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Timestamp: 2019-04-24 07:53:22+00:00

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A synthetic perfluoroalkyl-tagged lactosyl glycolipid has been shown to form lipid microdomains in fluid phospholipid bilayers. When embedded in the membranes of phospholipid vesicles, this glycolipid was trans-sialylated by soluble T. cruzi trans-sialidase (TcTS) to give a perfluoroalkyl-tagged glycolipid that displayed the ganglioside GM3 epitope, with up to 35% trans-sialylation from fetuin after 18 h. Following sialylation, vesicles bearing this Neu5Ac(α2-3)Gal(β1-4)Glc sequence in their “glycocalyx” were recognised and agglomerated by the lectin M. amurensis leukoagglutinin. Monitoring TcTS-mediated trans-sialylation by HPLC over the first 6 h revealed that enzymatic transformation of bilayer-embedded substrate was much slower than that of a soluble lactosyl substrate. Furthermore, clustering of the lactose-capped glycolipid into “acceptor” microdomains did not increase the rate of sialic acid transfer from fetuin by soluble TcTS, instead producing slight inhibition.
Fig. 1 (a) A sialic acid residue is transferred to phase separating Lac lipid 1 from fetuin by TcTS, producing Neu5Ac(α2-3)Gal(β1-4)Glc capped lipid 2. GlcNAc lipid 3 is a phase-separating non-substrate comparison. (b) Lac lipid 1 is dispersed across the membrane in liquid disordered (ld) bilayers like dimyristoyl phosphatidylcholine (DMPC) at 37 °C but (c) phase separates to form lipid microdomains (pale blue pyrenyl excimer fluorescence) in liquid ordered (lo, DMPC–cholesterol) or solid ordered membranes (so, DPPC) at 37 °C.
We have developed a pyrene-perfluoroalkyl membrane anchor that can form functionalised fluid microdomains in bilayers that are in liquid ordered (lo) or solid ordered (so) states.28 Appending lactose onto this pyrene-perfluoroalkyl membrane anchor to create “acceptor” glycolipid 1 (Fig. 1) will provide two insights: how sialylation by TcTS is affected by (a) substrate insertion into a bilayer and (b) clustering within that bilayer. Successful sialylation will afford a lipid bearing the ganglioside GM3 epitope (Neu5Ac(α2-3)Gal(β1-4)Glc) in a process analogous to GM3 biosynthesis from lactosylceramide, and may provide phospholipid vesicles with a synthetic sialylated glycocalyx. Rates of reaction can be compared with our previous assays of soluble enzyme activity20 and may provide insight into how secreted TcTS might act on lactosylceramide rafts.
Herein we describe the synthesis of perfluoroalkyl-tagged lactose-lipid 1 (Fig. 1) and studies of the TcTS mediated transfer of sialic acid onto the lactose headgroup of 1. The effect of clustering lipid 1 into microdomains on the rate of sialic acid transfer by soluble TcTS was also assessed.
Synthetic perfluoroalkyl-tagged glycolipids like 1 are readily available by coupling amine-terminated glycosides to acid-terminated membrane anchors (Scheme 1).
Scheme 1 Synthesis of lactose-capped lipid 1.
The key acetyl-protected 2-aminoethyl lactoside 7 is available via both the azide and CBz protection routes.29 Conversion of lactose peracetate to the bromide allowed activation by metal salts using the Koenigs–Knorr method.30 Using silver carbonate as an activator gave numerous side-products, so the more reactive Hg(CN)2–HgBr2 mixture was used with CBz-protected ethanolamine. A reasonable yield of the CBz-protected lactose derivative was obtained with an α : β anomeric ratio of 1 : 1.7. The required β-anomer 5 was recovered using column chromatography and then deprotected by hydrogenation to give 7. Similarly, employing 2-azidoethanol with Hg(CN)2–HgBr2 gave the azido-terminated lactose derivative, which could be hydrogenated to 7. Forming the amide linkage between the saccharide and lipid components was achieved using N,N′-dicyclohexylcarbodiimide (DCC) to form the N-hydroxysuccinimide (NHS) active ester. To avoid O- to N-acetyl transfer, acid activation was performed in parallel with hydrogenation, with the resulting amine immediately added to the activated NHS ester. The acetate protecting groups were then cleaved using Zemplén conditions31 to give lipid 1. Both the CBz and azido routes gave similar final yields (14% and 12% respectively from heptaacetylactosyl bromide).
The pyrenyl fluorophore in 1 has two functions: it allows the direct visualisation of 1 in vesicles by fluorescence microscopy and it forms excited dimers (excimers) at high local concentrations. The ratio of excimer (460 nm) to monomer (395 nm) fluorescence emission (the E/M ratio) is directly indicative of the local concentration of pyrene moieties and the rate of collision between them,32 so increases in the E/M ratio indicate the formation of microdomains containing 1. The E/M ratio also provides important information about the behaviour of lactose fluorolipid 1 in solution, such as the critical aggregation concentration (CAC), and in bilayers, like the exchange rate between outer and inner leaflets (“flip–flop”). To measure the CAC of 1, a suspension of 1 in pH 7.4 phosphate buffer (20 μM 1, 37 °C) was formed by sonication of a thin lipid film. A high E/M ratio of 20 ± 1 indicated aggregates were present at 20 μM, but serial dilution revealed a CAC of 49.5 nM. This value is ∼3-fold higher than similar glycolipids like GlcNAc-lipid 3, possibly due to increased aqueous solubility afforded by the additional sugar in the headgroup.
Lactose fluorolipid 1 should mix with liquid disordered (ld) phase bilayers but phase separate from so and lo bilayers.28a Lipid 1 was incorporated into large unilamellar phospholipid vesicles (LUVs, 800 nm diameter) with three different compositions: dimyristoyl phosphatidylcholine only (DMPC); dipalmitoyl phosphatidylcholine (DPPC); a 1 : 1 mix of DMPC and cholesterol (DMPC–chol). These compositions were chosen because at 37 °C these bilayers are in fluid ld, so and lo phases respectively. Vesicles were formed via extrusion of a buffered aqueous suspension of lipid 1 and the appropriate phospholipid mixture through 800 nm polycarbonate membranes above the bilayer melting temperature (Tm). As previously for GlcNAc-lipid 3,20 the maximum incorporation of 1 in each bilayer composition was determined using UV-visible spectroscopy, and was found to be 9.5% mol mol−11 in DMPC, 8.7% mol mol−11 in DPPC and 8.5% mol mol−11 in DMPC–chol.
The fluorescence emission spectra of these LUV suspensions were recorded at 37 °C. As expected, Lac-lipid 1 exhibited little phase separation in DMPC membranes at a low 1% mol mol−1 loading (E/M = 0.14) but phase-separated from DMPC–chol and DPPC (E/M = 1.3 and 1.0 respectively). At the maximum loading in DMPC (9.5% mol mol−1), Lac-lipid 1 had a higher E/M of 0.80 ± 0.15 due to the higher rate of interpyrene collision at this 10-fold higher loading. The maximum loadings in DMPC–chol (8.5% mol mol−1) and DPPC (8.7% mol mol−1) both showed extensive clustering of Lac-lipid 1 (E/M = 3.9 ± 0.2 and 3.0 ± 0.4 respectively). As for 3,20 giant unilamellar vesicles (GUVs) were then used to directly visualise microdomains of Lac-lipid 1. At ∼9% mol mol−1 loading, microdomains were observed in GUVs composed of either DMPC–chol or DPPC, but only weak uniformly distributed excimer emission could be observed in DMPC, in good agreement with the E/M values measured in LUVs (Fig. 2).
Fig. 2 (a) Epi-fluorescence microscopy image of a DMPC GUV with 9.5% mol mol−11. (b) Fluorescence emission spectrum from DMPC LUVs with 9.5% mol mol−11. (c) Epi-fluorescence microscopy image of a DPPC GUV with 8.7% mol mol−11, arrow indicates microdomain, white dots show GUV outline. (d) Fluorescence emission spectrum from DPPC LUVs with 8.7% mol mol−11. Scale bars 40 μm.
To contextualise rates of enzymatic transformation, the flip–flop rate for 1 was estimated. Following previous methodology,20 Lac-lipid 1 was added to blank LUVs composed of DMPC, DMPC–chol or DPPC to give a loading of 1% mol mol−1. Immediately after addition, the E/M ratio dropped from 20 (buffer) to 0.5 in DMPC, 1.5 in DMPC–chol and 1.3 in DPPC as the lipids inserted into the bilayers. A subsequent slower decline in E/M provided the outer-to-inner leaflet flip–flop half-lives, which were approximately t½ = 1.5 h in DMPC, t½ = 7 h in DMPC–chol and t½ = 5 h in DPPC. These half-lives indicate slower flip–flop through ordered bilayers and also show that flip–flop takes longer for 1 than for GlcNAc-lipid 3 (t½ = 1 h in DMPC and 4 h in DMPC–chol),20 possibly due to the extra hydrophilic saccharide unit prohibiting transit through the hydrophobic core of the bilayer. These flip–flop half-lives indicate that over short periods (<1 h) only the outer leaflet of 1 is available to the enzyme, but near complete sialylation should be possible after overnight incubation.
The transformation of 1 into 2 by TcTS/fetuin at 37 °C was assayed by HPLC and MS using procedures developed previously.20 LUVs (800 nm diameter) composed of DMPC, DMPC–chol and DPPC containing a target loading of ∼9% mol mol−1 Lac-lipid 1 (200 μM 1) were synthesised by extrusion. The maximum loading of 1 in the vesicle membranes was employed, as preliminary experiments had indicated that at 1% mol mol−1 loadings the amount of product formed was too small to be accurately measured (1% mol mol−1 corresponds to 20 μM 1 in the buffer volume). The vesicle suspensions (100 μL, ∼0.18 mM 1) were then incubated with fetuin and TcTS for 18 h. Two reagent concentrations were assessed: either 10 mg mL−1 fetuin (equivalent to 1.25 mM sialic acid33) with ∼39 nM TcTS, or 50 mg mL−1 fetuin (equivalent to 6.3 mM sialic acid) with ∼195 nM TcTS. The vesicles were stable under the reaction conditions, which was aided by the lack of any co-solvent or surfactant in the enzyme buffer. In both cases there was an excess of sialic acid available for transfer from fetuin.
After overnight incubation with 10 mg mL−1 fetuin and ∼39 nM TcTS, both MALDI-ToF/ToF MS and HPLC indicated partial sialylation of Lac-lipid 1. MALDI-ToF/ToF MS analysis showed a small peak at m/z 1438 that corresponded to the di-sodium sialylated product. The low intensity of the peak suggested poor conversion – peak height comparison gave 7–9% conversion after overnight reaction – but concerns about product decomposition under MS conditions complicated the analysis.34 HPLC and LC/MS proved to be better analytical methods, with good resolution of two pyrene-containing peaks. Sialylation produced the more hydrophilic lipid 2 with a retention time of ∼14 min, compared to ∼17 min for the starting Lac-lipid 1 (Fig. 3a). Using a standard HPLC method,20 the fraction of 1 converted to 2 was determined from the peak areas of the starting lipid 1 and sialylated product 2, revealing low levels of sialylation (∼8%) that agreed with the values from the MALDI-ToF mass spectra. However, increasing the amount of fetuin and TcTS five-fold gave a 3- to 4-fold increase in sialylation to 20–35% (Fig. 3a and b), clearly showing that TcTS can transform membrane-bound lactosyl-lipids into sialyl-terminated trisaccharide glycolipids in situ. Although a larger excess of fetuin may have driven the reaction further towards product 2, these assays show that Lac-lipid 1 is a competent substrate for TcTS despite its highly unnatural structure.
Fig. 3 (a, b) Conversion of 1 in DMPC vesicles (9.5% mol mol−1) to 2 after fetuin/TcTS treatment (50 mg mL−1 and 195 nM): (a) HPLC traces (abs. at 346 nm). (b) Partial MALDI-ToF/ToF MS spectrum. Product peaks in blue are [1 + Na]+ (m/z 1123) and [2 + 2Na]+ (m/z 1438). Phospholipid peaks (*) are [2DMPC + H]+ (m/z 1356), [2DMPC + Na]+ (m/z 1378) and [2DMPC + Na + K]+ (m/z 1417). (c, d) Epi-fluorescence micrographs of DMPC LUVs (800 nm) with 9.5% mol mol−11 after fetuin/TcTS treatment (50 mg mL−1 and 195 nM): (c) dispersed vesicles in the absence of fluorescein labelled Maackia amurensis leukoagglutinin (FITC-MAL) (d) agglutinated vesicles after FITC-MAL addition, showing pyrenyl (left) and fluorescein (right) emission. Scale bar 20 μm.
The sialylation of 1 was verified using lectin-mediated vesicle agglutination. The legume Maackia amurensis produces the well-studied lectin M. amurensis leukoagglutinin (MAL), which is known to selectively bind sialic acid terminated oligosaccharides with an α(2-3) glycosidic linkage to galactose.35 Fluorescein labelled MAL (FITC-MAL) was used to confirm enzymatic sialylation of 1 in vesicles. MAL should selectively bind to the enzymatically sialylated product 2 but not 1, and as an agglutinin with multiple sialic acid binding sites (K ∼ 1.1 × 106 M−1 for α(2,3)-sialyl LacNAc35a), vesicle aggregation would be expected if enzymatic sialylation had occurred. No aggregation was observed in the presence of FITC-MAL (20 μg mL−1) for DMPC vesicles bearing 9.5% 1 in their membrane (Fig. 3c). However after vesicle incubation with TcTS and fetuin as described above (18 h), the addition of FITC-MAL produced large aggregates, with fluorescence microscopy showing co-localisation of FITC and pyrene fluorescence (Fig. 3d). Aggregation by FITC-MAL shows these sialylated vesicles now expose the Neu5Ac(α2-3)Gal(β1-4)Glc recognition epitope in their artificial “glycocalyx”.
Interestingly, after 18 h the extent of Lac-lipid 1 sialylation differed little between the vesicle compositions (DMPC, DMPC–chol or DPPC). At 1 mg of fetuin and 39 nM TcTS, conversions were 8.5% (DMPC), 7.4% (DMPC–chol) and 7.6% (DPPC), rising to 35% (DMPC), 21% (DMPC–chol) and 30% (DMPC–chol) if 5 mg of fetuin and 195 nM TcTS were employed. With the proviso that after 18 h these reactions may be approaching equilibrium, these observations suggested that TcTS was not sensitive to substrate clustering, with the dispersed lipid (1 in DMPC) giving slightly higher conversions than the lipid 1 in microdomains (1 in DMPC–chol or DPPC).
To assess the initial rate of sialylation by TcTS, the time course for the conversion of 1 into 2 was monitored. The addition of 50 mg mL−1 fetuin (equivalent to 6.3 mM sialic acid) and 195 nM TcTS gave the highest total conversions after 18 h, so these conditions were employed to study the rate of production of 2 over the first 7 h (Fig. 4), which largely involves reaction of 1 in the outer leaflet of the bilayer.
Fig. 4 (a, b) Schemes showing the conversion of vesicle-bound 1 to 2 by TcTS and fetuin (F) when 1 is (a) dispersed or (b) clustered. (c) Rate of conversion of vesicle-bound 1 to 2 by TcTS/fetuin (195 nM and 50 mg mL−1): 9.5% mol mol−11 in DMPC vesicles (■); 8.7% mol mol−11 in DPPC ( ); 8.5% mol mol−11 in DMPC–chol ( ). The extent of clustering is indicated by the E/M values at 37 °C in each case. Curve fits are to guide the eye, standard error bars are shown.
The HPLC data clearly showed that, unlike the transformation of GlcNac fluorolipid 3 into a Gal-GlcNAc fluorolipid by β4GalT1/UDP-Gal, the rate of transformation of 1 into 2 by TcTS/fetuin in any of the membrane compositions DMPC, DMPC–chol or DPPC was similar, with slight inhibition observed under these conditions when lipid 1 was in microdomains. This is despite the clear difference in the extent of clustering of 1 in each of these membrane compositions, as indicated by the E/M values and fluorescence microscopy. In all cases the reaction rate at the membrane was much slower than in solution, with the reaction of benzyl lactose under the same conditions complete within 1 h. Using fluorogenic substrate 4-methyl-umbelliferyl-N-acetylneuraminic acid (MUNANA) as the sialic acid donor revealed that MUNANA hydrolysis was ∼34-fold faster than formation of 2 over the first 3 h (see ESI†), confirming the low reactivity of bilayer-embedded 1 with TcTS.
Unlike β4GalT1, secondary interactions of TcTS with “acceptor” substrate rich surfaces do not seem to be significant enough to enhance enzymatic activity. This difference suggests that enzyme structure could be a key factor that determines if substrate clustering in microdomains enhances the initial rate of enzymatic transformations. However other factors could also play a role. For example, during later periods of TcTS activity, when a significant amount of lipid 2 has been produced, the sialylated product could also be a donor substrate for TcTS. A decrease in the net rate of production of 2 might result, which may be more pronounced when both 1 and 2 are in close proximity within microdomains.
TcTS has been shown to successfully catalyse sialic acid transfer onto the headgroup of synthetic lactosyl-capped fluorolipid 1, creating a phase-separating glycolipid that displayed the GM3 epitope. This transfer was carried out on membrane-embedded 1, producing sialylated phospholipid vesicles that were recognised by the lectin Maackia amurensis leukoagglutinin. Unlike related studies using soluble β4GalT1, glycolipid clustering in the bilayer did not increase the rate of reaction with soluble TcTS, but instead produced slight inhibition.
The use of vesicles as a medium for biocatalytic syntheses can offer advantages over organic solvents when coupling lipophilic and hydrophilic substrates.42 The liposomal products from chemoenzymatic transformations can also have biotechnological applications, for example chemoenzymatically sialylated liposomes may target cells overexpressing Siglecs43 or be masked from the immune response.4,44 Furthermore, using TcTS in conjunction with β4GalT1 could mimic the biosynthesis of GM3 from glycosylceramide in vivo,45 and investigations into the use of chemoenzymatic cascades are ongoing.
We thank Prof. A. C. C. Frasch, Universidad Nacional General San Martín, Argentina, for kindly providing the TcTS clone. J.E.R.M. thanks CONACyT-México for funding (scholarship reference 214433/309655). F.C. and G.T.N. thank the BBSRC for providing studentship funding.
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