Discovery of Glycosyltransferases Using Carbohydrate Arrays and Mass Spectrometry

Reaction screening

We analyzed the genomes of several microorganisms to identify putative glycosyltransferases to include in the screen. In this way, we emphasize that the SAMDI method can be used in a non-biased screen to identify unanticipated transferase activities, though we note that this approach can also be used to analyze GTs that are taken from a single species or directed at a mechanistic question. Some fraction of these enzymes may not catalyze glycosylation reactions—they could, for example, have hydrolase activity or act on acceptors that use other functional groups as the nucleophile—and would not be active in the present screen. To speed the analysis, we used a high throughput screening platform based on metal plates having an array of 384 gold-coated islands in the standard microtiter plate geometry ¹⁵ . We prepared 24 oligosaccharide acceptors (Supplementary Methods, Supplementary Table 1 and Supplementary Fig. 1–4) and immobilized individual acceptors on the gold features by either forming monolayers from carbohydrate-terminated alkanethiol reagents, or performing immobilization reactions of a thiol-functionalized carbohydrate with a maleimide-terminated monolayer, or an azido-functionalized carbohydrate with an alkyne-terminated monolayer (Supplementary Fig. 5). Our use of multiple strategies for immobilizing the acceptors owes to the availability of, or efficient access to, the carbohydrate reagents. The choice of chemistries used to link the acceptor to the monolayer should not have a significant influence on the glycosylation reaction since GTs generally display selectivity for the terminal carbohydrate unit of the acceptor. We therefore expect the activities that are discovered in the screen to not be compromised by the choice of the immobilization reaction; in support of this idea, we discuss later the use of a homogeneous format to verify the activities of GTs found in this work.

An example of the screening assay is shown in Figure 1 and starts with a monolayer to which the acceptor lactose is immobilized. A SAMDI spectrum of the monolayer showed a peak at m/z 1296 that corresponds to the mixed disulfide with a single lactose group. The peak at m/z 693 represented the tri(ethylene glycol) disulfide. The monolayer was treated with bovine α1,3-galactosyltransferase (GGTA1) and analyzed by SAMDI to reveal a peak at m/z 1458 that corresponds to the mixed disulfide containing the trisaccharide that resulted from enzymatic galactosylation of lactose ¹⁶ . The absence of a peak at m/z 1296 demonstrated that the enzymatic reaction was essentially complete. We applied this assay to screen 85 GTs (Supplementary Results, Supplementary Table 4)—including 76 putative bacterial enzymes that had not been previously characterized—with the goal of identifying new GT activities.

Because traditional methods for protein expression and purification are tedious, time-consuming, and can compromise protein activity, we used an in vitro expression system to rapidly prepare the GTs, which were then assayed in unpurified form (Supplementary Fig. 7). Each individual protein was first mixed with one of the 7 sugar donors dissolved in one of four buffers—which were selected to vary the divalent metal ion and pH—and then applied to individual gold islands presenting the sugar acceptor (Fig. 1). The four buffers were selected because of their common use in enzyme assays and include different divalent ions and values of pH (see Methods).

From the 57,120 reactions tested in the screen, forty-four showed new glycosylation products (Supplementary Table 5). Included in these hits were glycosylation activities for four previously uncharacterized enzymes (Fig. 2). Two of these, BF0009 (GT80) and BF0614 (GT84), are from Bacteroides fragilis, and catalyze the GalNAcylation of β-glucose and cellobiose, and the galactosylation of N-acetylglucosamine (GlcNAc), respectively (Fig. 2a–c). The two others, HD0466 (GT24) and AAF28363.1 (GT09), are from Haemophilus ducreyi and transfer GlcNAc from UDP-GlcNAc to lactose and to GlcNAc, respectively (Fig. 2d–e). We also found that two known galactosyltransferases could utilize donors that had not been reported previously. The GTs GGTA1 (GT02) and LgtC (GT06), in addition to utilizing the UDP-Gal donor were also found to glycosylate their substrates with UDP-Glc for the former and with UDP-Glc and GDP-Man for the latter (Fig. 2f and Supplementary Fig. 8).

To elaborate on one example, the protein expressed from the gene BF0009 (GT80) was mixed with the donor UDP-GalNAc and applied to a spot presenting β-glucose. The SAMDI spectrum revealed a new peak at m/z 1337 (Fig. 2a), which was 203 Dalton greater than the peak for the acceptor-terminated alkyl disulfide (m/z 1134) and is consistent with the addition of GalNAc to the acceptor. To estimate the yield with which the GTs were expressed in active form, we included nine known GTs in the screen and found that six were active (Supplementary Table 5). We believe that this fraction provides a fair estimate of the yield for the expression of the other putative enzymes examined in this work, though we have not determined whether the inactivity arises from improper folding of the enzymes, requirement for cofactors or regulatory domains, or an inaccessibility of the immobilized substrates.

Identification of the glycosidic linkages in the products

The SAMDI screen is effective at identifying combinations of GT, donor and acceptor substrates that generate new glycosidic linkages, but the use of mass spectrometry does not provide information on the regio- and stereo-chemical structure of the linkage. To characterize the products, we expressed the four new enzymes in E. coli BL21(DE3) cells and used them in preparative glycosylation reactions to generate milligram quantities of the products, which we then characterized using 1D and 2D NMR (Fig. 2, Supplementary Table 2 and Supplementary Results). We found that: BF0009 (GT80) catalyzes the formation of aβ1,3 linkage between GalNAc and glucose; BF0614 (GT84) joins galactose and GlcNAc through a β1,4 bond; HD0466 (GT24) joins GlcNAc and lactose through a β1,3 linkage; and AAF28363.1 (GT09) creates aβ1,4 linkage between two GlcNAc residues.

Biochemical characterizations of the new GTs

We selected three GTs that gave new activities in the screen and characterized their kinetic parameters. These experiments used a ‘pull-down’ format wherein reactions were performed in solution, quenched and then applied to a monolayer to allow the substrate and product to undergo immobilization prior to analysis by SAMDI ¹⁷ . This method avoids perturbations that may arise from presentation of the ligands at the surface. We used an azido-modified oligosaccharide as the acceptor and a monolayer presenting terminal alkyne group to selectively immobilize the substrate and product of the reaction (Supplementary Table 3 and Supplementary Fig. 9). Previous work has shown that the SAMDI method provides quantitative information for the rates of enzyme-catalyzed reactions, including reactions of GTs ^17–18 . In one example, we characterized the BF0009-mediated transfer of UDP-GalNAc to an azido-glucose substrate by performing a series of reactions with the glucose azide and UDP-GalNAc present at several concentrations and for several reaction times (Supplementary Fig. 9c). The yields were determined by integrating the areas of the mass peaks for the product and acceptor substrate (Supplementary Fig. 9b and Supplementary Fig. 10). Kinetic parameters were determined from double reciprocal plots (Supplementary Fig. 11) ^19–20 and are summarized in Table 1. We also performed this experiment using a radiolabeled assay and found that those results agreed with our determination of kinetic parameters using the SAMDI pull-down assay (Supplementary Table 6). These experiments revealed several features of the GTs. The value of K_b for the UDP-Gal donor is an order magnitude lower with the human enzyme, reflecting a stronger interaction between the human homologue and the donor. In another example, HD0466 and the closely relatedβ1,3-N-acetylglucosaminyltransferase from Neisseria meningitidis (LgtA) ³ each interact with the donor with similar affinities and perform the glycosylation reaction with similar values of V_max.

Many GTs display conserved folds and structural motifs that bind, and require, divalent metal ions for activity. We therefore also investigated the metal-dependent activities of the GTs that were found in this work (Supplementary Fig. 6 and Supplementary Table 7) ²² . The addition of EDTA resulted in a loss of activity for each of the three enzymes described above, consistent with a requirement for metal ions. The three GTs were most active in the presence of Mn²⁺ and Mg²⁺, but were also active with other divalent metal ions. For example, BF0009 displayed the highest activity with Mg²⁺, retained activity when Mn²⁺, Co²⁺ or Mo²⁺ ions were present in the buffer, had only minor activity when Cu²⁺ or Ni²⁺ were present, while Ca²⁺, Fe²⁺ or Zn²⁺ elicit no activity. Comparison of HD0466 and LgtA surprisingly showed differences in metal dependence, with HD0466 having higher activity in the presence of Mg²⁺ relative to Mn²⁺ whereas LgtA showed the reversed trend.

In vitro expression of putative glycosyltransferases

The sources of materials including bacteria strains, genomic DNAs, plasmids, and growth conditions can be found in Supplementary Methods. The in vitro expression of the putative GTs was performed using the Expressway Cell-Free E. coli expression system as described by the vendor. In brief, 2 μg of plasmid was used for each 100 μL volume of the reaction volume, and the reactions were incubated at 37 °C for 4 hours. Insoluble particulates were removed by centrifugation and the supernatant containing soluble protein was used immediately in glycosylation reactions. A vector provided by the manufacturer (harboring a non-GT gene) was used as a positive control for protein expression, and the empty-vector pMCSG7 was used as a negative control and gave no detectable endogenous GT activity. The expression of the target enzymes were confirmed by western blot using mouse anti-His antibody and anti-mouse IgG horseradish peroxidase secondary antibody (Supplementary Fig. 7). The films were developed using the ECL with western blot detection reagent.

Reaction screening

Reactions were performed on metal plates having a 24 by 16 array of gold islands modified with monolayers. The preparation of the oligosaccharide-terminated monolayers is discussed in detail in the Supplementary Methods. Each sugar nucleotide donor was dissolved in one of the four buffer systems (0.75 mM, 4 μL) and combined with protein (2 μL) in a 384 well plate. The buffers used in this work were the following: Tris-HCl (50 mM, pH 8.0) and MnCl₂ (10 mM); sodium cacodylate (100 mM, pH 6.0) and MnCl₂ (10 mM); Tris-HCl (50 mM, pH 7.5) and CaCl₂ (10 mM); HEPES (50 mM, pH 7.5). The resulting reaction mixtures were then transferred to individual gold islands on a 384 metal plate presenting one of the 24 sugar acceptors. Liquid handling was performed by a Tecan Freedom Evo 200 robot. We estimate that enzymes were present at concentrations in the low nM range. The plates were kept in humidified chambers at 37 °C for 2 hours, and then rinsed with water followed by ethanol. The plates were dried under nitrogen, treated with a solution of 2,4,6-trihydroxyacetophenone matrix (THAP, 5 mg mL⁻¹, 0.5 mL per plate) and analyzed by SAMDI mass spectrometry. A 355 nm Nd:YAG laser was used as the desorption/ionization source with an accelerating voltage of 20 kV and extraction delay time of 50 ns. All spectra were acquired automatically using positive reflector mode. The combinations of GT, donor and acceptor substrate that gave a glycosylation reaction are summarized in Supplementary Table 5. The new enzymes were expressed again in E. coli, and were used to synthesize oligosaccharide products at preparative scale. See Supplementary Methods for the details of enzyme expression, oligosaccharide preparation and structure characterization. LgtC and GGTA1 were also expressed in E. coli and their activities with UDP-Glc and GDP-Man (for LgtC), and UDP-Glc (for GGTA1) were confirmed in a SAMDI assay.

Determining kinetic parameters of GTs using SAMDI

To obtain kinetic constants for the new GTs, reactions were performed in solution and then the substrate and product were immobilized to a monolayer by way of a Click reaction ¹⁸ . Reactions were initiated in 384 well plates and quenched through the addition of EDTA and then applied to monolayer and analyzed by SAMDI, according to the following protocols. Each reaction contained one of the purified enzymes, the sugar donor, the acceptor, Tris-HCl (50 mM, pH 8.0) and MnCl₂ (10 mM) in a total volume of 10 μL. The enzymes were used at the following concentrations: BF0009, 0.14 mg mL⁻¹; BF0614, 0.3 mg mL⁻¹; HD0466, 0.3 mg mL⁻¹. The concentrations of donors ranged from 100 μM to 5 mM and those of the acceptors ranged from 250 μM to 5 mM. For each set of concentrations, the reactions were carried out for times ranging from 2 to 30 min at intervals of 2 to 3 min and terminated by adding a mixture of cold ethanol and EDTA (10 mM, 20 μL). Each reaction mixture was then applied to an individual gold circle of the array (in a volume of 2 μL) that was modified with the alkyne-terminated monolayer. An aqueous solution (1μL per reaction) containing CuBr (2 mM) and triethylamine (0.5 mM) was applied to each circle and the reactions were incubated at room temperature from 30 min to 6 hours, depending on the concentrations of the azido sugars. The completion of the reactions was monitored by SAMDI. The slide was then rinsed with water, followed by ethanol, and dried under nitrogen. For quantification, the extent of glycosylation (R) was determined from the peak intensities for product (I_p) and acceptor substrate (I_s) on the SAMDI spectra using the relation: R = I_p/(I_p+I_s). We confirmed that the measured ratio reflects the actual ratio of the two azido sugars in solution by performing a calibration experiment, which is discussed in detail in the Supplementary Methods and Supplementary Fig. 10. The yield of the glycosylation, calculated from the equation in Supplementary Fig. 9b, was plotted against the reaction time. The linear region of the plot was fitted to obtain the slope which represented the initial velocity (v₀). Double reciprocal plots of initial velocities are plotted in Supplementary Fig. 11. For these plots, the donors were the variable substrates and the acceptors were the constant substrates. The data were fit to equation (1), which has been used to describe bisubstrate enzyme kinetics ^19–20 .

In this equation, [A] is the concentration of the acceptor, [B] is the concentration of the donor, V_max is the maximum velocity, K_a and K_b represent the cognate Michaelis constants for substrates A and B, respectively and K_ia is the dissociation constant of the substrate A. The metal activity studies were carried out in a similar fashion to obtain initial velocities and the details can be found in Supplementary Methods.

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Supplementary Materials