel 60, solvent system b) showed a spectrum of about 10bands, withsome radioactivity migrating at the solvent front. An identical pattern was obtained in a [3H]palmiticacid labeling experiment. The chloroform/methanol(2:l) extracted trypanosome residue was then dried under nitrogen and extracted with methanol/pyridine/water (2:l:l). The MPW extract typically contained about 10 times the radioactivity of the last CM extract. Methanol/pyridine/water has been previously used (Ferguson et aE., 1985a) as a solvent for the C-terminal amino acid-linked glycolipid obtained by Pronase digestion of mfVSG. Analysis of the MPW extractby HPLC gave a pattern of three peaks (Pl-P3) with relative intensities P1