Fluorescent mannosides serve as acceptor substrates for glycosyltransferase and sugar-1-phosphate transferase activities in Euglena gracilis membranes

Synthetic hexynyl α-D-mannopyranoside and its α-1,6-linked disaccharide counterpart were fluorescently labelled through CuAAC click chemistry with 3-azido-7-hydroxycoumarin. The resulting triazolyl-coumarin adducts, which were amenable to analysis by TLC, HPLC and mass spectrometry, proved to be acceptor substrates for α-1,6-ManT activities in mycobacterial membranes, as well as α- and β-GalT activities in trypanosomal membranes, benchmarking the potential of the fluorescent acceptor approach against earlier radiochemical assays. Following on to explore the glycobiology of the benign protozoan alga Euglena gracilis, α-1,3- and α-1,2-ManT activities were detected in membrane preparations, along with GlcT, Glc-P-T and GlcNAc-P-T activities. These studies serve to demonstrate the potential of readily accessible fluorescent glycans as substrates for exploring carbohydrate active enzymes.


TLC analyses
The lowest amount of fluorescent α-Man-1,6-α-Man-HCT (2) compound that could be detected on the TLC plates using UV-light were determined in the series of experiments. For this purpose, aqueous solution of 2 in a wide range of concentrations was prepared, and 2 µL aliquots were loaded onto a glass-backed TLC plate containing no fluorescent indicator. The TLC plate was eluted with CH 2 Cl 2 :MeOH (9:1) solvent system and then visualised with a mid-wave length UV light for fluorescence detection (Fig. S1A) and then stained with orcinol for standard carbohydrate detection (Fig. S1B). 1 The results indicated that the fluorescent method of detection for 2 is ca. 100 fold more sensitive than orcinol staining, allowing detection of fluorescent mannoside acceptor at 25 ng or 40 pmols for compound 2. Value obtained with fluorescent method of detection is lower compared to a typical limits of detection using radiolabelling.

Solution analyses
In order to achieve quantification of fluorescently labelled glycoside products obtained from the enzymatic biotransformation the detection limits of α-Man-1,6-α-Man-HCT (2) were examined in solution. Again a range of concentrations were prepared at the pH level with the highest fluorescence intensity observed, for 2 at pH 9 in universal buffer (0.04 M H 3 BO 3 , H 3 PO 4 , and CH 3 COOH). The fluorescence intensity was measured as the concentration of each compound decreased (Fig. S2). The analysis of results showed that concentrations as low as 0.06 μM could be detected using a fluorometer. The reported results demonstrated that both compounds acted as acceptor substrates for these enzymes, leading to formation of two radiolabelled mannoside products, as evaluated by HPTLC and autoradiography.
The nature of α-1,6-glycosidic linkage in these assays has been confirmed through exo-glycosidase digestion and permethylation analysis by GC-MS, which indicated formation of a major α-1,6-linked trisaccharide and minor α-1,6-linked tetrasaccharide radiolabelled products. Therefore, in order to benchmark our fluorescence-based methodologies we were looking to obtain similar results.

Linkage analysis through exo-mannosidase digestions
The newly formed linkages in fluorescent trimer and tetramer were assessed through the application of exomannosidase digestion with a hydrolase of well-defined α-1,6-specificity. For this purpose, both enzymatic reactions were scaled up to obtain sufficient amount of fluorescent products to be analysed. The purification of fluorescent trimer and tetramer from α-Man-1,6-α-Man-HCT (2) was attempted by a normal phase HPLC method. Disappointingly, the obtained fractions contained mixed products as some interactions of coumarinbased fluorophore with the amino column were observed, which resulted in tailing of peaks for all fluorescent compounds. HPLC purification of the reaction mixture from α-Man-1,6-α-Man-HCT (2) gave two mixed fractions. The first semi-purified fraction contained mixed trisaccharide product, a faint amount of α-Man-1,6-α-Man-HCT (2) and product of hydrolysis α-Man-HCT (1) (Fig. S5, lane 1). The second semi-purified fraction contained tetrasaccharide product, small amount of trisaccharide as well as 2 and 1 (Fig. S5, lane 3). Both fractions were subjected to exo-mannosidase digestion with Xanthomonas manihotis α-1,6-mannosidase (Fig. S5, lane 2 and 4). TLC analysis indicated that hydrolysis of trisaccharide and tetrasaccharide products with α-1,6-mannosidase resulted in formation of α-Man-HCT (1). The obtained data is in agreement with the data obtained from the radiolableed assays and also indicates that fluorescent α-Man-1,6-α-Man-HCT (2) can act as an alternative acceptor substrate for α-1,6 ManT present in M.

Fluorescence-based assay to probe galactosyltransferase activities responsible for α-galactosylation of GPI anchors in Trypanosoma brucei
Results from a study that investigated substrate specificity of chemically synthesised compounds for galactosyltransferases (GalT) responsible for the decoration of GPI anchor were used to further validate and benchmark our fluorescence-based assay. 5 In the published radiolabelled assays, three acceptor substrates, representing approximately 90, 40 and 10 %, respectively of these galactosylated products. The type of the glycosidic bond has been investigated in the case of only one acceptor, octyl 6-O-α-D-manopyranosyl-1-thioα-D-mannopyranoside, because the main aim of that study was to detect α-GalT activity in T. brucei membranes. The type of glycosidic linkage in this assay has been determined by a combination of tandem negative-ion ESI MS and periodate oxidation followed by NaBH 4 reduction and indicated formation of a major product with α-1,3-linkage and two minor products with α-1,2 and α-1,4-linkages.

TLC results from the fluorescence-based assay
In this round of validation, the main aim was to demonstrate that the fluorescent α-Man-1,6-α-Man-HCT (2), already used in benchmarking against M. smegmatis radiolabelled assays can equally act as acceptor substrate for galactosyltransferases involved in the decoration of the core structure of GPI anchors in T.
brucei microsomal membranes. The examined mannoside acceptor substrate 2 was incubated with UDP-Gal in the presence of trypanosome membranes under the same conditions as used in the literature radiolabelled assay. 5 Bloodstream Trypanosoma brucei (strain 427 variant MITat 1.4) were obtained from the infected rats 6 centrifuged, washed with ice-cold buffer and lysed using hypotonic buffer. 7 A cell-free lysate was then prepared as described in the literature. 5 TLC analysis showed formation of one fluorescent band with R f value of expected trisaccharide product with a low conversion as judged by TLC (Fig. S6). formation of trisaccharide product as followed from a similar fluorescent intensity when compared to previous TLC analyses (Fig. S6 vs Fig. S7 A). On the other hand, the increase in the amount of enzyme led to increase in hydrolysis of the starting acceptor resulting in increased fluorescent intensity of α-Man-HCT (1). The sequential addition of UDP-Gal donor resulted in an overall increase in acceptor substrate conversion as judged by the increase in intensity of fluorescent band of fluorescent trimer (Fig. S7 B).

Identification of fluorescent products by LC-MS
In order to confirm formation of trisaccharide compound the reaction mixture from α-Man-1,6-α-Man-HCT (2) was subjected to LC-MS analysis. The ion-extracted chromatogram of m/z 788.29 indicated presence of two peaks that were designated as trimer a (retention time 6.14 min) and trimer b (6.23 min) (Fig. S8 A).  (11), respectively (Fig. S8 B).

Linkage analysis through exo-galactosidase digestions
Assessment of the anomeric configuration of newly formed glycosidic linkages in trisaccharide was achieved through utilisation of enzymatic digestions with α-galactosidase from green coffee beans and β-galactosidase from Escherichia coli. The purification of trimer from starting 2 was attempted by a reverse phase HPLC method. The obtained fraction contained some starting material due to human error. The semi-purified fraction was subjected to exo-galactosidase digestion with α-galactosidase from green coffee beans and β-

TLC analysis of disaccharide 12a/b digestion with
Aspergillus saitoi α-1,2-mannosidase released a significant amount of fluorescent product with the same R f value as α-Man-HCT (1). However, a small amount of fluorescent disaccharide 12b remained unhydrolysed, which is indicative of the presence of another isomeric compound with a different linkage (Fig. S11A, lane 6 and Fig. S11B(ii)). In order to assess what type of glycosidic linkage the underlying mannoside possessed, it was subjected to further digestion with Xanthomonas manihotis α-1,2/3-mannosidase. In this case, both fluorescent products were digested. The disaccharide 12a was mainly converted into a fluorescent product with the same R f value as HCT (11)   Similarly, TLC analysis from jack bean α-mannosidase digestion of trisaccharide 13 revealed removal of three mannose residues releasing a fluorescent product with the same R f value as fluorescent HCT (11) aglycone (Fig. S12A, lane 2 and Fig. S12B(i)). Incubation of trisaccharide 13 with Xanthomonas manihotis α-1,6-mannosidase also had no effect on fluorescent compound therefore, excluding formation of the α-1,6-linkage (Fig. S12A, lane 4). The hydrolysis of 13 with Aspergillus saitoi α-1,2-mannosidase resulted in formation of only one fluorescent product with the same R f value as fluorescent α-Man-1,6-α-Man-HCT (2), thus indicating introduction of α-1,2-mannopyranosidic linkage in this product (Fig. S12, line 6, and Fig.   S12B (ii)).

Alkaline phosphatase digestion
To purified fluorescent product 15, 18 and 16, 19 (80 μM, 10 μL) were digested with 1 U of alkaline phosphatase in reaction buffer (50 mM, pH 8.0 Tris/HCl). The enzymatic reaction was mixed and incubated at 25 °C for 20 h in 20 μL of final volume. After incubation, reactions were terminated by boiling for 5 min.

Trifluoroacetic acid and alkaline phosphatase degradation
Purified fluorescent product 15, 18 and 16, 19 (60 μM, 10 μL) was treated with 40 mM of trifluoroacetic for 15 min at 100 °C. After drying, the sample was dissolved in 10 μL of water and digested with 1 U of alkaline phosphatase in reaction buffer (50 mM, pH 8.0 Tris/HCl). The enzymatic reaction was incubated at 25 °C for 20 h in 20 μL of final volume. After incubation reactions were terminated by boiling for 5 min.

Fluorescence-based assays to probe mannosyltransferase activities in Mycobacterium smegmatis
Buffers and incubation conditions from previously established cell-free radiolabelled assay were used to probe α-1,6-mannosyltransferase activities in fluorescence-based assay. 4 In the fluorescence-based assay 250 μL acceptor (2)

Fluorescence-based assays to probe galactosyltransferase activities in Trypanosoma brucei
Buffers and incubation conditions from previously established cell-free radiolabelled assay were used to probe galactosyltransferase activities in fluorescence-based assay. 5 The reaction buffer composition was as Aliquots of 20 μL were taken periodically and a mixture of CHCl 3 /MeOH (20 μL, 1:1, v/v) was added to each aliquot in order to stop the reaction. After taking the last aliquot (24 h or 36 h) the main reaction was stopped with CHCl 3 /MeOH (1:1, v/v) (40 μL). The resulting denatured T. brucei microsomal membranes were removed by several centrifugations (16000 g for 5 min) and washed with CHCl 3 /MeOH:H 2 O (10:6:1) (3 x 50 μL). The washings were combined and solvents dried under gentle stream of air. The residue was re-dissolved in deionised water and passed through a 0.45 μm PPTE filter, the filtrate was collected, and the sample was freeze-dried for storage.

Variations of enzyme concentration
Two fluorescence assays were performed as described above but with different amount of T. brucei microsomal membranes added. In the first assay of microsomal membranes were prepared from 1x10 7 cells and in the second assay from 5x10 8 cells. In both cases aliquots were taken after 2 h, 6 h, and 24 h, treated as described above and analysed by TLC (see Fig. S6 and Fig. S7A).

Increasing donor concentration
The fluorescence assay was performed as described above with addition of T. brucei microsomal membranes obtained from 1x10 8 cells. Aliquots (10 μL) were taken after incubation for 4 h, 8 h, 12 h, 24 h and 36 h and analysed by TLC (see Fig S7B). At the same time points (4 h, 8 h, 12 h, 24 h) the mixture was supplemented with 5 μL of UDP-Gal donor (4 mM).

Euglena gracilis growth medium composition
The composition of the medium used for growing Euglena gracilis culture in the dark was based on EG:JM medium (https://www.ccap.ac.uk/media/documents/EG_JM.pdf) and further modified as indicated in Table   2. To make EG medium all constituents stated in table 2 were dissolved in 1 L deionised water. JM medium was prepared by mixing 1 mL stock solutions listed in Table 2 and diluted to male 1 L solution. EG:JM medium consisted of 1:1 mixture of EG and JM media.