Glycobiology Advance Access originally published online on August 3, 2005
Glycobiology 2005 15(12):1302-1311; doi:10.1093/glycob/cwj018
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Synthesis of novel NBD-GM1 and NBD-GM2 for the transfer activity of GM2-activator protein by a FRET-based assay system
Kekulé-Institute für Organische Chemie und Biochemie, Gerhard-Domagk-Strasse 1, D-53121 Bonn, Germany
1 To whom correspondence should be addressed; e-mail: sandhoff{at}uni-bonn.de and schwarzmann{at}uni-bonn.de
2 Present address: National Cancer Institute, Frederick, MD 21702
Received on April 7, 2005; revised on July 6, 2005; accepted on July 18, 2005
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Abstract |
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The ganglioside-activator protein is an essential cofactor for the lysosomal degradation of ganglioside GM2 (GM2) by ß-hexosaminidase A. It mediates the interaction between the water-soluble exohydrolase and its membrane-embedded glycolipid substrate at the lipid-water interphase. Mutations in the gene encoding this glycoprotein result in a fatal neurological storage disorder, the AB variant of GM2-gangliosidosis. In order to efficiently and sensitively probe the glycolipid binding and membrane activity of this cofactor, we synthesized two new fluorescent glycosphingolipid (GSL) probes, 2-NBD-GM1 and 2-NBD-GM2. Both compounds were synthesized in a convergent and multistep synthesis starting from the respective gangliosides isolated from natural sources. The added functionality of 2-aminogangliosides allowed us to introduce the chromophore into the region between the polar head group and the hydrophobic anchor of the lipid. Both fluorescent glycolipids exhibited an extremely low off-rate in model membranes and displayed very efficient resonance energy transfer to rhodamine-dioleoyl phosphoglycerol ethanolamine (rhodamine-PE) as acceptor. The binding to GM2-activator protein (GM2AP) and the degrading enzyme was shown to be unaltered compared to their natural analogues. A novel fluorescence-resonance energy transfer (FRET) assay was developed to monitor in real time the protein-mediated intervesicular transfer of these lipids from donor to acceptor liposomes. The data obtained indicate that this rapid and robust system presented here should serve as a valuable tool to probe quantitatively and comprehensively the membrane activity of GM2AP and other sphingolipid activator proteins and facilitate further structure-function studies aimed at delineating independently the lipid- and the enzyme-binding mode of these essential cofactors.
Key words: 2-amino-gangliosides / 2-NBD-GM1 / 2-NBD-GM2 / fluorescence resonance energy transfer / G2-activator protein
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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The GM2-activator protein (GM2AP) is an essential cofactor for the degradation of ganglioside GM2 (GM2) by lysosomal ß-hexosaminidase A (Conzelmann and Sandhoff, 1979
). Its physiological significance is illustrated by the occurrence of a fatal neurological storage disorder, the AB variant of GM2-gangliosidosis, which results from its inherited deficiency (Conzelmann and Sandhoff, 1978). Similar in its function to the four other known sphingolipid activator proteins occurring in the lysosomal compartment, the saposins A, B, C, and D (Fürst and Sandhoff, 1992), GM2AP mediates the interaction between the water-soluble exohydrolase and its membrane-bound substrate at the lipid-water interphase. The mechanism by which the activator works is still a matter of conjecture. It is well known that GM2-activator binds avidly to a variety of anionic glycolipids in vitro (Hama et al., 1997), and that its membrane activity is critically influenced by several physiological parameters, including the lateral pressure of the lipid bilayer (Giehl et al., 1999), the pH (Bierfreund et al., 1999), the vesicle curvature, and the presence of negatively charged lysosomal lipids, especially bis(monoacylglycerol)phosphate (Werth et al., 2001). According to the liftase model of GM2AP function, this cofactor recognizes the ganglioside substrate within the membrane plane, complexes it, and lifts it out of the lipid bilayer, thereby giving the enzyme access to its carbohydrate headgroup (Meier et al., 1991; Sandhoff et al., 2001). The enzymatic degradation of the glycolipid substrate is then accompanied by a transient interaction between the enzyme and the activator protein (Yadao et al., 1997). It has also been suggested that GM2AP modifies the trisaccharide structure of GM2 by cleaving an intramolecular hydrogen bond between the terminal residues, thus facilitating the enzymatic attack on the N-acetyl-d-galactosamine (GalNAc) bond (Wu et al., 1994).
The crystal structure of unglycosylated, mature GM2AP expressed in Escherichia coli revealed a novel protein fold denoted ß-cup, consisting of an eight-stranded antiparallel ß-pleated sheet, which forms a central hydrophobic cavity. The dimensions of this pocket are such that it can accommodate the ceramide tail of GM2 (Wright et al., 2000). The structural analysis of lipid complexes of GM2AP (Wright et al., 2003) as well as of GM2AP in complex with platelet-activating factor (Wright et al., 2004), confirmed lipid binding within this cavity and revealed different lipid-binding modes within the apolar pocket. By photoaffinity labeling, we were able to identify a highly flexible surface loop of GM2AP as that part of the activator protein that directly interacts with the ganglioside substrate and to obtain further mechanistic insight into GM2 degradation (Wendeler et al., 2004).
All attempts to identify both the lipid- and the enzyme-binding region of GM2AP by site-directed mutagenesis and functional studies were so far hampered by the fact that the current assay system for GM2AP, which measures its stimulatory activity on the degradation rate of GM2 by ß-hexosaminidase A (Klima et al., 1993), reflects simultaneously both the interaction of GM2AP with GM2, as well as the interaction of the GM2AP-GM2 complex with the enzyme. Therefore, the molecular basis underlying the GM2AP deficiency in patients with fatal AB variant of GM2 gangliosidosis remained elusive. Previously presented systems to evaluate the membrane activity of GM2AP include the transfer of radioactive GM2 between donor and acceptor vesicles (Conzelmann et al., 1982; Meier et al., 1991), and a fluorescence-dequenching assay based on the self-quenching of the lipid-probe octadecylrhodamine (Smiljanic-Georgijev et al., 1997). Whereas the first method does not allow the continuous real-time monitoring of intervesicular lipid transfer and therefore impedes mechanistic studies, the latter system is not specific for ganglioside transfer activity and might not prove sensitive enough to reliably detect subtle differences in intervesicular transfer activity exhibited by GM2AP variants.
To investigate the glycolipid binding and transfer activity of GM2AP separated from its interaction with the enzyme, we developed a simple transfer assay that allowed rapid measurement of membrane activity of GM2AP and its variant forms. This assay is based on fluorescence-resonance energy transfer (FRET) from a fluorescent GM2 or ganglioside GM1 (GM1) (donor) to a quencher molecule (acceptor) within the same membrane (donor liposomes) and on an activator-mediated transfer of either fluorescent GM1 or GM2 from donor liposomes to acceptor liposomes that do not contain quencher molecules. It has been shown previously that both gangliosides are transferred almost equally well between membranes by GM2AP (Conzelmann et al., 1982). A prerequisite for the fluorescent gangliosides as well as for the quencher molecules to act as sensitive transfer probes is their low "off rate" (Brown et al., 1985) which impedes spontaneous transfer between membranes. As these fluorescent gangliosides are not commercially available, they had to be synthesized. The 7-nitrobenz-2-oxa-1,3-diazol (NBD) group was chosen as the donor fluorophore and the rhodamine group as a quenching fluorophore.
The structures of the two fluorescent ganglioside analogues 2-acetamido-2-deoxy-ß-d-galactopyranosyl-(1,4)-[
-d-neuraminyl-(2,3)]-ß-d-galactopyranosyl-(1,4)-ß-d-glucopyranosyl-(1,1)-(2S, 3R, 4E)-2-[(2-N-(7-nitrobenz-1,3-diazol-2-oxa-4-yl)-amino)-octadecanamido]-4-octadecen-1,3-diol (2-NBD-GM2) and ß-d-galactopyranosyl-(1,3)-2-acetamido-2-deoxy-ß-d-galactopyranosyl-(1,4)-[-d-neuraminyl-(2,3)]- ß-d-galactopyranosyl-(1,4)-ß-d-glucopyranosyl-(1,1)-(2S, 3R, 4E)-2-[(2-N-(7-nitrobenz-1,3-diazol-2-oxa-4-yl)-amino)-octadecanamido]-4-octadecen-1,3-diol (2-NBD-GM1) are shown in Figure 1.
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The fluorescent GM2 was recognized and degraded by ß-hexosaminidase A in the presence of GM2AP similar to its natural analogue, indicating that the fluorophore does not impede the glycolipid binding to both enzyme and activator protein.
Both fluorescent probes show the same ability as their naturally occurring analogues of being easily incorporated into model membranes where they exhibit an extremely low "off-rate." Their intervesicular transport between donor and acceptor liposomes by GM2AP could be sensitively monitored using real-time FRET measurements.
This rapid and simple assay system is a promising tool for probing in detail the membrane activity of GM2AP under various conditions and for further quantitative and mechanistic studies of the protein-mediated glycolipid transfer. Furthermore, it offers the possibility for structure-function studies aimed at delineating the residues critical for glycolipid binding and transfer activity.
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Results |
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Synthesis of 2-NBD-GM2 and 2-NBD-GM1
The fluorescent analogues of GM2 and GM1 were synthesized in a convergent and multistep synthesis starting from the respective gangliosides GM2 and GM1 isolated from postmortem human brain and from racemic 2-bromo-octadecanoic acid as outlined in Schemes 1 and 2. In the first step, deacetyl-lyso-GM2 and -GM1 were obtained by alkaline hydrolysis of gangliosides GM2 and GM1 purified from bovine brain as described previously (Neuenhofer et al., 1985
; Schwarzmann and Sandhoff, 1987). Briefly, the intermediate deacylated gangliosides GM2 and GM1 (mono-deacetyl-lyso-GM2 and -GM1) containing two amino groups were obtained by vigorous alkaline hydrolysis of GM1 with potassium hydroxide in methanol at 100°C. By this procedure, the fatty acyl as well as the acetyl group of the sialic acid residue are completely removed with very little hydrolysis of the acetamido group of the N-acetylgalactosaminyl moiety of GM2 and GM1 (Schwarzmann and Sandhoff, 1987). The amino group of the sphingosine residue is more basic than those of the neuraminic acid moiety. Thus, selective acylation of the free amino group of the sphingosine moiety of deacylated GM2 and deacylated GM1 could be achieved with fluorenylmethoxycarbonyl chloride (Fmoc chloride). The Fmoc-protected compounds were subsequently re-acetylated with acetic anhydride or [14C]acetic anhydride to yield, after removal of the protecting group, the unlabeled or radioactively labeled lysogangliosides. The protecting Fmoc group was removed by treating Fmoc-lysogangliosides with piperidine in N,N-dimethylformamide. At this step the synthesis converged.
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The lysogangliosides were then acylated with the N-succinimidyl ester of racemic 2-azido-octadecanoic acid to yield (2R/S)-azido-GM2 and -GM1. To avoid O-acylation of carbohydrate hydroxyl groups, the reaction was performed at low temperature in the presence of a large excess of methanol. The N-succinimidyl ester was obtained in good yield by treating 2-azido-octadecanoic acid with N-hydroxysuccinimide and equimolar amounts of dicyclohexylcarbodiimide in ethyl acetate. Racemic 2-azido-octadecanoic acid was prepared by treating racemic 2-bromo-octadecanoic acid with sodium azide in aqueous dioxane.
The conversion of an azido into a functional amino group is usually and easily accomplished by catalytic hydrogenation. However, for the reduction of the azido group in 2-azido-GM2 and -GM1, this hydrogenation was inappropriate as it also reduced the double bond in the sphingoid moiety. Therefore, dihydrogen sulfide in pyridine was the reducing agent of choice and led to (2R/S)-amino-GM2 and -GM1 in acceptable yields. At this stage it was also possible, by chromatography, to separate quite well (2S)-amino-GM2 from (2R)-amino-GM2. The separation of the two diastereomeric 2-amino-GM1 was hampered by the fact that this ganglioside analogue contained a sphingoid moiety of 18 as well as 20 C-atoms leading to a broad peak with the latter analogue eluting in the first part of the peak. Chromatographic separation of the respective azido compounds was not sufficient. Both chiral forms were expected as synthesis started from racemic 2-bromo-octadecanoic acid. For measurement of membrane activity of GM2AP and its variant forms with the analogue of GM1 it was, however, not necessary to separate the chiral forms as enzymatic studies showed that both (2S)-NBD-GM2 and (2R)-NBD-GM2 were degraded equally well by lysosomal ß-hexosaminidase A in the presence of GM2AP (data not shown).
The fluorescent analogues NBD-GM2 and NBD-GM1 were obtained by treating the respective 2-amino-gangliosides with NBD fluoride in N,N-dimethylformamide in the presence of an aprotic acid scavenger.
The products were purified by high performance liquid chromatography on prepacked silica gel columns Lobar either in an isocratic mode or using a two-step gradient of increasing polarity.
Product identity and purity were checked by thin-layer chromatography (TLC) and followed, if appropriate, by autoradiography and fluorescence. The structures of selected intermediates and of the final products were confirmed by ESI-TOF mass spectrometry.
To check whether the introduction of the NBD group altered the recognition and binding of the ganglioside by both the enzyme and the activator protein, the enzymatic degradation of both diastereomeric forms of 2-NBD-GM2 was tested in a micellar in vitro system as described (Conzelmann and Sandhoff, 1979). Both stereoisomers of 2-NBD-GM2 were found to be enzymatically degraded like the normal GM2 in the presence of GM2AP (data not shown), indicating that the NBD-group does not impede binding of the glycolipid to both the enzyme and the activator protein.
Assay design
Assay optimization involved assessment of the amount and ratio of donor and acceptor liposomes needed to provide a readily measurable fluorescence signal response over the time frame of the assay while minimizing the required quantities of the NBD-labeled glycolipid. Both FRET lipid probes were initially incorporated in equimolar amounts into large unilamellar donor vesicles whose composition was chosen such as to mimic most closely the conditions on intralysosomal membranes. As the presence of anionic lipids like bis(monoacylglycero)phosphate and phosphatidic acid was found to drastically stimulate the activity of GM2AP in surface plasmon resonance spectroscopy as well as the enzymatic degradation of GM2 (Werth et al., 2001), donor and acceptor liposomes contained 20 mol% phosphatidic acid. Initially, we also synthesized radioactively labeled NBD-GM1 and used this to quantitate most accurately the amount of fluorescent label incorporated into liposomes as well as the liposomal concentration in the assay mixture. To obtain a maximal possible increase in fluorescence, a five-fold excess of acceptor vesicles was employed in the assays.
For the donor vesicles containing either 2-NBD-GM2 or 2-NBD-GM1 as well as equimolar amounts of rhodamine-dioleoyl phosphoglycerol ethanolamine (rhodamine-PE), the NBD-fluorescence was quenched, showing the efficient resonance energy transfer between the 2-NBD-gangliosides and the rhodamine-PE in the model bilayer membrane. Upon addition of a five-fold excess of acceptor vesicles consisting of phosphatidylcholine, cholesterol, and phosphatidic acid, no change in the emission spectra was observed. This remained unaltered when emission scans were obtained after 90 min. Therefore, over the time course of the experiment, no spontaneous transfer of NBD-glycolipid between donor and acceptor vesicles could be detected in the absence of GM2AP, consistent with a negligible off-rate of the glycolipids from the donor membranes.
The addition of 3% Triton X-100 to the vesicle mixture resulted in an immediate increase in NBD-fluorescence to a value half of that maximal value observed for donor vesicles containing no quencher. This is in agreement with the complete solubilization of the liposomes by the detergent and the previously reported observation that Triton X-100 quenches NBD fluorescence in micellar systems by about 50% (Pagano et al., 1981).
FRET-based transfer assay for GM2AP
The addition of catalytic amounts of GM2AP (25–62.5 nM) to the vesicle mixture containing donor and acceptor liposomes produced an immediate increase in NBD fluorescence. As shown in Figure 2, the observed signal response in the initial phase of the reaction was dependent on the amount of GM2AP added. In contrast, the maximum value observed for the fluorescence intensity remained unchanged irrespective of the amount of GM2AP added. All curves showed saturation kinetics. The observed response in NBD fluorescence is consistent with the resonance transfer changes accompanying the transfer of NBD-GM1 or NBD-GM2 from the donor to the acceptor vesicles. Whereas the initial glycolipid transfer rate is dependent on the amount of GM2AP added, the net transfer equilibrium is apparently independent of the GM2AP concentration.
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Discussion |
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Fluorescence resonance energy transfer is well established as a convenient and nondestructive method providing valuable information about the structure and dynamics of macromolecules (Wu and Brandt, 1994
). Applied to the study of lipid trafficking and transport, it offers the advantage of continuous real-time monitoring of the transfer process (Mattjus et al., 1999; Corsico et al., 2004; Rao et al., 2004). Here, we present a synthetic approach to two new fluorescent ganglioside probes and their application in a novel assay system, which permits the investigation of the lipid transfer activity of GM2 activator protein.
As the NBD group attached to the methyl end of the acyl chain can significantly alter the orientation of the modified lipid within the membrane bilayer and might considerably change the interfacial properties of the membrane surface as well as the "off rate" of the modified lipid (Chattopadhyay and London, 1988; Chattopadhyay, 1990; Tremblay et al., 1998; Schwarzmann, 2001), we decided to introduce the NBD group close to the polar head region of the glycolipids. This proved possible through the synthesis of aminogangliosides and the exploitation of the added functionality to anchor the NBD-group. The chosen position of the fluorophore offers the advantage of neither perturbing the hydrophobic lipid bilayer core nor the polar oligosaccharides of gangliosides that are to be recognized by GM2AP. Also, this position of the NBD group was found to leave the binding of the modified lipid to both the activator protein and the degrading enzyme unaffected.
Recently, a FRET assay for a galactosylceramide transfer protein was described (Rao et al., 2004). In their assay, the authors used an anthryl vinyl group attached to the end of the acyl chain of galactosylceramide as the fluorescent donor. For the assay of GM2AP, this approach seemed not desirable as the bulky anthryl vinyl group may affect the lipid accommodation inside the hydrophobic protein pocket, thereby altering substrate-binding affinity and specificity. Furthermore, the precursor of NBD-gangliosides, that is, aminogangliosides, presented here have the potential of being immobilized on appropriate surfaces, which might facilitate the rapid screening of various parameters influencing the membrane transfer activity in microtiter-plate format. This class of compounds also offers the unique possibility of incorporating a third acyl chain into the lipid backbone, thereby drastically changing the hydrophobic properties of the gangliosides. This would allow to investigate whether the then impaired liftase function of GM2AP reduces the enzymatic ganglioside degradation, and to further delineate the mechanistic details of this reaction at the lipid-water interphase.
In donor vesicles containing equal amounts of NBD-ganglioside and rhodamine-PE, the NBD fluorescence was almost completely quenched, indicating efficient resonance energy transfer between the two fluorophores in model membranes. In the absence of GM2AP, no transfer of NBD-glycolipid between donor and acceptor vesicles could be detected.
GM2AP was shown to transfer 2-NBD-GM1 and -GM2 from the donor to acceptor vesicles. Whereas the initial glycolipid transfer rate was found to be dependent on the amount of GM2AP added, the net transfer equilibrium was independent of the GM2AP concentration.
The GM2 activator is an intensively investigated protein, however, many aspects of its mode of action are still unknown. In particular, no quantitative data are available describing the kinetic and thermodynamic characteristics of its lipid transfer properties. In addition, the protein regions responsible for the at least three distinct functions of GM2AP, the oligosaccharide recognition site, the lipid-binding site, and the contact region to the enzyme, remain elusive.
The assay presented here allows for the first time to investigate accurately the lipid-binding and -transfer activity of GM2AP separated from its interaction with the lysosomal enzyme ß-hexosaminidase A. It can be anticipated that this greatly facilitates structure-function studies aimed at delineating the lipid-binding and the enzyme-binding site. In addition, the membrane parameters that influence the kinetics of the transfer mediated by GM2AP can be probed in detail, and the continuous nature of FRET provides the opportunity for comprehensive quantitative mechanistic studies.
In conclusion, we describe a synthetic approach to new fluorescent glycosphingolipids (GSL) and their application as valuable tools in FRET-based assay systems for intervesicular transfer that can easily be adapted to the study of other sphingolipid activator proteins.
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Materials and methods |
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Materials
GM2 from postmortem human brain of a Tay-Sachs patient and GM1 from bovine brain were available in our laboratory. [1–14C]Acetic anhydride (spec. activity 4.22 TBq/mol) was obtained from Amersham-Buchler (Braunschweig, Germany), N,N'-dicyclohexylcarbodiimide, Fmoc chloride, N-hydroxysuccinimide, 7-nitrobenz-1,3-diazol-2-oxa-4-yl fluoride (NBDF), N,N-diisopropylethylamine and piperidine were obtained from Fluka (Buchs, Switzerland). LiChroprep RP-18, silica gel Si 60 (15–40 µm), prepacked silica gel chromatography columns Lobar, TLC plates with fluorescence indicator (silica gel Si 60 F254) were from E. Merck (Darmstadt, Germany). Phosphatidylcholine and phosphatidic acid (both dioleoyl) were from Sigma (Taufkirchen, Germany). All other reagents and chemicals used in this study were of the highest purity available.
ß-Hexosaminidases A was purified from postmortem human liver to apparent homogeneity as described (Schuette, 1999). Recombinant GM2AP was expressed in the baculovirus expression vector system and purified as reported previously (Wendeler et al., 2003).
Synthesis of NBD-GM1 and NBD-GM2
As the synthesis of lysogangliosides has been described in detail elsewhere (Schwarzmann and Sandhoff, 1987), this section will focus on the synthesis of the final product starting from lyso-GM2 except for the synthesis of radiocarbon labeled lyso-GM1.
Synthesis of (2R/S)-NBD-[14C]GM1
N-Fmoc-lyso-[14C]GM1
N-Fmoc-deacetyl-lyso-GM1 (10.3 mg, about 7 µmol) (prepared as described previously, Schwarzmann and Sandhoff, 1987) was dissolved in dimethylformamide (300 µL) and N,N-diisopropylethylamine (7 µL). [1–14C]Acetic anhydride (4.38 µmol, 18.5 MBq, spec. activity 4.22 TBq/mol) in benzene (50 µL) was added and the mixture kept at 20°C overnight. As the reaction was not complete, further 18.5 MBq of acetic anhydride was added. After 10 h, the reaction was monitored by TLC with chloroform/methanol/15 mM calcium chloride (60:35:8, by vol.) as the mobile phase on TLC plates with fluorescence indicator. A little remaining educt (Rf = 0.13) and mainly product (Rf = 0.20) were detected by fluorescence quench and the product also by phosphoimaging of its radioactivity. N-Fmoc-lyso-[14C]GM1 was purified by chromatography on a prepacked silica gel column Lobar A with chloroform/methanol/water (60:35:8, by vol.) as eluant. The elution profile was monitored by TLC as above. All fractions containing the pure product were pooled and dried in a stream of nitrogen. The yield was 5.8 mg (55%). Electrospray ionization time of flight mass spectrometry (ESI-TOF MS) in the negative mode gave two peaks of double negatively charged species [M–2H]2– at m/z 750.8 and 764.8 with C18 and C20 sphingosine, respectively.
Lyso-[14C]GM1
Fmoc-lyso-[14C]GM1 (5.8 mg, 3.85 µmol) was kept for 20 h at 20°C in a mixture of methanol (500 µL), dimethylformamide (200 µL) and piperidine (300 µL). TLC with chloroform/methanol/15 mM calcium chloride (60:35:8, by vol.) as the mobile phase demonstrated that the educt (Rf = 0.20) had completely disappeared. Instead a double band (Rf = 0.12 and 0.14) showed the formation of radioactive lyso-GM1 with C18 and C20 sphingosine moiety, respectively. This was expected as the parent GM1 from bovine brain contained both C18 and C20 sphingosine in almost equal amounts. Solvents were removed azeotropically in a nitrogen jet with several additions of benzene, and the residue (about 90% yield) was used without further purification in the next synthetic step.
(2R/S)-Azido-[14C]GM1
Lyso-[14C]GM1 (about 3.5 µmol) was N-acylated with the N-succinimidyl derivative of (2R/S)-azido-octadecanoic acid (a total of 9 µmol) in a mixture of dimethylformamide (100 µL), N,N-diisopropylethylamine (10 µL), and methanol (10 µL). The addition of the azido ester was at 0°C, and the reaction monitored by TLC with chloroform/methanol/15 mM calcium chloride (60:35:8, by vol.) as the mobile phase. When a new product (Rf = 0.21) had appeared on the expense of lyso-GM1 solvents were evaporated and the residue purified by chromatography on a prepacked silica gel column Lobar A with chloroform/methanol/water (65:25:4, by vol.; 66 mL) followed by chloroform/methanol/water (60:35:8, by vol.; 90 mL) as eluant. Radioactive fractions containing the pure product (Rf = 0.21) were collected and dried to yield 2.1 µmol 2-Azido-[14C]GM1 (3.3 mg, 60%).
(2R/S)-Amino-[14C]GM1
(2R/S)-Azido-[14C]GM1 (3.3 mg, 2.1 µmol) was treated for 12 h with hydrogen sulfide (0.5 M) in pyridine. The reaction mixture was dried azeotropically in a nitrogen jet with several additions of benzene, and the residue (about 65% yield as determined by radioactivity) was purified by chromatography on a prepacked silica gel column Lobar A with chloroform/methanol/2.5 M ammonia (60:35:8, by vol.) as eluant. The elution profile was monitored by TLC with chloroform/methanol/water (60:35:8, by vol.) as the mobile phase. All fractions containing the pure radioactive product (Rf = 0.16) were pooled and dried in a stream of nitrogen. After purification the yield was 1.8 mg (54%).
(2R/S)-NBD-[14C]GM1
To (2R/S)-amino-[14C]GM1 (1.8 mg, 1.1 µmol) dissolved in a mixture of DMF (100 µL), methanol (100 µL) and N,N-diisopropylethylamine (15 µL) NBD fluoride (4.1 mg, 22 µmol) was added at 0°C. The mixture was kept at 0°C for 40 min and then for 12 h at 20°C. After drying in a nitrogen jet, the residue was purified by chromatography on a prepacked silica gel column Lobar A with 45 mL of chloroform/methanol/water (65:25:4, by vol.) followed by 160 mL of chloroform/methanol/water (60:35:8, by vol.) as eluent. The elution profile was monitored by TLC with chloroform/methanol/water (60:35:8, by vol.) as the mobile phase. All fluorescent and radioactive fractions containing the pure product (Rf = 0.22 to Rf = 0.19) were collected separately, dried in a stream of nitrogen and then dissolved in methanol for analysis. Total yield as determined by radioactivity was 0.5 µmol (45%). ESI-TOF MS in the negative mode of early fractions (Rf = 0.23) of (2R/S)-NBD-[14C]GM1 gave one peak of double negatively charged species [M–2H]2– at m/z 875.93 (calculated 875.96) representing the molecule with C20 sphingosine moiety, whereas a later fraction (Rf = 0.21) of (2R/S)-NBD-[14C]GM1 with mainly C18 sphingosine residue yielded one peak of double negatively charged species [M–2H]2– at m/z 861.95 (calculated 861.93). A clear-cut separation of the two chiral forms was not observed.
(2R)-NBD-GM2 and (2S)-NBD-GM2
(2R/S)-Azido-GM2
Lyso-GM2 containing exclusively C18 sphingosine (5.6 mg, 5 µmol) was N-acylated with the N-succinimidoyl derivative of (2R/S)-azido-octadecanoic acid (a total of 12.5 µmol) in a mixture of dimethylformamide (100 µL), N,N-diisopropylethylamine (10 µL) and methanol (10 µL). The addition of the azido ester in ethyl acetate (100 µL) was at 0°C and the reaction monitored by TLC with chloroform/methanol/15 mM calcium chloride (60:35:8, by vol.) as the mobile phase. After 12 h, at 4°C a new product (Rf = 0.25) had appeared on the expense of lyso-GM2 (Rf = 0.14). Then solvents were evaporated and the residue purified by chromatography on a prepacked silica gel column Lobar A with chloroform/methanol/water (65:25:4, by vol.; 45 mL) followed by chloroform/methanol/water (60:35:8, by vol.; 105 mL) as eluant. Fractions containing the pure product (Rf = 0.25) were collected and dried to yield 3.5 µmol 2-azido-GM2 (5.0 mg, 70%). Characterization was by ESI-TOF MS in the positive mode and yielded the sodium salt of [M+Na]+ at m/z 1469.90 (calculated 1469.82) and double charged sodium salt of [M+2Na]2+ at m/z 746.39 (calculated 746.41).
(2R/S)-Amino-GM2
2-Azido-GM2 (5.0 mg, 3.5 µmol) was treated for 12 h with hydrogen sulphide (0.5 M) in pyridine. When an aliquot showed the disappearance of the azido compound and the appearance of two new products (Rf = 0.23 and 0.21), the reaction mixture was dried azeotropically in a nitrogen jet with several additions of benzene, and the residue was purified by chromatography on a prepacked silica gel column Lobar A with chloroform/methanol/2.5 M ammonia (60:35:8, by vol.) as eluant. The elution profile was monitored by TLC with chloroform/methanol/water (60:35:8, by vol.) as the mobile phase. All amine-reactive fractions containing product (Rf = 0.23) and (Rf = 0.21), respectively, were pooled separately and dried in a stream of nitrogen. The yield was about 25% (1.2 mg to 1.3 mg, 0.88 µmol) for each pool. Characterization was by ESI-TOF MS in the positive mode and yielded [M+Na]+ at m/z 1421.79 (calculated 1421.83), [M+H]+ at m/z 1399.80 (calculated 1399.83) and double charged [M+2Na]2+ at m/z 722.41 (calculated 722.40).
(2R)-NBD-GM2 and (2S)-NBD-GM2
To each isolated pool of the above 2R- and 2S-amino-GM2 (1.2 mg, 0.88 µmol) dissolved in a mixture of dimethylformamide (100 µL), methanol (50 µL) and N,N-diisopropylethylamine (5 µL) solid NBD fluoride (3.0 mg, 16 µmol) was added at 0°C. The mixture was kept at 0°C for 40 min and then for 12 h at 20°C. After drying in a nitrogen jet, the respective residue was purified by preparative chromatography on thin-layer plates (20 by 20 cm) with chloroform/methanol/15 mM calcium chloride (60:35:8, by vol.) as the mobile phase. Fluorescent bands containing the pure product with Rf = 0.28 and Rf = 0.25, respectively, were scraped off the plates and extracted from silica gel with mixtures of chloroform and methanol. After drying, each sample was freed of salt and silicic acid by reversed phase chromatography on LiChroprep RP-18 as described (Williams and McCluer, 1980). Elution was performed with chloroform/methanol (1:1, by vol.). After drying in a stream of nitrogen, the samples were then dissolved in methanol for analysis. Total yield as determined by fluorescence with reference to known amounts of NBD-C6-ceramide was about 0.35 µmol (0.55 mg, 40%). ESI-TOF MS in negative ion mode of one chiral form of 2-NBD-GM2 (Rf = 0.28) yielded [M–H]– at m/z 1560.70 (calculated 1560.83) and double negatively charged species [M–2H]2– at m/z 779.83 (calculated 779.92). For the other chiral form of 2-NBD-GM2 (Rf = 0.25) [M–H]– at m/z 1560.75 and [M–2H]2– at m/z 779.86 was obtained. ESI-TOF MS for both samples was also performed in the positive ion mode and gave the sodium salt of [M+2Na]2+ at m/z 814.89 (calculated 814.90). In addition [M-sialic acid + Na]+ at m/z 1293.78 was also found. Owing to the small amount it could not yet be determined which of the two samples is 2R-NBD-GM2 and which is 2S-NBD-GM2.
Vesicle preparation
Donor vesicles were prepared with the following composition: Phosphatidylcholine (56 mol%), cholesterol (20 mol%), phosphatidic acid (20 mol%), either NBD-GM2 or NBD-GM1 (2 mol%), and rhodamine-PE (2 mol%). Acceptor vesicles consisted of phosphatidylcholine (60 mol%), cholesterol (20 mol%), and phosphatidic acid (20 mol%). Appropriate amounts of lipids from stock solutions were mixed and dried under nitrogen. The lipid mixture was then hydrated in citrate buffer (40 mM, pH 4.2) and this dispersion subjected to 10 freeze-thaw cycles. Large unilamellar vesicles were then prepared by extrusion 21 times through two polycarbonate filters with a pore size of 100 nm mounted in tandem in a mini-extruder (Liposofast, Avestin, Mannheim, Germany). This preparation contained in addition to unilamellar also some bi- and trilamellar vesicles as revealed by electron microscopy of negatively stained preparations (data not shown).
FRET-based transfer assay
The final donor vesicle concentration in the assay mixture was 20 nmol lipid per mL, the concentration of acceptor vesicles 100 nmol lipid per mL in a total volume of 320 µL in 50 mM citrate buffer, pH 4.2.
The transfer of NBD-GM1 or NBD-GM2 was started by the addition of catalytic amounts of GM2AP (25–62.5 nM) to the vesicle mixture. Fluorescence measurements in a quartz cuvette were preformed in a Shimadzu RF 5000 instrument (Kyoto, Japan) with an excitation wavelength of 480 nm. NBD emission was measured at 522 nm. Transfer was monitored at 28°C. Under our experimental conditions, photobleaching was negligible as for each time point (Figure 2) the shutter was opened for 3–4 s only resulting in a total of 39–52 s of illumination. With continuous illumination photobleaching over 1 h was linear with a loss of fluorescence of less than two relative fluorescence units per minute.
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Acknowledgments |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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We thank Dr. Christina Schuette, Goettingen, for purified ß-hexosaminidase A, and the Deutsche Forschungsgemeinschaft, SFB 284 and SFB 645, for financial support. We are indebted to Dr. Bernadette Breiden for carefully reading the manuscript.
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Abbreviations |
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ESI-TOF MS, electrospray ionization time of flight mass spectrometry; Fmoc, fluorenylmethyloxycarbonyl; FRET, fluorescence resonance energy transfer; GM2AP, GM2-activator protein; GM1, ganglioside GM1; GM2, ganglioside GM2; GSL, glycosphingolipid; NBD, 7-nitrobenz-2-oxa-1,3-diazol; PE, dioleoyl phosphoglycerol ethanolamine; TLC, thin-layer chromatography
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Bierfreund, U., Lemm, T., Hoffmann, A., Uhlhorn-Dierks, G., Childs, R.A., Yuen, C.T., Feizi, T., and Sandhoff, K. (1999) Recombinant GM2-activator protein stimulates in vivo degradation of GA2 in GM2 gangliosidosis AB variant fibroblasts but exhibits no detectable binding of GA2 in an in vitro assay. Neurochem. Res., 24, 295–300.[Medline]
Brown, R.E., Stephenson, F.A., Markello, T., Barenholz, Y., and Thompson, T.E. (1985) Properties of a specific glycolipid transfer protein from bovine brain. Chem. Phys. Lipids, 38, 79–93.[CrossRef][Medline]
Chattopadhyay, A. (1990) Chemistry and biology of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-labeled lipids: fluorescent probes of biological and model membranes. Chem. Phys. Lipids, 53, 1–15.[CrossRef][Medline]
Chattopadhyay, A. and London, E. (1988) Spectroscopic and ionization properties of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-labeled lipids in model membranes. Biochim. Biophys. Acta, 938, 24–34.[Medline]
Conzelmann, E. and Sandhoff, K. (1978) AB variant of infantile GM2 gangliosidosis: deficiency of a factor necessary for stimulation of hexosaminidase A-catalyzed degradation of ganglioside GM2 and glycolipid GA2. Proc. Natl. Acad. Sci. U. S. A., 75, 3979–3983.
Conzelmann, E. and Sandhoff, K. (1979) Purification and characterization of an activator protein for the degradation of GM2 and GA2 by hexosaminidase A. Hoppe Seylers Z. Physiol. Chem., 360, 1837–1849.[Web of Science][Medline]
Conzelmann, E., Burg, J., Stephan, G., and Sandhoff, K. (1982) Complexing of glycolipids and their transfer between membranes by the activator protein for degradation of lysosomal ganglioside GM2. Eur. J. Biochem., 123, 455–464.[Web of Science][Medline]
Corsico, B., Lious, H.L., and Storch, J. (2004) The -helical domain of liver fatty acid binding protein is responsible for the diffusion-mediated transfer of fatty acids to phospholipid membranes. Biochemistry, 43, 3600–3607.[CrossRef][Medline]
Fürst, W. and Sandhoff, K. (1992) Activator proteins and topology of lysosomal sphingolipid catabolism. Biochim. Biophys. Acta, 1126, 1–16.[Medline]
Giehl, A., Lemm, T., Bartelsen, O., Sandhoff, K., and Blume, A. (1999) Interaction of the GM2-activator protein with phospholipid-ganglioside bilayer membranes and with monolayers at the air-water interface. Eur. J. Biochem., 261, 650–658.[Web of Science][Medline]
Hama, Y., Li, Y.-T., and Li, S.-C. (1997) Interaction of the GM2 activator with glycosphingolipids. J. Biol. Chem., 272, 2828–2833.
Klima, H., Klein, A., van Echten, G., Schwarzmann, G., and Sandhoff, K. (1993) Over-expression of a functionally active human GM2-activator protein in Escherichia coli. Biochem. J., 292, 571–576.
Mattjus, P., Molotkowsky, J.G., Smaby, J.M., and Brown, R.E. (1999) A fluorescence resonance energy transfer approach for monitoring protein-mediated glycolipid transfer between vesicle membranes. Anal. Biochem., 268, 297–304.[CrossRef][Medline]
Meier, E.M., Schwarzmann, G., Fürst, W., and Sandhoff, K. (1991) The human GM2 activator protein: a substrate specific cofactor of hexosaminidase A. J. Biol. Chem., 266, 1879–1887.
Neuenhofer, S., Schwarzmann, G., Egge, H., and Sandhoff, K. (1985) Synthesis of lysogangliosides. Biochemistry, 24, 525–532.[CrossRef][Medline]
Pagano, R.E., Martin, O.C., Schroit, A.J., and Struck, D.K. (1981) Formation of asymmetric phospholipid membranes via spontaneous transfer of fluorescent lipid analogues between vesicle populations. Biochemistry, 20, 4920–4927.[CrossRef][Medline]
Rao, C.S., Lin, X., Pike, H.M., Molotkowsky, J.G., and Brown, R.E. (2004) Glycolipid transfer protein mediated transfer of glycosphingolipids between membranes: a model for action based on kinetic and thermodynamic analyses. Biochemistry, 43, 13805–13815.[CrossRef][Medline]
Sandhoff, K., Kolter, T., and Harzer, K. (2001) Sphingolipid activator proteins. In Scriver, C., Beudet, A.L., Sly, W.S., and Valle, D. (eds), The Metabolic and Molecular Basis of Inherited Disease, 8th ed. David Mc Graw Hill, NY, pp. 3371–3389.
Schuette, C.G. (1999) Structure analysis of ß-hexosaminidase B and GM2 activator protein with protein-chemical methods and MALDI-mass spectrometry. PhD Thesis, University of Bonn, Germany.
Schwarzmann, G. (2001) Uptake and metabolism of exogenous glycosphingolipids by cultured cells. Semin. Cell Dev. Biol., 12, 163–171.[CrossRef][Medline]
Schwarzmann, G. and Sandhoff, K. (1987) Lysogangliosides: synthesis and use in preparing labeled gangliosides. Methods Enzymol., 138, 319–341.[Web of Science][Medline]
Smiljanic-Georgijev, N., Rigat, B., Xie, B., Wang, W., and Mahuran, D.J. (1997) Characterization of the affinity of the GM2 activator protein for glycolipids by a fluorescence dequenching assay. Biochim. Biophys. Acta, 1339, 192–202.[CrossRef][Medline]
Tremblay, J.M., Voziyan, P.A., Helmkamp, G.M., and Yarbrough, L.R. (1998) The C-terminus of phosphatidylinositol transfer protein modulates membrane interactions and transfer activity but not phospholipid binding. Biochim. Biophys. Acta, 1389, 91–100.[Medline]
Wendeler, M., Lemm, T., Weisgerber, J., Hoernschemeyer, J., Bartelsen, O., Schepers, U., and Sandhoff, K. (2003) Expression of recombinant human GM2-activator protein in insect cells: purification and characterization by mass spectrometry. Protein Expr. Purif., 27, 259–266.[CrossRef][Medline]
Wendeler, M., Hoernschemeyer, J., Hoffmann, D., Kolter, T., Schwarzmann, G., and Sandhoff, K. (2004) Photoaffinity labelling of the human GM2-activator protein. Mechanistic insight into ganglioside GM2 degradation. Eur. J. Biochem., 271, 614–627.[Medline]
Werth, N., Schuette, C.G., Wilkening, G., Lemm, T., and Sandhoff, K. (2001) Degradation of membrane-bound ganglioside GM2 by ß-hexosaminidase A. Stimulation by GM2-activator protein and lysosomal lipids. J. Biol. Chem., 276, 12685–12690.
Williams, M. and McCluer, R. (1980) The use of Sep-Pak C18 cartridges during the isolation of gangliosides. J. Neurochem., 35, 266–269.[Web of Science][Medline]
Wright, C.S., Li, S.C., and Rastinejad, F. (2000) Crystal structure of human GM2-activator protein with a novel ß-cup topology. J. Mol. Biol., 304, 411–422.[CrossRef][Web of Science][Medline]
Wright, C.S., Zhao, Q., and Rastinejad, F. (2003) Structural analysis of lipid complexes of GM2-activator protein. J. Mol. Biol., 331, 951–964.[CrossRef][Web of Science][Medline]
Wright, C.S., Mi, L.-Z., and Rastinejad, F. (2004) Evidence for lipid packaging in the crystal structure of the GM2-activator complex with platelet activating Factor. J. Mol. Biol., 342, 585–592.[CrossRef][Web of Science][Medline]
Wu, P. and Brandt, L. (1994) Resonance energy transfer: methods and applications. Anal. Biochem., 218, 1–13.[CrossRef][Web of Science][Medline]
Wu, Y.Y., Lockyer, J.M., Sugiyama, E., Pavlova, N.V., Li, Y.T., and Li, S.C. (1994) Expression and specificity of human GM2 activator protein. J. Biol. Chem., 269, 16276–16283.
Yadao, F., Hechtman, P., and Kaplan, F. (1997) Formation of a ternary complex between GM2 activator protein, GM2 ganglioside and hexosaminidase A. Biochim. Biophys. Acta, 1340, 45–52.[CrossRef][Medline]
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