A robust and selective method for the quantification of glycosylphosphatidylinositols in biological samples

James I. MacRae and Michael A.J. Ferguson1

Division of Biological Chemistry and Molecular Microbiology, School of Life Sciences, The Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, Scotland


1 To whom correspondence should be addressed; e-mail: m.a.j.ferguson{at}dundee.ac.uk

Received on September 25, 2003; revised on September 9, 2004; accepted on September 10, 2004


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
We have developed an assay for the quantification of glycosylphosphatidylinositol (GPI)-anchored glycoconjugates. The method is based on nitrous acid deamination and sodium borodeuteride reduction of the glucosamine residue, common to all GPI structures, to yield [1-2H]-2,5-anhydromannitol. Following acid methanolysis and trimethylsilyl derivatization, detection is by selected ion monitoring gas chromatography-mass spectrometry. The unnatural inositol isomer scyllo-inositol is used as an internal standard and the [1-2H]-2,5-anhydromannitol trimethylsilyl derivative is detected by following a characteristic electron-impact fragment ion at m/z 273. This method is more selective for GPIs than assays based on measuring myo-inositol content, which are often confounded by contaminating inositol-phospholipids. We show that the method can be applied to measure total GPI content in crude total lipid extracts and even in whole trypanosome ghosts. The method was applied to whole cell lysates of wild-type, GPI-deficient, and glycosaminoglycan-deficient CHO cells. The data revealed that proteoglycans did not interfere with total glucosamine estimates but that there are background levels of non-GPI and nonproteoglycan glucosamine-containing material in CHO cells.

Key words: glucosamine / glycosylphosphatidylinositol / inositol / trypanosome


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Glycosylphosphatidylinositol (GPI)-anchored glycoproteins are cell surface structures found in all eukaryotes (Ferguson et al., 1999Go). The GPI anchor is an alternative method of membrane localisation to hydrophobic transmembrane domains. The GPI family of structures are classified as those that contain the motif: Man{alpha}1-4GlcNH2{alpha}1-6myo-inositol-1-PO4-lipid (McConville and Ferguson, 1993Go). In addition, free GPI structures sharing this motif but not attached to protein, such as the glycoinositolphospholipids (GIPLs) and lipophosphoglycans, are abundant molecules in protozoal species like the trypanosomatids (Ferguson, 1999Go; Guha-Niyogi et al., 2001Go). GPI-anchored glycoproteins are topical because they are components and markers of lipid-ordered membrane microdomains or lipid rafts (Simons and Inkonen, 1997Go). Such microdomains are believed to play a role in protein targeting and signal transduction (Anderson and Jacobson, 2002Go). Similarly, the immunomodulatory and virulence-factor properties of certain parasite GPIs make these molecules of interest (Almeida et al., 2000Go; Schofield et al., 2002Go).

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, 1993Go). 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 10–100 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., 1999Go). 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., 1989Go) encoded by the PIG-L/GPI12 genes (Chang et al., 2002Go; Nakamura et al., 1997Go; Watanabe et al., 1999Go). This is an obligatory step in GPI biosynthesis (Sharma et al., 1997Go; Smith et al., 2001Go), 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., 1999Go). 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, 1992Go). 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, 1993Go). The application of this method to both highly purified and crude GPI preparations is described.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Reaction chemistry
Some consideration of the type of sample should be made before embarking on the analysis. The key decisions relate to (1) sample purity and (2) sample origin. With respect to sample purity, crude samples of lipid extracts or whole cells require an additional step after alkaline hydrolysis and before deamination to remove debris. With respect to sample origin, the GPIs of certain trypanosomes, like Trypanosoma cruzi and T. dionisii, have relatively acid-stable substituents of ethanolamine phosphate (EtNP) or 2-aminoethylphosphonate (AEP) attached to the 6-position of the GlcN residue (Branquinha et al., 1999Go; Lederkremer et al., 1991Go; Previato et al., 1990Go). Therefore, GPI samples from these organisms require an additional aqueous HF dephosphorylation step after deamination/reduction. The reaction scheme for the complete protocol, including the dephosphorylation step, is shown in Figure 1.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 1. Reaction scheme for the generation of AHM from GPIs. A generic GPI structure (± an EtNP substituent on the GlcN residue) is shown undergoing (1) mild alkaline hydrolysis; (2) nitrous acid deamination/sodium boro-deuteride reduction; (3) aqueous HF dephosphorylation (an optional step for samples with substituents on the GlcN residue); (4) acid methanolysis; (5) TMS derivatization. R can be H for free GPIs or protein.

 
The first step, after mixing the sample with a known quantity of scyllo-inositol internal standard, is mild alkaline hydrolysis with ammonium hydroxide. This removes ester-linked fatty acids and renders many (but not ceramide-based) GPIs water-soluble. This treatment also removes any Schiff base adducts that may have formed on the GlcN amino group during sample handling. It also solubilizes crude lipid and whole cell preparations by saponification of the major phospholipids.

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.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 2. Representative selected ion monitoring GC-MS chromatogram showing the detection of AHM and scyllo-inositol. The sample was derived from a mixture of 400 pmol GlcN and 50 pmol scyllo-inositol processed by protocol A.

 
A plot of the area of the m/z 273 AHM peak divided by the area of the m/z 318 scyllo-inositol peak against the amount of GlcN (Figure 3) shows (1) that the method is linear over the range 50 to 3200 pmol GlcN and (2) that the inclusion of step 3, aqueous HF dephosphorylation, does not significantly affect the measurements.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 3. Linear range of the assay. Triplicate samples of various amounts of GlcN were mixed with 50 pmol of scyllo-inositol and processed by protocol A (solid line) or protocol B (dotted line). The area of the m/z 273 (AHM) peak divided by the area of the m/z 318 (scyllo-inositol) peak is plotted against the amount of GlcN.

 
Validation of the method with authentic GPI standards
The method was applied to GPI-anchored mucins and to the GIPLs of T. cruzi (Lederkremer et al., 1991Go; Serrano et al., 1995Go). These chromatographically purified fractions (see Materials and methods) were quantified by myo-inositol content using the traditional selected ion monitoring GC-MS analysis using a [1,2,3,4,5,6-2H]-myo-inositol internal standard (Ferguson, 1993Go; Sherman et al., 1977Go) (Table I). Aliquots of the same samples were then subjected to our new method, with the inclusion of the aqueous HF step (protocol B) due to the presence of EtNP or AEP on the GlcN residue in T. cruzi GPIs. The amounts of AHM, and thus GPI, were calculated from the GC-MS data (Table I). The two methods were in good agreement with each other and with the expected amounts based on literature values. These data suggest that the new method is suitable for quantifying purified GPI-containing samples.


View this table:
[in this window]
[in a new window]
 
Table I. Comparison of AHM and inositol quantifications and published data

 
Application of the method to nonmammalian crude biological samples
To test the usefulness of the method for measuring GPIs in nonmammalian crude biological samples, we used samples from the tsetse fly–transmitted protozoan parasite T. brucei, the causative agent of African sleeping sickness in humans and nagana in cattle. This organism is one of the best studied in terms of GPI structure, content, and biosynthesis (Ferguson, 1999Go). T. brucei expresses different GPI-anchored glycoconjugates when living in the bloodstream of the infected host and in the midgut of the infected vector. Thus the bloodstream form of the parasite expresses a dense coat of variant surface glycoprotein (VSG), whereas the procyclic form expresses a coat of procyclins.

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., 1985Go). 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.



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 4. GC-MS analyses of procyclic form T. brucei cell ghosts. (A) Total ion chromatogram (linear scanning m/z 40–650) of the AHM, methyl glycoside, and inositol TMS derivatives produced from procyclic form T. brucei cell ghosts using protocol C. (B) Selected ion monitoring (m/z 273, 9–19 min and m/z 318, 19–47 min) chromatogram of the same sample.

 
The results of the AHM analyses for bloodstream form T. brucei total lipids and for procyclic form T. brucei ghosts are summarized in Table I. The total lipid extract is free of GPI-anchored VSG and other proteins, and the AHM figure therefore represents the total GPI precursor levels in these cells. The total GPI content of this fraction has not been measured previously, but it is more than double the prior estimates for the principal GPI precursors glycolipid A (Doering et al., 1994Go) plus glycolipid C (Güther et al., 1996Go). However, the total GPI pool will include earlier GPI precursors and galactosylated forms of glycolipid A (Mayor et al., 1992Go). In addition, GPI levels may reflect the metabolic state of the trypanosomes as they do in mammalian cells (Schubert et al., 1993Go). The parasites used in this study were in the logarithmic phase of growth in culture, whereas previous estimates of glycolipids A and C were made on cells approaching stationary phase in vivo. The results of the AHM analyses for the procyclic form T. brucei ghosts are also significantly (60%) higher than our previous estimate for GPI-anchored procyclin. However, in this case, the discrepancy may be due to the presence of the recently discovered large (nonprotein-linked) GIPLs in procyclic cells (Lillico et al., 2003Go; Vassella et al., 2003Go). In any case, the figures obtained using the AHM method appear to be reasonable and most likely represent the most reliable figures.

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., 1988Go), and a CHO cell GlcNAc-PI de-N-acetylase (PIG-L) deficient mutant (that lacks GPIs; Nakamura et al., 1997Go) 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.


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
In this article, we describe protocols for GPI quantification. The method relies on the presence of a single non-N-acetylated GlcN residue per GPI molecule and the ability to convert this to the unnatural sugar 2,5-anydromannose through nitrous acid deamination. The method is sensitive and linear over a wide range (50–3200 pmol) and, uniquely, can be applied to crude samples, including total lipid extracts and even whole trypanosome cell ghosts. In this regard, it is superior to selected ion monitoring GC-MS analysis for myo-inositol content that (1) has a useful range of 5–100 pmol (the upper limit being imposed by the need to keep the ratio of sample inositol:deuterated inositol internal standard within 1:5) and (2) cannot be used on crude samples due to the abundance of non-GPI inositol-phospholipids.

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., 1999Go; Serrano et al., 1995Go), 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., 1988Go).

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Protocols for the conversion of GPI GlcN residues to free [1-2H]-AHM
Protocol A and protocol B. An internal standard of scyllo-inositol (Calbiochem, Darmstadt, Germany) (50 pmol; 50 µl of a 1 µM stock solution) is added to each of triplicate samples in 1.5-ml screw-top Eppendorf tubes and dried in a Speed Vac concentrator. To determine the molar response factor for AHM versus scyllo-inositol for each batch of samples, triplicate samples of 50 pmol scyllo-inositol and 50 pmol D-glucosamine hydrochloride (Sigma, St. Louis, MO) were prepared and processed in parallel.

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, 100–200 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 20–30 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 9–19 min, hexoses using m/z 204 and 217 over the range 9–19 min, and inositols using m/z 305 and 318 over the range 19–47 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)Go and Goldman et al. (1990)Go. 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, 1989Go) 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 sulfate–polyacrylamide 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, 1979Go) 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, 1964Go) 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., 1995Go). The separate GPI-anchored mucins and GIPL fractions were purified a second time by octyl-Sepharose chromatography. Sample purity was assessed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Invitrogen, Carlsbad, CA, precast 4–12% 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.


    Acknowledgements
 
We thank Alvaro Acosta-Serrano for T. brucei procyclic cells and helpful discussions, Lucia Güther for T. brucei bloodstream form lipid extracts, Catherine Merry for providing the pgsA-745 CHO cells, Taroh Kinoshita for providing the PIG-L-deficient CHO cells, and Terry Smith and Angela Mehlert for helpful discussions about GC-MS. This study was supported by the Wellcome Trust (program grant 071463). J.M. thanks the States of Guernsey for a doctoral studentship.


    Abbreviations
 
AEP, 2-aminoethylphosphonate; AHM, 2,5-anydromannitol; CHO, Chinese hamster ovary; EtNP, ethanolamine phosphate; GC-MS, gas chromatography-mass spectrometry; GIPL, glycoinositolphospholipid; GPI, glycosylphosphatidylinositol; HI-FCS, heat-inactivated fetal calf serum; PI, phosphatidylinositol; TMS, trimethylsilyl


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Almeida, I.C., Camargo, M.M., Procopio, D.O., Silva, L.S., Mehlert, A., Travassos, L.R., Gazzinelli, R.T., and Ferguson, M.A. (2000) Highly purified glycosylphosphatidylinositols from Trypanosoma cruzi are potent proinflammatory agents. EMBO J., 19, 1476–1485.[Abstract/Free Full Text]

Anderson, R.G. and Jacobson, K. (2002) A role for lipid shells in targeting proteins to caveolae, rafts, and other lipid domains. Science, 296, 1821–1825.[Abstract/Free Full Text]

Branquinha, M.H., Vermelho, A.B., Almeida, I.C., Mehlert, A., Ferguson, M.A. (1999) Structural studies on the polar glycoinositol phospholipids of Trypanosoma (Schizotrypanum) dionisii from bats. Mol. Biochem. Parasitol., 102, 179–189.[CrossRef][ISI][Medline]

Brun, R. and Schönenberger, M. (1979) Cultivation and in vitro cloning of procyclic culture forms of Trypanosoma brucei in semi-defined medium. Acta Tropica, 36, 289–292.[ISI][Medline]

Camargo, E.P. (1964) Growth and differentiation in Trypanosoma cruzi. I. Origin of metacyclic trypanosomes in liquid media. Rev. Inst. Med. Trop. Sao Paulo, 12, 93–100.[Medline]

Chang, T., Milne, K.G., Guther, M.L., Smith, T.K., and Ferguson, M.A. (2002) Cloning of Trypanosoma brucei and Leishmania major genes encoding the GlcNAc-phosphatidylinositol de-N-acetylase of glycosylphosphatidylinositol biosynthesis that is essential to the African sleeping sickness parasite. J. Biol. Chem., 277, 50176–50182.[Abstract/Free Full Text]

Conrad, H.E. and Guo, Y. (1992) Structural analysis of periodate-oxidized heparin. Adv. Exp. Med. Biol., 313, 31–36.[Medline]

Doering, T.L., Masterson, W.J., Englund, P.T., and Hart, G.W. (1989) Biosynthesis of the glycosyl-phosphatidylinositol membrane anchor of the trypanosome variant surface glycoprotein: origin of the non-acetylated glucosamine. J. Biol. Chem., 264, 11168–11173.[Abstract/Free Full Text]

Doering, T.L., Pessin, M.S., Hart, G.W., Raben, D.M., and Englund, P.T. (1994) The fatty acids in unremodelled trypanosome glycosyl-phosphatidylinositols. Biochem. J., 299, 741–746.[ISI][Medline]

Ferguson, M.A. (1993) GPI membrane anchors: isolation and analysis. In Fukuda, M. and Kobata, A. (Eds.), Glycobiology: a practical approach. IRL Press, Oxford, pp. 349–383.

Ferguson, M.A. (1997) The surface glycoconjugates of trypanosomatid parasites. Philos. Trans. R. Soc. Lond. B. Biol. Sci., 352, 1295–1302.[CrossRef][ISI][Medline]

Ferguson, M.A. (1999) The structure, biosynthesis and functions of glycosylphosphatidylinositol anchors, and the contributions of trypanosome research. J. Cell Sci., 112, 2799–2809.[Abstract/Free Full Text]

Ferguson, M.A.J., Haldar, K., and Cross, G.A.M. (1985) Trypanosoma brucei variant surface glycoprotein has a sn-1,2-dimyristyl glycerol membrane anchor at its COOH-terminus. J. Biol. Chem., 260, 4963–4968.[Abstract]

Ferguson, M.A., Homans, S.W., Dwek, R.A., and Rademacher, T.W. (1988) Glycosyl-phosphatidylinositol moiety that anchors Trypanosoma brucei variant surface glycoprotein to the membrane. Science, 239, 753–759.[ISI][Medline]

Ferguson, M.A., Brimacombe, J.S., Brown, J.R., Crossman, A., Dix, A., Field, R.A., Guther, M.L., Milne, K.G., Sharma, D.K., and Smith, T.K. (1999) The GPI biosynthetic pathway as a therapeutic target for African sleeping sickness. Biochem. Biophys. Acta, 1455, 327–340.[ISI][Medline]

Goldman, H.D., Hsu, F.F., and Sherman, W.R. (1990) Studies on the permethylation/dephosphorylation of inositol polyphosphates: an approach to a more sensitive assay. Biomed. Environ. Mass Spectrom., 19, 771–776.[ISI][Medline]

Guha-Niyogi, A., Sullivan, D.R., and Turco, S.J. (2001) Glycoconjugate structures of parasitic protozoa. Glycobiology, 11, 45–59.[CrossRef]

Güther, M.L., Treumann, A., and Ferguson, M.A. (1996) Molecular species analysis and quantification of the glycosylphosphatidylinositol intermediate glycolipid C from Trypanosoma brucei. Mol. Biochem. Parasitol., 77, 137–145.[CrossRef][ISI][Medline]

Hirumi, H. and Hirumi, K. (1989) Continuous cultivation of Trypanosoma brucei blood stream forms in a medium containing a low concentration of serum protein without feeder cell layers. J. Parasitol., 75, 985–989.[ISI][Medline]

Lederkremer, R.M., Lima, C., Ramirez, M.I., Ferguson, M.A.J., Homans, S.W., and Thomas-Oates, J. (1991) Complete structure of the glycan of lipopeptidophosphoglycan from Trypanosoma cruzi epimastigotes. J. Biol. Chem., 266, 23670–23675.[Abstract/Free Full Text]

Lillico, S., Field, M.C., Blundell, P., Coombs, G.H., and Mottram, J.C. (2003) Essential roles for GPI-anchored proteins in African trypanosomes revealed using mutants deficient in GPI8. Mol. Biol. Cell, 14, 1182–1194.[Abstract/Free Full Text]

Mayor, S., Menon, A.K., and Cross, G.A. (1992) Galactose-containing glycosylphosphatidylinositols in Trypanosoma brucei. J. Biol. Chem., 267, 754–761.[Abstract/Free Full Text]

McConville, M.J. and Ferguson, M.A.J. (1993) The structure, biosynthesis and function of glycosylated-phosphatidylinositols in the parasitic protozoa and higher eukaryotes. Biochem, J., 294, 305–324.[ISI][Medline]

Murphy-Ullrich, J.E., Westrick, L.G., Esko, J.D., and Mosher, D.F. (1988) Altered metabolism of thrombospondin by Chinese hamster ovary cells defective in glycosaminoglycan synthesis. J. Biol. Chem., 263, 6400–6406.[Abstract/Free Full Text]

Nakamura, N., Inoue, N., Watanabe, R., Takahashi, M., Takeda, J., Stevens, V.L., and Kinoshita, T. (1997) Expression cloning of PIG-L, a candidate N-acetylglucosaminyl-phosphatidylinositol deacetylase. J. Biol. Chem., 272, 15834–15840.[Abstract/Free Full Text]

Previato, J.O., Gorin, P.A.J., Mazurek, M., Xavier, M.T., Fournet, B., Wieruszesk, J.M., and Mendonca-Previato, L. (1990) Primary structure of the oligosaccharide chain of lipopeptidophosphoglycan of epimastigote forms of Trypanosoma cruzi. J. Biol. Chem., 265, 2518–2526.[Abstract/Free Full Text]

Schofield, L., Hewitt, M.C., Evans, K., Siomos, M.A., and Seeberger, P.H. (2002) Synthetic GPI as a candidate anti-toxic vaccine in a model of malaria. Nature, 418, 785–789.[CrossRef][ISI][Medline]

Schubert, J., Schmidt, R.E., and Medof, M.E. (1993) Regulation of glycoinositol phospholipid anchor assembly in human lymphocytes. Absent mannolipid synthesis in affected T and natural killer cell lines from paroxysmal nocturnal hemoglobinuria patients. J. Biol. Chem., 268, 6281–6287.[Abstract/Free Full Text]

Serrano, A.A., Schenkman, S., Yoshida, N., Mehlert, A., Richardson, J.M., and Ferguson, M.A. (1995) The lipid structure of the glycosylphosphatidylinositol-anchored mucin-like sialic acid acceptors of Trypanosoma cruzi changes during parasite differentiation from epimastigotes to infective metacyclic trypomastigote forms. J. Biol. Chem., 270, 27244–27253.[Abstract/Free Full Text]

Sharma, D.K., Smith, T.K., Crossman, A., Brimacombe, J.S., and Ferguson, M.A. (1997) Substrate specificity of the N- acetylglucosaminyl-phosphatidylinositol de-N-acetylase of glycosylphosphatidylinositol membrane anchor biosynthesis in African trypanosomes and human cells. Biochem. J., 328, 171–177.[ISI][Medline]

Sherman, W.R., Packman, P.M., Laird, M.H., and Boshans, R.L. (1977) Measurement of myo-inositol in single cells and defined areas of the nervous system by selected ion monitoring. Anal. Biochem., 78, 119–131.[CrossRef][ISI][Medline]

Simons, K. and Ikonen, E. (1997) Functional rafts in cell membranes. Nature, 387, 569–572.[CrossRef][ISI][Medline]

Smith, T.K., Crossman, A., Borissow, C.N., Paterson, M.J., Dix, A., Brimacombe, J.S., and Ferguson, M.A. (2001) Specificity of GlcNAc-PI de-N-acetylase of GPI biosynthesis and synthesis of parasite-specific suicide substrate inhibitors. EMBO J., 20, 3322–3332.[Abstract/Free Full Text]

Treumann, A., Zitzmann, N., Hulsmeier, A., Prescott, A.R., Almond, A., Sheehan, J., and Ferguson, M.A. (1997) Structural characterisation of two forms of procyclic acidic repetitive protein expressed by procyclic forms of Trypanosoma brucei. J. Mol. Biol., 269, 529–547.[CrossRef][ISI][Medline]

Vassella, E., Butikofer, P., Engstler, M., Jelk, J., and Roditi, I. (2003) Procyclin null mutants of Trypanosoma brucei express free glycosylphosphatidylinositols on their surface. Mol. Biol. Cell, 14, 1308–1318.[CrossRef][ISI][Medline]

Watanabe, R., Ohishi, K., Maeda, Y., Nakamura, N., and Kinoshita, T. (1999) Mammalian PIG-L and its yeast homologue Gpi12p are N-acetylglucosaminylphosphatidylinositol de-N-acetylases essential in glycosylphosphatidylinositol biosynthesis. Biochem. J., 339, 185–192.[CrossRef][ISI][Medline]