Sequential Glycosylation of Proteins with Substrate-Specific N-Glycosyltransferases

Abstract

Protein glycosylation is a common post-translational modification that influences the functions and properties of proteins. Despite advances in methods to produce defined glycoproteins by chemoenzymatic elaboration of monosaccharides, the understanding and engineering of glycoproteins remain challenging, in part, due to the difficulty of site-specifically controlling glycosylation at each of several positions within a protein. Here, we address this limitation by discovering and exploiting the unique, conditionally orthogonal peptide acceptor specificities of N-glycosyltransferases (NGTs). We used cell-free protein synthesis and mass spectrometry of self-assembled monolayers to rapidly screen 41 putative NGTs and rigorously characterize the unique acceptor sequence preferences of four NGT variants using 1254 acceptor peptides and 8306 reaction conditions. We then used the optimized NGT-acceptor sequence pairs to sequentially install monosaccharides at four sites within one target protein. This strategy to site-specifically control the installation of N-linked monosaccharides for elaboration to a variety of functional N-glycans overcomes a major limitation in synthesizing defined glycoproteins for research and therapeutic applications.

Figure 1

Site-specific control of glycosylation by sequential enzymatic addition of glycans. The GlycoSCORES workflow was used to screen 41 putative NGTs and discover differences in peptide acceptor specificities. Conditionally orthogonal NGT-substrate sequence pairs were further optimized, such that the first NGT only glycosylates one of the four substrate sequences, the second NGT glycosylates one of the remaining three substrate sequences, and the third NGT only glycosylates one of the remaining two substrate sequences, with the last substrate sequence glycosylated by the fourth NGT. The four acceptor sites were then incorporated as GlycTags into a single protein. Sequential treatment of the protein with the four NGTs gave stepwise glycosylation of each of the four glycosylation sites. Glycans, modification sites, and NGTs have been color coded for illustration purposes.

Figure 2

GlycoSCORES screening of NGT homologues for unique peptide specificities. (a) Screening for active NGTs from putative bacterial glycosyltransferases. Forty-one putative glycosyltransferases from the CAZY database were screened against six representative peptide substrates for N-glucosyltransferase activity. The phylogenetic tree is rooted with human OGT (black dot) as the outgroup. Six enzymes showed N-glucosyltransferase activity, with strong activity from ApNGT, EcNGT, HdNGT, HiNGT, and MhNGT. Relative MS intensities of peptide substrates and glucose modified peptide products are shown when treated with 5% (v/v) CFPS NGTs and 2.5 mM UDP-Glc at 30 °C for 21 h (n = 1). (b) Scheme for GlycoSCORES workflow for discovery and characterization of the peptide specificities for the NGTs. NGTs were produced in CFPS and mixed with UDP-Glc sugar donor and cysteine-containing peptide substrates, which were then immobilized to a maleimide functionalized self-assembled monolayer and characterized by SAMDI-MS. (c) Peptide acceptor specificity comparison of HiNGT (gray), EcNGT (orange), ApNGT (blue), and ApNGT^Q469A (red) using a peptide array of X_–1NX₊₁TRC sequence. Expanded X_–1NX₊₁(T/S)RC substrate libraries with numerical annotation are shown in Supplementary Figure 4. Enzyme concentrations were controlled to obtain a maximum yield of ∼85% in each heatmap to facilitate the comparison of specificities. The heatmap for ApNGT is taken from a previous report. Each heatmap shows percentage glucose modification from n = 1 experiment using the conditions: 0.42 μM CFPS HiNGT or 0.75 μM CFPS EcNGT, 30 °C for 21 h; 0.055 μM CFPS ApNGT or 0.014 μM CFPS ApNGT^Q469A, 30 °C for 1 h.

Figure 3

Optimization of conditionally orthogonal GlycTags for NGTs. (a) Screening to improve the conditional orthogonality of NGT-GlycTag pairs for HiNGT, EcNGT, ApNGT, and ApNGT^Q469A using an X_–1NX₊₁(T/S)RC peptide library. Peptides were screened with HiNGT, EcNGT, ApNGT, and then ApNGT^Q469A, respectively. Sequences are arranged by decreasing conditional orthogonality (differences in modification between specific NGT and the sum of preceding NGTs). The sequences are also divided into regions that are preferred by ApNGT^Q469A only (red), ApNGT but not EcNGT or HiNGT (blue), EcNGT but not HiNGT (orange), and HiNGT (gray). GlycTags showing strong conditional orthogonality which were identified in each region used for additional optimization are highlighted (colored boxes). Heatmaps with experimental conditions and numerical annotation from n = 2 separate peptide IVG reactions are shown in Supplementary Figure 7. (b) Conditional orthogonality screening of selected sequences from a which were resynthesized with 19 amino acids in the X_–2 position in 5-mer X_–2(X_–1NX₊₁T/S)RC library. GlycTags identified from each region and used for additional optimization are highlighted. (c) Conditional orthogonality screening of selected sequences from (b) which were resynthesized with 19 amino acids in the X_–3 position. Heatmaps with experimental conditions and numerical annotation from n = 1 separate peptide IVG reactions for (b) and (c) are shown in Supplementary Figures 8 and 9, respectively. The corresponding colors of sequences originating from previous screens are shown above the heatmaps. A set of four GlycTags with >95% conditional orthogonality was selected for site-specific control of sequential glycosylation at four sites within one target protein.

Figure 4

Optimized GlycTag sequences showing conditional orthogonality as peptides and within a single Im7 target protein. (a) The conditional orthogonality of optimized 6-mer GlycTags. HiNGT, EcNGT, ApNGT, and ApNGT^Q469A modification of selected GlycTags from Figure 3c under optimized conditions were resynthesized and reanalyzed by GlycoSCORES with purified NGTs. The modification efficiency of each peptide was calculated using individually measured RIFs (Supplementary Table 4). Experimental conditions (n = 3 individual IVGs): 0.2 μM HiNGT or 0.67 μM EcNGT, 30 °C for 21 h; 0.45 μM ApNGT or 0.1 μM ApNGT^Q469A, 30 °C for 3 h. (b) Optimized 6-mer GlycTags were inserted into the N-terminus, C-terminus, and two exposed loops of the glycosylation model protein Im7, with flanking sequences of RATT-GlycTag-RAGG to facilitate trypsinization and quantitative LC-qTOF analysis. 10 μM of purified 4gIm7 bearing all four optimized GlycTags was reacted with 2.5 mM UDP-Glc and various concentrations of each purified NGT for 4 h in n = 3 IVG reactions. After modification, 4gIm7 was purified from the reaction, treated with trypsin, and analyzed by LC-qTOF. The modification efficiency (occupancy) of each site was calculated using individually measured RIFs (shown in Supplementary Table 5). A vertical dotted line in each graph denotes optimal conditions for each NGT which provide the greatest conditional orthogonality. Representative LC chromatograms and related MS spectra are presented in Supplementary Figure 13. (c) The conditional orthogonality of each optimized 6-mer GlycTag within 4gIm7 under optimized conditions. Bar graphs with errors for heatmaps in (a) and (c) are shown in Supplementary Figure 10. Similar modification patterns were observed for peptides and GlycTags within engineered 4gIm7.

Figure 5

Site-specific control of sequential glycosylation at four distinct GlycTag sequences within a single Im7 protein. (a) A workflow for site-specific control of glycosylation at four sequences within Im7 by sequential addition of NGT enzymes. (b) Representative deconvoluted MS spectra for intact 4gIm7 of n = 3 IVG reactions showing SUMO-4gIm7 after each NGT treatment, cleaved by UIp1 and analyzed by LC-qTOF. Two μM purified SUMO-4gIm7 was incubated with 0.3 μM HiNGT and then immobilized to magnetic beads and sequentially washed and treated with 0.3 μM EcNGT, 0.4 μM ApNGT, and then 2 μM ApNGT^Q469A. Reactions (n = 3) were performed for 4 h at 30 °C containing 2.5 mM UDP-Glc and monitored by elution and LC-qTOF after each wash step. These data show that the dominant forms of 4gIm7 are those modified with one glucose per NGT treatment. The optimization of reaction conditions for SUMO-4gIm7 are shown in Supplementary Figure 15. One representative LC chromatogram and related deconvoluted MS spectra are presented in Supplementary Figure 16. (c) A bar graph of site-occupancy at each of the four GlycTags in the same n = 3 experiments in (b) after each NGT treatment, cleavage by trypsin, and analysis by LC-qTOF. Each GlycTag was mainly glycosylated by its corresponding NGT.