Division of Biological Chemistry and Molecular Microbiology, School of Life Sciences, The Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, Scotland
Received on September 25, 2003; revised on September 9, 2004; accepted on September 10, 2004
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Abstract |
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Key words: glucosamine / glycosylphosphatidylinositol / inositol / trypanosome
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Introduction |
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The current method of choice for quantification of GPIs is gas chromatography-mass spectrometry (GC-MS) analysis of the myo-inositol content of highly purified GPI-anchored glycoproteins or free GPIs (Ferguson, 1993). In this method, the sample is mixed with an internal standard of hexa-deuterated myo-inositol and subjected to strong acid hydrolysis to liberate myo-inositol from any structure. This is followed by trimethylsilyl (TMS) derivatization. The resulting inositol-TMS6 derivatives are then analysed by GC-MS using selected ion monitoring for the characteristic m/z 305 and 318 fragment ions of inositol-TMS6 and the corresponding m/z 307 and 321 ions for the deuterated internal standard. This method is robust, accurate, and sensitive (useful range 10100 pmol) but relies on the absence of other myo-inositol-containing molecules, such as phosphatidylinositol (PI), lyso-PI, and inositol-phosphoceramide. These can be difficult to quantitatively remove from GPI-anchored structures. Other methods that have been employed, such as phosphate or mannose content, are equally susceptible to common biological contaminants, such as phospholipids and dolichol cycle intermediates, respectively. Thus, estimates of GPIs are frequently overestimates and the less abundant a particular GPI-containing molecule is, the harder it is to obtain an accurate estimate of its concentration.
In this present work, we describe a method of GPI quantification that exploits the presence of a single non-N-acetylated glucosamine residue in all GPI structures (Ferguson et al., 1999). The origin of this residue is the second step of GPI biosynthesis: the enzymatic de-N-acetylation of GlcNAc-PI to GlcN-PI by the GlcNAc-PI de-N-acetylase (E.C. 3.1.1.69) (Doering et al., 1989
) encoded by the PIG-L/GPI12 genes (Chang et al., 2002
; Nakamura et al., 1997
; Watanabe et al., 1999
). This is an obligatory step in GPI biosynthesis (Sharma et al., 1997
; Smith et al., 2001
), and consequently all GPIs contain this feature. Some GPIs contain additional hexosamine residues, but these are invariably N-acetyl-hexosamines (GlcNAc or GalNAc) (Ferguson et al., 1999
). A useful property of non-N-acetylated GlcN is its ability to react with nitrous acid generated in situ under mild conditions (pH 4.0, room temperature) and to be almost quantitatively converted to 2,5-anhydromannose (Conrad and Guo, 1992
). This is an unnatural sugar that will not be found as a biological contaminant. To stabilize this exocyclic aldehyde-bearing sugar, we reduce it with sodium borodeuteride to yield [1-2H]-2,5-anhydromannitol (AHM), a stable compound that is easily detected as its TMS derivative by GC-MS (Ferguson, 1993
). The application of this method to both highly purified and crude GPI preparations is described.
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Results |
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The second step exploits the fact that non-N-acetylated/non-N-sulfated GlcN is unique to GPIs in many extracts (see Discussion). Thus nitrous acid treatment of the GlcN residue at pH 4.0 creates a diazonium ion that rearranges via a ring contraction to the unnatural sugar 2,5-anhydromannose and liberates N2 gas. Subsequent reduction with sodium borodeuteride coverts this sugar to [1-2H]-AHM.
The third (optional) step is dephosphorylation using cold 48% aqueous HF to remove any EtNP or AEP substituents that might be attached to the 6-position of the AHM. This step will also remove the phosphoglyceride from the 1-position of the myo-inositol residue, allowing simultaneous detection of this component in the final analysis.
The fourth step is methanolysis, using methanolic HCl, which liberates the AHM and generates 1-O-methyl-glycosides of any other monosaccharides present in the sample.
The fifth step is TMS derivatization of hydroxyl groups to render the products volatile for GC-MS analysis.
Range and linearity of the method
A set of triplicate samples containing 50 pmol scyllo-inositol and 50, 100, 200, 400, 800, 1600, and 3200 pmol GlcN were subjected to protocols A and to B (see Materials and methods) and analysed by GC-MS. The selected ion monitoring GC-MS program detects the m/z 273 fragment ion of the deutero-reduced AHM-TMS4 derivative at about 16.3 min and the m/z 318 fragment ion of the scyllo-inositol-TMS6 derivative at about 22.6 min; see Figure 2 for a representative chromatogram.
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We used our method to estimate the total copy number of GPI precursor molecules in bloodstream form T. brucei and the total number of all GPI molecules in the procyclic form of T. brucei. Washed bloodstream-form parasites were subjected to osmotic shock to remove the majority of the VSG molecules through the action of endogenous GPI-specific phospholipase C (Ferguson et al., 1985). The washed cell ghosts were extracted with chloroform/methanol/water, and the soluble fraction was dried and repartitioned between water and butan-1-ol. The butan-1-ol phase was backwashed with water and used as the total lipid extract. Triplicate aliquots of this extract corresponding to 5 x 107 cells were analysed for GPI content using protocol C; protocol C is identical to protocol A except that it includes an extra step to remove water-insoluble material after the alkaline hydrolysis step (see Materials and methods). Washed procyclic cells were also lysed by osmotic shock, but in this case, the washed cell ghosts were simply freeze-dried prior to analysis by protocol C.
The total ion current chromatogram of a GC-MS trace of the processed procyclic cell ghosts is shown in Figure 4A. This shows the presence of Rib, Xyl, Man, Gal, Glc, and myo-inositol, as well as AHM from deaminated/reduced GPIs, and the scyllo-inositol internal standard. The selected ion monitoring chromatogram (Figure 4B) illustrates the selectivity provided by this type of analysis and reinforces the extremely high quantity of myo-inositol in the sample that would preclude GPI quantification by conventional inositol analysis.
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Application of the method to mammalian crude biological samples
Chinese hamster ovary (CHO) cells were used to assess the application of the method to mammalian cells. In particular we wished to address whether the presence of glycosaminoglycans, which contain small amounts of non-N-acetylated glucosamine, might affect the results. To assess this, we analyzed whole cell lysates of wild-type CHO cells, glycosaminoglycan-deficient CHO cell mutant pgsA-745 (Murphy-Ullrich et al., 1988), and a CHO cell GlcNAc-PI de-N-acetylase (PIG-L) deficient mutant (that lacks GPIs; Nakamura et al., 1997
) for total glucosamine content using protocol C (Table I). Triplicate samples from 107 CHO cells were used. The figures for the wild-type cells and proteoglycan-deficient cells are indistinguishable, suggesting that the presence of proteoglycans does not significantly contribute to the measurement. However, the GPI-deficient cells have a significant background (50% of wild type) level of glucosamine of unknown origin. This suggests that the method may not be appropriate for very crude mammalian cell preparations without a GPI-minus control.
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Discussion |
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When sample GPIs have or are suspected to have EtNP or AEP attached to the GlcN residue of the glycan core, as found in T. cruzi and T. dionisii GPIs (Branquinha et al., 1999; Serrano et al., 1995
), an additional step of aqueous HF dephosphorylation is required. We have shown that this treatment does not significantly affect the efficiency of the conversion of GlcN to AHM (Figure 3). However, it is important that this step comes after deamination/reduction because it causes partial de-N-acetylation of HexNAc residues (Ferguson et al., 1988
).
The method was less useful with whole mammalian cell lysates due to the presence of significant background levels of non-GPI and non-proteoglycan glucosamine (Table I). However, subtraction of this background from the wild-type value suggests that CHO cells contain 2.3 ± 0.6 x 107 GPIs per cell. Analysis of the total lipid extract of CHO cells revealed that the copy number of GPI intermediates per cell was negligible (data not shown). Assuming CHO cells have a diameter of 35 µm and a surface area of around 4000 µm2, this suggests a density of 6000 GPI-anchored proteins per µm2. This figure can be compared with that of T. brucei bloodstream form parasites of
66,000 GPI-anchored glycoproteins per µm2.
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Materials and methods |
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Step 1: alkaline hydrolysis. Each sample is resuspended in 100 µl concentrated aqueous NH3/40% propan-1-ol (1:1, v/v) and incubated for >16 h at room temperature. Samples are then dried under a stream of nitrogen.
Step 2: deamination and reduction. The samples are redissolved in 15 µl 300 mM sodium acetate buffer (this buffer is 300 mM with respect to sodium acetate and adjusted to pH 4.0 with glacial acetic acid). To each sample, 10 µl freshly prepared 1 M sodium nitrite is added and incubated at room temperature for 3 h. Following nitrous acid deamination, 5 µl of 400 mM boric acid is added followed by x µl 2 M sodium hydroxide and 20 µl freshly prepared 1 M sodium borodeuteride (Aldrich, Milwaukee, WI). (Note: The amount of 2 M sodium hydroxide to be used is determined empirically for each batch of samples by measuring the number of ml required to adjust 6 ml 300 mM sodium acetate buffer [pH 4.0], 4 ml 1 M sodium nitrite, and 2 ml 400 mM boric acid to pH 10.5. This number [in ml] is multiplied by 5 to give the amount x in µl.) The samples are allowed to reduce at 4°C for 16 h, with the lids left lightly screwed to allow the release of deuterium gas. Each sample is acidified with 100 µl 1 M acetic acid and applied to a disposable 0.4-ml column of AG50WX12 resin (Bio-Rad, Hercules, CA) that has been converted to the H+ form by washing with 0.5 M HCl followed by >20 column volumes of water, according to the manufacturer's instructions. The desodiated samples are eluted with five column volumes of water into a glass tube and dried by rotary evaporation. Boric acid is removed by coevaporation three times with 100 µl 2% acetic acid in methanol and once with 200 µl methanol. Samples are redissolved in 50 µl water.
Step 3: aqueous HF dephosphorylation (protocol B only). If the samples are suspected to contain GlcN residues that are substituted with EtNP or AEP groups, then they are transferred to Eppendorf tubes, dried in a Speed Vac, and incubated with 50 µl ice-cold 48% aqueous HF (VWR/Merck, Darmstadt, Germany) for >16 h at 0°C. After drying in a Speed Vac, samples are redissolved in 50 µl water.
Step 4: methanolysis. Sample solutions are transferred to glass capillary tubes (Drummond Scientific, Broomall, PA, 100200 µl) that have been heated to 400°C for 4 h in a furnace to destroy organic contaminants and flame-sealed at one end to create a microtube. The samples are dried in a Speed Vac and 50 µl 0.5 M HCl acid in dry methanol (Supelco, Dorset, UK) is added to each tube. After flame-sealing, the tubes are incubated at 95°C for 4 h in a heating block. The tubes are scored with a glass knife and broken open, and 10 µl pyridine and 10 µl acetic anhydride are added to neutralize the HCl and re-N-acetylate any HexNAc residues. After 30 min, the samples are dried in a Speed Vac and dried again from 20 µl dry methanol.
Step 5: TMS derivatization. Finally, 15 µl fresh TMS reagent (l-trimethylchlorosilane/hexamethyldisilazane/dry pyridine [1:3:10, v/v/v]) is added and the tubes are sealed with Teflon tape. After 2030 min at room temperature, 1-µl aliquots are analyzed by GC-MS.
Protocol C. Protocol C is used for crude preparations like whole lipid extracts and whole cell ghosts. The protocol is identical to protocol A except that after step 1, samples are resuspended in 100 µl water with vortexing and incubation in a sonicating waterbath for 3 x 10 min. The suspension is then centrifuged in a microfuge at 13,200 rpm for 10 min to pellet debris. The upper 50 µl of the supernatant is transferred to a clean Eppendorf tube and dried in a Speed Vac concentrator before commencement of the remainder of the protocol.
Selected ion monitoring GC-MS for [1-2H]-AHM, methyl glycosides, and inositol TMS derivatives
GC-MS was performed with a Hewlett-Packard 6890-5973 system. Splitless injection (injection temperature 280°C) onto a 30 m x 0.25 mm HP5 (Agilent, Wilmington, DE) column was used, using helium as the carrier gas. The initial oven temperature was 80°C (2 min), followed by temperature gradients to 140°C at 30°C/min, from 140°C to 250°C at 5°C/min, and from 250°C to 265°C at 15°C/min. The final temperature was held for 10 min.
Typical elution times (±0.2 min, allowing for different columns) are: AHM, 16.4 min; Man, 16.9 min; Gal, 17.7 min; Glc, 18.9 min; scyllo-inositol, 22.7 min; myo-inositol, 23.9 min; GlcNAc, 23.0 min.
The electron impact ionization/quadrupole mass detector was programmed to detect [1-2H]-AHM using m/z 273 over the range 919 min, hexoses using m/z 204 and 217 over the range 919 min, and inositols using m/z 305 and 318 over the range 1947 min. A dwell time of 50 ms was used for each ion.
Quantification of AHM (and GPI) is made using the formula: AHM (pmol) = (area m/z 273 peak at 16.4 ± 0.2 min/area m/z 318 peak at 22.7 ± 0.2 min) x (1/AHMMRRFsI) x 50; where AHMMRRFsI is the molar relative response factor determined from the mean value of: area m/z 273 peak at 16.4 ± 0.2 min/area m/z 318 peak at 22.7 ± 0.2 min, for the GlcN/scyllo-inositol, 1:1 (mol/mol), standards. The multiplier 50 assumes that 50 pmol scyllo-inositol internal standard was used.
Analysis of myo-inositol content by GC-MS
Samples are mixed with an internal standard of 20 pmol [1,2,3,4,5,6-2H]-myo-inositol and subjected to strong acid hydrolysis, TMS derivatization, and selected ion monitoring GC-MS analysis, as described in Ferguson (1993) and Goldman et al. (1990)
. Quantification of myo-inositol is made by comparing the areas of the m/z 318 (sample) and m/z 321 (internal standard) ions.
Bloodstream form T. brucei total lipid extract
Bloodstream form T. brucei (strain 427) was grown in HMI-9 medium (Hirumi and Hirumi, 1989) supplemented with 10% heat-inactivated fetal calf serum (HI-FCS; PAA Laboratories, Somerset, UK) and the antibiotic G418 (Roche, Indianapolis, IN) to a final concentration of 2.5 µg/ml, at 37°C and 5% CO2. Cells were collected at mid-log phase and resuspended in 150 µl phosphate-buffered saline, counted using a hemocytometer, and transferred to a glass vial. Total lipids were extracted from 5 x 107 cells by the addition of 1 ml chloroform/methanol (1:1, v/v) to give a final ratio of chloroform/methanol/water of 10:10:3, v/v/v. After vortexing and sonication, the extracts were centrifuged at 800 x g to pellet protein and other cellular debris. The supernatant was removed and transferred to an Eppendorf tube. To check that no protein remained in the sample, 10% was analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and Coomassie blue staining. The remaining sample was dried under nitrogen and partitioned using 1 ml butan-1-ol/water (2:1, v/v). The butan-1-ol phase containing the lipid extract was washed four times with water, removed, and dried in a Speed Vac concentrator.
Procyclic form T. brucei cell ghosts
Procyclic T. brucei (strain 427) was grown at 28°C in SDM-79 medium (Brun and Schonenberger, 1979) supplemented with 10% HI-FCS and 100 U/ml each of penicillin and streptomycin (Gibco, Rockville, MD). Cells were collected at mid-log phase and washed three times with ice-cold phosphate buffered saline. Cells were counted using a hemocytometer, and aliquots of 5 x107 cells were centrifuged at 13,200 rpm in a microfuge for 10 min. The cell pellet was resuspended in 1 ml ice-cold water to lyse the cells and centrifuged at 4°C for 10 min at 13,200 rpm in a microfuge. The supernatant was removed and the membrane pellets were snap-frozen and freeze-dried.
Epimastigote form T. cruzi glycoconjugates
Epimastigotes of T. cruzi CL-Brener strain were grown at 28°C in liver-infusion tryptose medium (Camargo, 1964) supplemented with 10% HI-FCS. Glycoconjugates were extracted from 2.6 x 109 cells using organic solvents and purified by octyl-Sepharose column (100 x 5 mm) chromatography, as previously described (Serrano et al., 1995
). The separate GPI-anchored mucins and GIPL fractions were purified a second time by octyl-Sepharose chromatography. Sample purity was assessed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (Invitrogen, Carlsbad, CA, precast 412% gel) and carbohydrate silver staining.
Whole CHO cell lysates
Wild-type and mutant CHO cells were cultured in 75 cm2 tissue culture flasks at 37°C in F-12 (Ham) medium (Gibco) containing 10% FCS. Confluent cells were washed with phosphate buffered saline and harvested with 4 ml trypsin-EDTA (Gibco), 20 min, 37°C. The cell suspension was centrifuged, and the cell pellet was resuspended in 1 ml ice-cold water to lyse the cells and centrifuged at 4°C for 10 min at 13,200 rpm in a microfuge. The supernatant was removed and the membrane pellets were snap-frozen and freeze-dried.
Carbohydrate silver stain
After electrophoresis, the gel was washed with water to remove excess sodium dodecyl sulfate and incubated at room temperature for 10 min in 50% methanol/12% trichloroacetic acid/2% CuCl2. The gel was rinsed and incubated in 10% methanol/5% acetic acid for 5 min, rinsed, and incubated in fresh 0.05% w/v potassium permanganate for 5 min. The gel was rinsed again, incubated in 10% methanol/5% acetic acid for 5 min, rinsed, and incubated in dry ethanol for 10 min. The gel was then washed with water for 5 min and incubated with 0.1% w/v silver nitrate for 5 min, followed by 10% w/v potassium carbonate for 1 min and 1% w/v potassium carbonate for a few minutes, until components are detected as orange-brown bands. The gel was then washed and stored in water to stop overdevelopment.
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Acknowledgements |
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Abbreviations |
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