Glycobiology

Materials

S-Citronellol, DE-52 cellulose, monensin, valinomycin, ionomycin, carbonylcyanide p-trifluoromethoxy-phenylhydrazone (FCCP), carbonylcyanide m-chlorophenylhydrazone (CCCP), ATP, and [gamma]-S-ATP were purchased from Sigma Chemical Co. (St. Louis, MO). Phosphorus trichloride oxide was obtained from Alfa Products (Danvers, MA). Trichloroacetonitrile was obtained from Aldrich Chemical Co. (Milwaukee, WI). [U-¹⁴C]Glucose (360 mCi/mmol) was obtained from American Radiolabelled Chemicals (St. Louis, MO). UPD-[1-³H]glucose was synthesized enzymatically as described previously (Rush and Waechter, 1998). [2-³H]Man 6-P was synthesized enzymatically and purified as described previously (Rush and Waechter, 1992). Millipore HA (0.45 µm) filter disks were purchased from Baxter Scientific Products (Obetz, OH). Whatman GF/C glass microfiber filter disks were obtained from American Scientific Products (McGaw Park, IL). Econosafe Liquid Scintillation counting cocktail is a product of Research Products International Corp. (Mount Prospect, IL). All other chemicals and reagents were purchased from standard commercial sources.

Synthesis of ([omega],c)Dol₁₅, ([omega],t,t)Dol₂₀, and ([omega],t,c)Dol₂₀

([omega],t,t)Dol₂₀ and ([omega],t,c)Dol₂₀were prepared as described previously (Jaenicke and Siegmund, 1989) using geranyl-4-tolyl sulfone and either 8-chloro-(6-c)-citronellylbenzyl ether or 8-chloro-(6-t)-citronellylbenzyl ether as building blocks for the synthesis. For the synthesis of ([omega],c) Dol₁₅, 1 g of (6-benzyloxy-4-methyl-(c)-4-hexen-1-yl)triphenylphosphonium iodide, prepared according to Sato et al. (1983), was dissolved in methanol and shaken overnight at room temperature under a 35 psi hydrogen atmosphere in the presence of 100 mg PtO₂. After filtration over Celite, the filtrate was concentrated under reduced pressure to yield 1g of a yellowish oil that was directly used for the following Wittig reaction. The reaction product, (6-benzyloxy-4-methyl-1-hexyl)-triphenyl-phosphonium iodide was dissolved in 5 ml anhydrous tetrahydrofuran and stirred for 10 min at -78°C after the addition of 1.16 ml (1.85 mmol, 1.1 eq.) of 1.6 M n-butyl-lithium in hexane. After 10 min, 0.235 g (1.1 eq.) of 6-methyl-5-hepten-2-one (Aldrich) was added dropwise and the reaction was stirred overnight at room temperature. The reaction mixture turned light brown, and TLC analysis indicated that no starting material was left. After the addition of saturated ammonium chloride solution, the mixture was decanted and extracted twice with ether. The combined organic layers were washed once with brine, then dried briefly over MgSO₄ and concentrated under reduced pressure to give an oily solid, which was purified on a short path silica gel column equilibrated in hexane and eluted stepwise with 2%, then 3% ethyl acetate to yield 0.27 g of the benzyl ether of ([omega],c)Dol₁₅ (51%, based on phosphonium salt). The benzyl group was removed by adding 0.3 g (0.95 mmol) of the benzyl ether in 5 ml anhydrous tetrahydrofuran to a solution of 110 mg (4.77 mmol, 5 eq.) Na in NH₃ at -78°C. After 30 min at -33°C (refluxing NH₃), solid ammonium chloride was added until the blue color disappeared and ammonia was allowed to evaporate over a period of ~45 min. The residue was evaporated under reduced pressure and purified over a short path silica gel column with ethyl acetate/hexane (1:3) to yield 177 mg (82.7%) of a colorless oil. Progress of reactions was monitored by thin layer chromatography on silica gel in either ethyl acetate/hexane (1:9) for the Wittig reaction or ethyl acetate/hexane (1:2) for the removal of the benzyl group. Reaction products were analyzed by ¹H-NMR and the trans/cis ratios of the double bonds were established by integration of the vinylic methyl shifts at 1.6 ppm according to Jaenicke and Siegmund (1989). ([omega],c)Dol₁₅, ([omega],t,t)Dol₂₀, and ([omega],t,c)Dol₂₀ were chemically phosphorylated as described below for citronellol.

Synthesis and purification of short chain, water-soluble analogs of Glc-P-Dol

Citronellyl phosphate was synthesized by phosphorylation of citronellol using trichloroacetonitrile and phosphorus trichloride oxide (Danilov and Chojnacki, 1981) and purified as described previously (Rush et al., 1993). Enzymatic reactions for the synthesis of Glc-P-Dol₁₀ contained 10 mM MgCl₂, 50 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 2 mM Cit-P, 5 mM 2-mercaptoethanol, 1 mM sodium orthovanadate, 20-80 µM UDP-[³H]Glc (1-500 c.p.m./pmol), and hen oviduct microsomes (0.75 mg protein) in a total volume of 0.5 ml. Following incubation for 1 h at 37°C, the reaction was centrifuged(100,000 × g, 10 min) in a Beckman TL-100 tabletop ultracentrifuge and the supernatant removed. The resulting membrane pellet was washed two times with 0.25 ml of water. The supernatants were combined and loaded onto an 8 ml column of DE-52 cellulose. After rinsing the ion-exchange column with 5 column volumes of distilled water, Glc-P-Dol₁₀ was eluted with a 50 ml gradient (0-0.5 M) of NH₄HCO₃. The fractions containing Glc-P-Dol₁₀ were combined, concentrated by rotary evaporation under reduced pressure at 30°C and desalted by gel-filtration chromatography on a Bio-Gel P-2 column (1.5 cm × 40 cm) eluted with distilled water. The fractions containing Glc-P-Dol₁₀ were again combined, concentrated and stored at -20°C until use.

Enzymatic reactions for the synthesis of ([omega],c)-Glc-P-Dol₁₅, ([omega],t,t)-Glc-P-Dol₂₀, and ([omega],t,c)-Glc-P-Dol₂₀ contained 10 mM MgCl₂, 50 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 5 mM 2-mercaptoethanol, 1 mM sodium orthovanadate, 20-80 µM UDP-[1-³H]Glc (5-500 c.p.m./pmol), hen oviduct microsomes (1-10 mg protein) and the appropriate isoprenyl monophosphate substrate (0.2 mM) in a total volume of 0.5-4 ml. Following incubation for 2 h at 37°C, the reaction was stopped by the addition of 5 ml of CH₃OH. The precipitated protein was sedimented by centrifugation (1000 × g, 5 min) and the supernatant removed and placed on ice. The pellet was then resuspended twice with 2 ml of CHCl₃/CH₃OH/H₂O (10:10:3) and recentrifuged, and the supernatants were combined with the CH₃OH layer. The organic extract was then supplemented with additional CHCl₃ to give a final composition of CHCl₃/CH₃OH/H₂O (3:2:1) and mixed vigorously. The two phases were separated by a brief centrifugation and the aqueous (upper) layer was removed. The organic (lower) layer was then reextracted with 5 ml of CHCl₃/CH₃OH/H₂O (3:48:47). The aqueous layers were combined, evaporated to dryness under reduced pressure at 35°C by rotary evaporation, redissolved in 1 ml water, and transferred with two rinses to a glass centrifuge tube. This solution was then mixed with 1 ml of water-saturated butanol and centrifuged briefly. The butanol phase was collected and the aqueous phase was re-extracted with 1 ml of water-saturated butanol. The butanol phases were combined, washed once with butanol-saturated water, and dried under a stream of N₂. Contaminating phospholipids were next destroyed by deacylation at 0°C, 30 min in toluene/CH₃OH (1:1) containing 0.1 M KOH. The reaction mixture was neutralized with glacial acetic acid and dried under a stream of N₂. The deacylated mixture was desalted by partitioning with water-saturated butanol, as described previously, and the Glc-P-Dols were purified by ion-exchange chromatography on DE-52 cellulose as described by Waechter and Scher (1981). Following purification by DE-52 cellulose chromatography, Glc-P-Dols were again desalted by butanol/water partitioning and purified further by preparative thin layer chromatography on Silica Gel G developed in CHCl₃/CH₃OH/H₂O/NH₄OH (65:35:4:1). Zones containing radioactive Glc-P-Dols were located with a Bioscan 200 Imaging System, excised from the thin layer plate and eluted from the silica gel with CHCl₃/CH₃OH/H₂O (10:10:3). Purified water soluble Glc-P-Dolichols were then dried, dissolved in water and stored at -20°C until use. The concentration of various water-soluble analogs was determined by phosphate analysis as described previously (Bartlett, 1959).

Electrospray-ionization tandem mass spectrometry of water-soluble Glc-P-Dols

Negative-ion electrospray-ionization mass spectrometry was performed with a Finnigan-MAT (San Jose, CA) TSQ 7000 triple quadrupole instrument using direct injection as previously described (Woluka et al., 1998). Samples were dissolved in acetonitrile-H₂O (1:1) and infused into the electrospray source at a flow rate of 3 µl/min. In tandem mass spectrometry experiments, the pressure of the target gas (xenon) was 0.6 mTorr and the collision energy was maintained at 15 or 20 eV.

Preparation of microsomes from rat liver and pig brain

For transport studies with the water-soluble analog of Glc-P-Dol, ER-enriched microsomal vesicles were prepared from rat liver essentially as described by Coleman and Bell (1978). The membrane vesicles were judged to be >95% intact as assessed by mannose 6-phosphate (Man 6-P) phosphatase latency (see below). Pig brain microsomal vesicles were prepared as described in the preceding publication (Rush and Waechter, 1998).

Preparation of microsomal fractions and mitochondria from rat liver

Light (Golgi-enriched) and heavy (ER-enriched) microsomes and mitochondria were prepared from rat liver by minor modifications of the methods of Leelavathi et al. (1970) and Fleischer and Kervina (1974) exactly as described previously (Rush and Waechter, 1995). Cross-contamination of the subcellular fractions from rat liver was assessed by measuring the established marker enzyme activities: ER/glucose 6-phosphatase (Rush and Waechter, 1992); Golgi/galactosyltransferase (Fleischer and Smigel, 1978) and mitochondria/succinate dehydrogenase (Pennington, 1961).

Isolation of bovine red blood cells

Bovine red blood cells were collected into 5 mM EDTA and sedimented by centrifugation at 1000 × g, 10 min 4°C. The buffy coat (white cells) was removed by aspiration and the red cells were resuspended in Dulbecco's phosphate-buffered saline (PBS) and stored on ice until used for transport assays.

Assay of Glc-P-Dol₁₀ transport by microsomal fractions from rat liver or pig brain

Assay mixtures for the measurement of [³H]Glc-P-Dol₁₀ uptake contained 10 mM Tris-Cl (pH 7.4), 0.25 M sucrose, the appropriate concentration of [³H]Glc-P-Dol₁₀ (5-500 c.p.m./pmol) and brain or liver microsomes (150-500 µg membrane protein) in a total volume of 10-20 µl. Following incubation at 19-23°C for 30 sec, [³H]Glc-P-Dol₁₀ transport was stopped by the addition of 0.5 ml of ice-cold 0.25 M sucrose, 10 mM Tris-HCl (pH 7.4), and the amount of [³H]Glc-P-Dol₁₀ transported was determined by a filtration assay as described by Bishop and Bell (1985). The diluted assay mixtures were quickly transferred to a chilled filtration manifold equipped with a Millipore HA (0.45 µm) filter disk and suction-filtered. The disks were then washed with an additional 10 ml of ice-cold 0.25 M sucrose, 10 mM Tris-HCl (pH 7.4). Filtration was usually completed in less than 30 sec. The Millipore filter was then transferred to a 20 ml scintillation vial and the amount of radioactivity retained on the filter determined by scintillation spectrometry in a Packard 2100TR Scintillation Spectrometer after the addition of 1.0 ml of 1% SDS and 10 ml of Econosafe liquid scintillation cocktail.

Measurement of glucose transport by erythrocytes and liver ER-enriched microsomes

Glucose transport assay mixtures contained 10 mM Tris-HCl (pH 7.4), 0.25 M sucrose, 0.5 mM [¹⁴C]glucose (52 c.p.m./pmol) and the indicated amount of either bovine erythrocytes or microsomal vesicles from rat liver (0-120 nmol membrane phospholipid) in a total volume of 50 µl. Following incubation at 19-23°C for 1 min, transport reactions were diluted with 0.5 ml of ice-cold 10 mM Tris-HCl (pH 7.4), 0.25 M sucrose. Glucose transport was determined exactly as described above for Glc-P-Dol₁₀ except that Whatman GF/C glass-fiber filter disks were used for the measurement of transport by human erythrocytes.

Assessment of the integrity of ER-enriched and Golgi-enriched vesicles and mitochondria

The integrity of liver ER microsomes was determined by measuring Man 6-P phosphatase latency using [³H]Man 6-P as substrate (Rush and Waechter, 1992). The intactness of pig brain microsomes was assessed by assaying for glucosidase I/II latency (Rush and Waechter, 1998). The integrity of rat liver Golgi vesicles was determined by measuring the latency of rat liver galactosyltransferase to proteolytic digestion with trypsin. Mitochondrial integrity was estimated by measuring the sensitivity of succinate dependent reduction of potassium ferricyanide to inhibition by antimycin A as described by Klingenberg (1979).

Preparation of synthetic PC-liposomes

Synthetic liposomes were prepared using liposome kit No. L-4012 (Sigma Chemical Co.) containing egg phosphatidylcholine, dicetylphosphate, and cholesterol in a molar ratio of 7:2:1. Liposomes were formed by sonication in 10 mM Tris-HCl (pH 7.4), 0.25 M sucrose and dialyzed against the same solution overnight before use as described previously (Rush and Waechter, 1995).

Measurement of uptake of [³H]Glc-P-Cit by synthetic PC-liposomes.

Uptake of [³H]Glc-P-Cit by synthetic PC-liposomes was determined by gel filtration on a column (0.5 × 20 cm) of Sephacryl S-300 equilibrated in 10 mM Tris-HCl pH 8.0, 0.25 M sucrose as described previously (Rush and Waechter, 1995).

Assay of Glc-P-Dol_10-20:Glc_0-2Man₉GlcNAc₂-P-P-Dol GlcTase activity in sealed pig brain vesicles

Incubation mixtures contained 50 mM Tris-HCl (pH 8), 0.2 M sucrose, 10 mM EDTA, 2 mg/ml Triton X-100, 0.75 mg of pig brain microsomal protein, and the indicated concentration of [³H]Glc-P-Dol_10-20 (500 c.p.m./pmol) in a total volume of 0.1 ml. Following incubation for 2 min at 37°C the incorporation of [³H]glucose into Glc_1-3Man₉GlcNAc₂-P-P-Dol was determined by a multiple extraction procedure (Waechter and Scher, 1978). For the study described in Table III, reaction mixtures containing 50 mM Tris-HCl (pH 8), 0.2 M sucrose, 10 mM EDTA, 0.75 mg of pig brain microsomal protein, and 0.2 µM [³H]Glc-P-Dol₂₀ (500 c.p.m./pmol) in a total volume of 0.1 ml were incubated for 3 min at 37°C in the presence or absence of Triton X-100 (0.5 mg/ml).

Characterization of lipid-linked [³H]oligosaccharide products formed in Glc-P-Dol-mediated GlcTase reactions

The lipid-bound oligosaccharide products of the enzymatic reactions were released by mild acid hydrolysis at 50°C, 30 min, in 80% tetrahydrofuran containing 0.1 M HCl (Lucas et al., 1975). The hydrolysate was neutralized by the addition of 1 N NaOH, dried under N₂, and desalted by gel-filtration chromatography on a Sephadex G-10 column (1.5 X 30 cm) equilibrated in distilled water. The desalted oligosaccharides were characterized by high pH anion-exchange chromatography using a Dionex BioLC HPAEC system equipped with an analytical CarboPac PA1 column (0.4 × 25 cm) equilibrated in 0.25 M NaOH and eluted with a sodium acetate gradient (0-200 mM, 35 min) at a flow rate of 1 ml/min (Cooper and Rohrer, 1994). Fractions of 0.5 ml were collected, neutralized with glacial acetic acid and analyzed for radioactivity by scintillation spectrometry in a Packard Tri-Carb 2100TR liquid scintillation spectrometer. The chain lengths of the enzymatically labeled oligosaccharides were determined by comparing their elution positions with defined oligosaccharide standards and an exogenously added internal standard with the composition Man₉GlcNAc₁.

Analytic methods

Protein concentrations were determined by the method of Rodriquez-Vico et al. (1989) using a protein assay reagent (BCA, Pierce, Rockford, IL). Lipid-phosphorus was determined by the method of Bartlett (1959).