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Bump-and-Hole Engineering Identifies Specific Substrates of Glycosyltransferases in Living Cells

Summary Studying posttranslational modifications classically relies on experimental strategies that oversimplify the complex biosynthetic machineries of living cells. Protein glycosylation contributes to essential biological processes, but correlating glycan structure, underlying protein, and disease-relevant biosynthetic regulation is currently elusive. Here, we engineer living cells to tag glycans with editable chemical functionalities while providing information on biosynthesis, physiological context, and glycan fine structure. We introduce a non-natural substrate biosynthetic pathway and use engineered glycosyltransferases to incorporate chemically tagged sugars into the cell surface glycome of the living cell. We apply the strategy to a particularly redundant yet disease-relevant human glycosyltransferase family, the polypeptide N-acetylgalactosaminyl transferases. This approach bestows a gain-of-chemical-functionality modification on cells, where the products of individual glycosyltransferases can be selectively characterized or manipulated to understand glycan contribution to major physiological processes.


INTRODUCTION
Posttranslational modifications expand the structural diversity of proteins but are notoriously difficult to study in living systems. Most modifications are refractory to direct genetic manipulation and require reductionist strategies such as in vitro systems or simplified cells. Glycans are the prime example for this; the human glycome is constructed by the combinatorial activity of more than 250 glycosyltransferases (GTs) with both hierarchical and competing activities. On the cell surface, glycans play a central role in modulating signal transduction, cell-cell interactions, and biophysical properties of the plasma membrane (Varki, 2017;Varki et al., 2017). Yet, we still lack the methodology to selectively visualize, modify, or sequence either a certain glycan subtype or the product of a certain GT. In a synthetic biology approach, individual GTs could be engineered to accommodate a chemical-functionality that is not found in native substrates and not accommodated by other GTs. This ''bump-and-hole'' tactic has been applied to a range of enzymes, including but not limited to kinases, methyl transferases, and ADP-ribosyltransferases (Besanceney-Webler et al., 2011;Alaimo et al., 2001;Carter-O'Connell et al., 2014;Gibson et al., 2016;Islam et al., 2011Islam et al., , 2018. We have recently developed the first GT bump-and-hole system that was applicable to multiple members of a GT family in vitro (Choi et al., 2019). However, application in the living cell has always been a substantial technical challenge for most bump-and-hole-systems; the nucleotide-based substrate analog must be delivered across the plasma membrane and into the Golgi compartment, and the cell must stably express the correctly localized and folded mutant enzyme. Bump-andhole engineering is particularly attractive to deconvolve GT families of multiple homologous isoenzymes, as the complex interplay of these isoenzymes in the secretory pathway cannot be probed in sufficient detail in in vitro assays.
One of the largest GT families in the human genome is the polypeptide N-acetylgalactosaminyl (GalNAc) transferase family (Gal-NAc-T1.T20, abbreviated T1.T20). Transferring GalNAc to Ser/Thr side chains, GalNAc-Ts initiate abundant O-linked glycosylation in the secretory pathway ( Figure 1A) (Bennett et al., 2012;Hang and Bertozzi, 2005). O-GalNAc glycosylation can differ based on cell type or activation stage, and clear disease phenotypes are associated with glycan aberration (Antonopoulos et al., 2012;Radhakrishnan et al., 2014). Unsurprisingly, GalNAc-T expression is often associated with tumorigenesis, sometimes with opposite effects on different types of cancer (Peng et al., 2012;Gaziel-Sovran et al., 2011). However, the absence of a glycosylation consensus sequence and the variability of glycan elaboration render O-GalNAc glycans challenging to study by mass spectrometry (MS)-based glycoproteomics. Thus, the protein substrate specificity of each isoenzyme is poorly studied and largely built on data inferred from synthetic peptide libraries (de Las Rivas et al., 2019). So-called SimpleCells that do not elaborate O-GalNAc glycans make glycoproteins that are easier to enrich and study by MS (Steentoft et al., 2011Schjoldager et al., 2012;Narimatsu et al., 2019). Knock-outs (KOs) of single GalNAc-Ts in the SimpleCell background have been profiled by glycoproteomics (Schjoldager et al., 2012(Schjoldager et al., , 2015. Similarly, glycopeptides from cells with wild-type (WT) O-glycan elaboration and titratable GalNAc-T knockin have been enzymatically simplified to allow for enrichment and MS (Hintze et al., 2018). These studies have revealed comprehensive glycoproteomics datasets but suffer from a loss of glycan elaboration and laborious genome engineering required for targeted knockin. Further, the activity of GalNAc-T isoenzymes is both redundant and competitive, such that the compensation and/or shift of glycosylation sites occurs upon KO (Schjoldager et al., 2015;Hintze et al., 2018). A gain-of-chemical-functionality strategy to visualize the products of a particular GT on the living cell is currently unavailable.
Here, we equipped living cells with the ability to tag the protein substrates of individual GalNAc-Ts with chemical, editable functionalities. We made use of the fact that a double mutation (''BH,'' I253A/L310A double mutant in GalNAc-T2; Figure 1B) re-programmed GalNAc-Ts to accept alkyne-containing uridine diphosphate (UDP)-GalNAc analogs such as compounds 1-4 instead of the native substrate UDP-GalNAc ( Figure 1B; Choi et al., 2019). We endowed living cells with the capacity to biosynthesize a UDP-GalNAc analog that was complementary to engineered BH-GalNAc-Ts. Further, we showed that BH-GalNAc-Ts emulate WT-GalNAc-Ts with regard to structure, localization, and protein substrate specificity. This approach bestowed a bioorthogonal tag (Sletten and Bertozzi, 2009;Prescher et al., 2004;Hang et al., 2003;Zaro et al., 2011;Woo et al., 2015) on the protein substrates of distinct GalNAc-T isoenzymes while displaying the complexity of glycan elaboration in the secretory pathway ( Figure 1C). Precision glycome engineering has widespread applications in biomarker discovery, GT profiling, and targeted cell surface engineering.

Structural Basis for GalNAc-T Engineering
As bump-and-hole engineering of a GT family in living cells has no precedent, we first set out to understand the structural implications of this approach. All GalNAc-Ts are type II transmembrane proteins with luminal GT and lectin domains connected through a flexible linker (Bennett et al., 2012;Hang and Bertozzi, 2005;Ten Hagen et al., 2003). We crystallized the luminal part of BH-T2 in complex with the native ligands Mn 2+ , UDP, and the substrate peptide EA2 (PTTDSTTPAPTTK) at 1.8-Å resolution. Comparison of BH-T2 and WT-T2 (PDB: 2FFU) revealed complete conservation of both the three-dimensional enzyme architecture and bound ligand structure (Figures 2A and S1A-S1C; Table 1; Fritz et al., 2006). In BH-T2, the interdomain linker adopts an extended conformation previously found in the catalytically active WT enzyme (Lira-Navarrete et al., 2014. The mutant A253 and A310 side chains in the BH enzyme are congruent with the WT side chains I253 and L310, respectively ( Figure 2B). Two glycine residues, G308 and G309, are slightly shifted by 1.2 Å and 1.7 Å (C a distances), respectively, likely to account for the changes elsewhere in the active site. BH-T2 thus retains the native structural properties of the WT-GalNAc-T enzyme.
A co-crystal structure of BH-T2, Mn 2+ , and UDP-GalNAc analog 1 at 3.1-Å resolution helped us visualize how the BH-T2 active site mutations affect enzyme-substrate binding. In comparison with a corresponding WT-T2/UDP-GalNAc/Mn 2+ /EA2 complex (PDB: 4D0T), UDP-sugar binding is completely conserved (Figures S1B and S1C; Table S1; Lira-Navarrete et al., 2014). BH-T2 indeed contains a hole that accommodates the alkyne side chain bump in UDP-GalNAc analog 1 ( Figure 2C). The formation of additional van der Waals interactions between the enzyme and substrate explain why the K M of BH-T2 toward 1 (2.6 mM) is approximately 10-fold lower than the K M of WT-T2 toward UDP-GalNAc (30 mM) (Choi et al., 2019). In turn, the fact that the k cat of BH-T2/1 (0.158 s À1 ) is approximately 5-fold lower than the k cat of WT-T2/UDP-GalNAc (0.813 s À1 ) indicates that these additional interactions may slightly slow the ring distortion that is needed to mediate the nucleophilic attack (Lira-Navarrete et al., 2014).
To corroborate our structural interpretation that GalNAc-T bump-and-hole engineering does not substantially alter substrate peptide binding, we profiled the substrate specificities of two engineered GalNAc-Ts: BH-T1 and BH-T2 (Choi et al., 2019;Gerken et al., 2006Gerken et al., , 2011Ji et al., 2018;Revoredo et al., 2016). A peptide library containing a single Thr residue with randomized neighboring residues was used in in vitro glycosylation experiments and analyzed by self-assembled monolayers for matrix-assisted desorption/ionization MS (SAMDI-MS; Figures 2D and S1D; Kightlinger et al., 2018). We found differences in substrate specificity between BH-T1 and BH-T2 that were similar to the differences seen in the corresponding WT-Gal-NAc-Ts: T1 generally preferred hydrophobic amino acids at À1 position and Glu, Ile, Pro, Val, or Trp at +1 position of the Thr glycan acceptor, whereas T2 showed a preference for Pro, Ala, Ser, and Thr at À1 position ( Figure S1D; Kightlinger et al., 2018). We note that BH-GalNAc-Ts glycosylated fewer peptides than the corresponding WT enzymes; we therefore repeated the experiment on a smaller peptide subset that allowed a more detailed view on peptide substrate fine specificities and found that glycosylation profiles by BH-GalNAc-Ts matched the corresponding WT-GalNAc-Ts' profiles ( Figure S1D). These data indicate that bump-and-hole engineering faithfully reports on GalNAc-T isoenzyme activity in vitro with simple peptide substrates containing a single acceptor Thr, a crucial prerequisite for a cellular bump-and-hole system. (A) Crystal structure of BH-T2 at 1.8 Å superposed with WT-T2 (PDB: 2FFU). Bound EA2 substrate peptide is cyan (sticks), Mn 2+ is magenta (sphere), and UDP is gray (sticks). Ligands are taken from BH-T2. For superposition with WT-T2 ligands, see Figure S1A. (B) Superposition of the UDP-sugar binding site of BH-T2 and WT-T2. Electron density is rendered at 1 s and carved at 1.6 Å . (C) Depiction of UDP-GalNAc analog 1 in a co-crystal structure with BH-T2 at 3.1 Å and UDP-GalNAc in a co-crystal structure with WT-T2 (PDB: 4D0T) (Lira-Navarrete et al., 2014), as well as WT and mutated gatekeeper residues. (D) Substrate specificities of BH-T1 and BH-T2 as determined in an in vitro glycosylation assay with detection by SAMDI-MS. For comparison with WT-GalNAc-T glycosylation, see Figure S1. Data are from one representative out of two independent experiments. See also Figure S1D and Table 1. ll Article BH-GalNAc-Ts Glycosylate Membrane Proteins We next sought to confirm that BH-GalNAc-Ts localize to the Golgi compartment and glycosylate proteins in a membrane environment. Full-length WT-and BH-T1 and T2 with a C-terminal vesicular stomatitis virus G protein (VSV-G) epitope tag exhibited doxycycline (Dox) dose-dependent expression under the control of a tetracycline-responsive promoter in stably transfected HepG2 cells (Figures 3A and S2A;Kowarz et al., 2015). All tested GalNAc-Ts co-localized with the Golgi marker giantin, confirming the native localization of engineered GalNAc-Ts (Figures 3B and S2B). To assess GalNAc-T activity, membrane protein fractions were prepared from WT-and BH-GalNAc-T-expressing HepG2 cells and used for in vitro glycosylation experiments. After incubation with alkyne-containing UDP-Gal-NAc analog 1, alkyne-tagged glycoproteins were derivatized with azide-biotin using Cu I -catalyzed [3+2] ''click'' cycloaddition and characterized on a streptavidin blot. Compounds 1, 2, and 3 were specific substrates for BH-but not WT-GalNAc-Ts. In the presence of these substrates, BH-T1 and BH-T2 labeled overlapping and also unique glycoprotein species (Figures 3C,S2C,and S2D). In contrast, treatment of lysates from WT-T1and WT-T2-expressing cells with compound 4 with a shorter alkyne chain led to substantial labeling, consistent with 4 being a substrate for WT-GalNAc-Ts (Choi et al., 2019;Zaro et al., 2011). These results were confirmed using soluble GalNAc-Ts and a membrane fraction of non-transfected cells ( Figure S2E). Importantly, profiling both enzyme activity and protein specificity of single GalNAc-T isoenzymes has been impossible to date even in cell lysates. We next targeted a GalNAc-T bump-andhole platform in the living cell.

Biosynthesis of a Bumped UDP-GalNAc Analog
Among the insightful bump-and-hole studies that have probed various enzyme families, few have been performed in living cells due to the inability to deliver negatively charged substrates across the plasma membrane (Carter-O'Connell et al., 2014;Gibson et al., 2016;Islam, 2018;Wang et al., 2013). GalNAc analogs have been fed to cells as membrane-permeable peracetylated precursors that are deprotected by esterases and converted to UDP-GalNAc analogs via the kinase GALK2 and the pyrophosphorylase AGX1 ( Figure 4A; Zaro et al., 2011;Woo et al., 2015;Boyce et al., 2011;Pouilly et al., 2012). By delivery of the corresponding protected GalNAc-1-phosphate analog, a GalNAc analog can bypass GALK2, such that only AGX1 is necessary for biosynthesis to the UDP-GalNAc analog. However, in accordance with previous findings (Pouilly et al., A lack of AGX1 activity toward synthetic N-acetylglucosamine (GlcNAc) analogs has previously prompted the engineering of AGX1 to recognize the corresponding GlcNAc-1-phosphate analog as a substrate (Yu et al., 2012). As WT-AGX1 accepts both GlcNAc-1-phosphate and GalNAc-1-phosphate as substrates, we investigated whether AGX1 mutants could biosynthesize UDP-GalNAc analogs 1, 2, and 3 from the corresponding GalNAc-1-phosphate analogs in the living cell. We mutated the gatekeeper residues Phe381 and Phe383 to Gly or Ala in FLAG-tagged AGX1 expression constructs (Yu et al., 2012). GalNAc-1-phosphate analogs were delivered to stably transfected HEK293T cells by virtue of caged precursors 5, 6, and 7. UDP-sugar biosynthesis was then determined by highperformance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) of cell lysates. Gly and Ala mutants of Phe383 efficiently biosynthesized bumped UDP-GalNAc analog 1, while neither Phe381 single mutants nor any Phe381/Phe383 double mutants produced 1 despite equal expression levels ( Figure S3A; Yu et al., 2012). In contrast to linear alkyne 1, neither of the methylated alkynes (2 or 3) was biosynthesized by engineered AGX1 F383A , hereby called mut-AGX1 ( Figures 5B and S3B). We thus concluded that bumped GalNAc-1-P(SATE) 2 precursor 5 can be used in conjunction with mut-AGX1 to deliver UDP-GalNAc analog 1 to the living cell and establish a GalNAc-T bump-and-hole system.
In HPAEC-PAD chromatograms of cells biosynthesizing UDP-GalNAc analog 1, we consistently found a satellite peak that eluted after 1 (arrowhead in Figure 4B). The epimerase UDPgalactose/UDP-GalNAc-4'-epimerase (GALE) maintains a cytosolic equilibrium between UDP-GalNAc and UDP-GlcNAc and has been shown to also accept azide-containing analogs (Boyce et al., 2011;Schulz et al., 2004). We found that GALE epimerizes UDP-GalNAc analog 1 to the corresponding UDP-GlcNAc analog 8 ( Figure 4A): first, HPAEC-MS confirmed that the satellite peak was caused by an isomer of UDP-GalNAc analog 1 with 328 m/z [M-2H] 2-. Second, CRISPR-mediated GALE-KO in the K-562 background abrogated the satellite peak . In contrast, epimerization was still observed in GALE-containing cells. Alkyne-containing UDP-GlcNAc analog 8 might result in the labeling of GlcNAc-containing glycans on the cell surface, such as N-linked glycans. We thus concluded that epimerization of 1 to 8 must be accounted for in glycoproteomics experiments (e.g., by enzymatic abrogation of cell surface N-glycans during sample processing).

A GT Bump-and-Hole System in Living Cells
With a GalNAc-T bump-and-hole system and a method for cellular substrate delivery of 1 in hand, we set out to probe isoenzyme-dependent glycosylation in the living cell. We cloned FLAG-tagged WT-or mut-AGX1 under a constitutive promoter into an expression vector containing VSV-G-tagged WT or BH versions of GalNAc-T1 or T2 under the control of a Dox-inducible promoter ( Figure 5A). This setup allowed us to systematically  (C) In vitro glycosylation of proteins in a membrane fraction by full-length GalNAc-Ts using UDP-GalNAc analogs. Data are from one representative out of two independent experiments. Experiments were repeated with the membrane fraction of non-transfected cells and soluble, purified GalNAc-Ts as an enzyme source. DIC, differential interference contrast; rtTA, reverse tetracycline transcriptional activator. See also Figure S2. ll Article assess any potential background protein labeling when UDP-GalNAc analog 1 could not be biosynthesized or when 1 was biosynthesized but BH-GalNAc-Ts were absent. To preclude any labeling due to epimerization of 1 to UDP-GlcNAc analog 8, we first used K-562 GALE-KO cells for labeling. Cells were supplemented with GalNAc to compensate for the loss of UDP-GalNAc biosynthesis ( Figure S3C). GalNAc-T expression was induced ( Figure S4A), cells were fed with caged GalNAc-1-phosphate analog 5, and the cell surface was reacted with clickable Alexa488-picolyl azide in the presence of a non-membrane-permissive Cu(I) complex (Besanceney-Webler et al., 2011;Uttamapinant et al., 2012). After gating for positive VSV-G signal, flow cytometry showed a more-than-15-fold fluorescence increase when both BH-T2 and substrate 1 were present over controls lacking either component ( Figures 5B and S4B). In GALE-containing cells, a more-than-2-fold-higher signal was still measured over control cells when a functional bump-and-hole system was present ( Figures S4C and S4D).
In order to better visualize the scope of protein labeling by our bump-and-hole system, we reacted cell surfaces with a clickable version of the infrared dye CF680 to profile labeled cell surface glycoproteins by in-gel fluorescence. We compared protein labeling patterns of GALE-containing K-562 cells stably expressing T1 or T2 constructs. Here, N-linked glycans were removed by PNGase F treatment prior to analysis to reduce background fluorescence ( Figure S4E). A profound band pattern was observed (B) HPAEC-PAD traces of extracts from HEK293T cells stably expressing WT-AGX1 or mut-AGX1 and fed with the indicated compounds. Dashed boxes indicate retention times of standards in separate reference runs. The product of potential epimerization of 1 by GALE, compound 8, is marked with an arrowhead. Data are of one experiment and were repeated for compound 5 in HEK293T cells transiently transfected with AGX1 constructs, as well as stably transfected K-562 cells. Insert: epimerization to 8 is suppressed in GALE-deficient K-562 cells expressing mut-AGX1 and fed with 5, but not cells carrying a control single guide RNA (sgRNA). A reference trace of compound 1 is shown. Data are of one representative out of two independent experiments. See also Figure S3.
when functional bump-and-hole pairs (5, mut-AGX1, and BH-GalNAc-Ts) were present ( Figures 5C and S4F). Mut-AGX1 was required for labeling, confirming that our experiments probe enzymatic glycosylation rather than non-specific protein modification (Qin et al., 2018). Fluorescence was of similar intensity as in cells treated with well-characterized alkynecontaining N-acetylneuraminic acid precursor Ac 4 ManNAl (Chang et al., 2009). Furthermore, the presence of BH-GalNAc-T protein was essential, as the omission of Dox induction prevented fluorescent labeling ( Figure S4F). Importantly, BH-T1 and BH-T2 produced slightly different band patterns, especially between 25 and 37 kDa. The most intense 110-kDa band migrated at slightly higher molecular weight when glycosylated by BH-T1 instead of BH-T2, indicating that T1 potentially labeled more sites of this particular protein. Digestion with the mucin-selective protease StcE completely removed this band in T2 labeled samples ( Figure S4G), confirming labeling of mucintype proteins that are rich in O-GalNAc glycosylation (Malaker et al., 2019). Discrete band patterns were obtained from in-gel fluorescence experiments when GALE-KO cells were used and a functional bump-and-hole pair was present. In contrast, pulsing the same cells with GlcNAc analog 9, a precursor of UDP-GlcNAc analog 8, led to a diffuse background whenever mut-AGX1 was present, but independent of the GalNAc-T construct used ( Figure S4H). We re-cloned GalNAc-T and AGX1 constructs into an expression vector with one constitutive promoter for both genes and showed that the labeling pattern after feeding 5 is similar to the pattern using the Dox-inducible constructs (Figure S5A). To profile the identity of labeled cell surface glycoproteins, we synthesized clickable, acid-cleavable biotin-picolyl azide 10 as an enrichment handle of cell surface glycoproteins ( Figure 5D). Following lysis and PNGase F treatment, O-ll Article glycoproteins were enriched and digested on-bead with trypsin to profile the corresponding unmodified peptides by MS. The myelogenous K-562 cell line expresses mucins and mucin-like proteins that are rich in O-GalNAc glycans such as CD36, CD43, CD45, Glycophorins A and C, and MUC18 (https:// www.proteinatlas.org/). We found all these proteins enriched (log prob > 5 for BH and < 5 for WT samples, or at least 40 units higher for BH than controls) from lysates of cells carrying functional T1 or T2 bump-and-hole systems (5, mut-AGX1, and BH-GalNAc-T) over control cells lacking any component (Figure S5B). It is noteworthy that we found mainly cell surface glycoproteins with abundant O-glycosylation in these experiments, probably because BH-GalNAc-Ts were in constant competition for protein substrates with the corresponding endogenous WT-GalNAc-Ts in these cells.
The use of GalNAc-T-KO SimpleCells has enabled the mapping of glycosylation sites that are introduced by several GalNAc-Ts including T1 and T2, but by design it is not suited to profile the diversity of glycan structures beyond the initiating GalNAc. Our bump-and-hole approach can contribute this information if the bumped GalNAc analog is recognized and extended by downstream GTs. To probe this, we used bumpand-hole pairs to profile the glycoproteome of T1-KO or T2-KO HepG2 cells (Schjoldager et al., 2015). Cells were transfected with GalNAc-T1 or T2 and Dox-induced before labeling with Gal-NAc-1-phosphate analog 5. As glycoproteins are abundant in the HepG2 secretome (Schjoldager et al., 2015), we clicked acid-labile enrichment handle 10 onto proteins in conditioned cell culture supernatants. Following on-bead tryptic digest to release non-glycosylated peptides, glycopeptides were liberated by acidic cleavage of the enrichment handle and analyzed by MS. We used the presence of a 491.2238 m/z ion in higherenergy collisional dissociation (HCD) as an indicator for a bumped glycan to trigger peptide sequencing by electron-transfer dissociation (ETD; Figures 5E and S5C-S5E). Following an automated search for glycopeptides containing the GalNAc analog alone or as a part of longer glycans, we obtained raw glycopeptide hits, which we manually validated. Seventy-seven spectra were found of BH-T2-modified glycopeptides that corresponded to 37 peptides with 27 glycosylation sites and the presence of up to 4 different glycan compositions per glycopeptide, the structures of which were inferred based on biosynthetic considerations (Data S1;Schjoldager et al., 2012). In contrast, four glycopepetides were found as modified by BH-T1. Cells fed with DMSO alone gave zero glycopeptide hits. WT-T1-and WT-T2-expressing cells fed with GalNAc-1phosphate analog 5 showed one and two glycopeptides, respectively. Several known glycosylation sites in the SimpleCell glycoproteomics dataset by Schjoldager et al. (2015) were confirmed herein, and new sites were revealed (Data S1). For ll Article instance, we found hitherto unannotated T2 glycosylation sites in the proteins ITIL2, AMBP, Complement C3 and C4, GPC3 and STC2, and Laminin subunit gamma-1, among others ( Figure 5E; Data S1) (Luo et al., 2005). T2 is involved in lipid homeostasis and was found to glycosylate certain apolipoproteins, including ApoE at Thr307 and Ser308, in HepG2 cells (Steentoft et al., 2013;Schjoldager et al., 2015). Both sites were confirmed herein and, additionally, found to be elaborated up to the trisaccharide Neu5Aca-Galb-GalNAc-Ser/Thr or tetrasaccharide Neu5Aca--Galb-(Neu5Aca)GalNAc-Ser/Thr structures (Data S1; Schjoldager et al., 2012). Elaboration of GalNAc-T isoenzyme-specific sites cannot be probed with conventional glycoproteomic techniques, highlighting the usefulness of our gain-of-chemical-functionality method to contribute another dimension to the glycoproteome. Our data further resolved ambiguity about the Oglycosylation of apolipoprotein A-1 (ApoA1) in the peptide sequence ATEHLSTLSEK (ApoA1 220-230 ): previous data showed that upon T1-KO, glycosylation of Thr221, Thr226, and Ser228 is slightly decreased, while upon T2-KO, all three sites had increased occupancy (Schjoldager et al., 2015). However, the reasons for these changes in glycosylation were elusive (Schjoldager et al., 2015). Our gain-of-chemical-functionality approach revealed that BH-T2 glycosylated Ser225, while BH-T1 glycosylated Thr221, indicating that T2-KO cells lose Ser225 glycosylation that is compensated by increased glycosylation at the other sites ( Figure 5F). We repeated the experiment using BH-GalNAc-Ts and treatment with caged GalNAc-1-phosphate analog 5 only and found similar results (Data S2). Such detailed analysis is facilitated by the chemical tagging strategy that emulates GalNAc-T activity as a result of our bump-and-hole approach.

DISCUSSION
The cellular glycoproteome is subject to disease-related alterations. Bump-and-hole engineering has been artfully employed to obtain insight into the protein substrates of single kinases (Alaimo et al., 2001;Bishop et al., 2000), ADP-ribosyltransferases (Carter-O'Connell et al., 2014;Gibson et al., 2016), and methyltransferases (Wang et al., 2013;Islam et al., 2013;Li et al., 2011), among others (Islam, 2019). Only a few of these systems have been established in the living cell, as cellular delivery of bumped substrates is rarely straightforward, especially when substrates are nucleotide based. Herein, we established the first GT bumpand-hole system in the living cell to profile GalNAc-Ts, the largest GT family in the human genome. The fascinating biology of GalNAc-Ts, with multiple isoenzymes and a profound cross-talk between glycosylation and proteolysis (Revoredo et al., 2016;King et al., 2018), prompted us to develop an isoenzyme-specific chemical reporter. This approach allowed us to expand our knowledge on T1 and T2 glycosylation sites by adding information on the nature of the mature glycans after extension by other GTs.
The era of quantitative biology demands great specificity in tools to probe cellular processes or expressed antigens (Purcell and Godula, 2019). Biological tools, including antibodies, lectins, engineered binding proteins, or silenced hydrolases, have been employed to profile or isolate cellular glycans (Malaker et al., 2019;Chen et al., 2017). While powerful probes, these proteins suffer from various drawbacks, including the dependence of binding on the structural context or the requirement to simplify glycans before performing binding studies. Additionally, all conventional strategies for glycan pull-down or visualization rely on post-glycosylation derivatization or binding. As a consequence, information on GT isoenzyme specificity is already lost or-in the context of KO cell lines-may have been obscured by partial redundancy. This ''specificity space'' is covered by our bump-and-hole approach that enables GT-and glycosite-specific labeling. As Gal-NAc-T engineering retains both three-dimensional structure and peptide glycosylation preferences, this strategy probes glycosylation in an unbiased fashion without context dependence. Thereby, we obtained for the first time information on both the site and structure of glycans initiated by a single GT isoenzyme in a single experiment.
Taken together, we now have a strategy in hand to specifically modify GT isoenzyme-dependent glycosylation sites in the living cell.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

EXPERIMENTAL MODEL AND SUBJECT DETAILS
All cell lines were mycoplasma-free, as confirmed using the MycoAlert Kit (Lonza, Basel, Switzerland). Female K-562 cells with stable expression of Streptococcus pyogenes Cas9 (K-562-spCas9) were a gift from Michael Bassik (Stanford University, USA) and propagated in RPMI (Thermo Fisher) with 10% (v/v) FBS, penicillin (100 U/mL) and streptomycin (100 mg/mL) at 37 C with 5% CO 2 . Expression of spCas9 was confirmed by western blot. K-562 GALE-KO cells were derived from K-562-spCas9 cells, and were grown under the same conditions, but with addition of 200 mM GalNAc and 20 mM galactose to the culture medium. GALE-KO cells were maintained in standard growth medium supplemented with 20 mM galactose and 200 mM GalNAc. GALE was not detectable by western blot using mouse anti-GALE sc-390407 (Santa Cruz Biotechnology, Dallas, USA) over at least 20 passages. Cell authentication was performed via STR profiling and species authentication. Female K-562 cells were a gift from Jonathan Weissman (University of California, San Francisco). Cells were grown in RPMI with 10% (v/v) FBS, penicillin (100 U/mL) and streptomycin (100 mg/mL). Cell authentication was performed via STR profiling and species authentication. HEK293T (ATCC CRL-3216) of unknown sex were from ATCC and grown in DMEM (Thermo Fisher) with 10% (v/v) FBS (Thermo Fisher), penicillin (100 U/mL) and streptomycin (100 mg/mL, GE Healthcare, Chicago, USA). Cell authentication was performed via STR profiling and species authentication. Freestyle TM 293-F cells of unknown sex were propagated in FreeStyle TM 293 Expression Medium (Thermo Fisher) with 10% (v/v) fetal bovine serum (FBS, Thermo Fisher) at 37 C and 8% CO 2 with orbital rotation at 135 rpm. Cells were not authenticated. HepG2 cells (ATCC HB-8065), HepG2-T1-KO and HepG2-T2-KO cells (a gift from Katrine T. Schjoldager and Hans H. Wandall, University of Copenhagen, Denmark) were propagated in low-glucose DMEM (Caisson Labs, Smithfield, USA) with 10% (v/v) FBS, penicillin (100 U/mL) and streptomycin (100 mg/mL). Cells were not authenticated.
Full length human GalNAc-T2 (EBI accession number LC043140.1) in pCMV-NTAP was a kind gift from Lawrence Tabak (National Institutes of Health, Bethesda, MD) (Choi et al., 2019). T2 was first sub-cloned with a C-terminal FLAG tag into a version of pFLAG-CMV-2 (Sigma Aldrich, St. Louis, USA) in which the N-terminal FLAG tag had been removed, using a two-step PCR elongation procedure with the primer T2_F1, T2_R1 (step 1) and T2_R2 (step 2), and a NotI/EcoRI restriction strategy. Site-directed mutagenesis (I253A/L310A) was performed at this step according to a published procedure (Choi et al., 2019). T2 constructs were cloned into pFLAG-CMV-5.1 (Sigma Aldrich) using the primers T2_F2 and T2-R3 and a NotI/BamHI strategy. These constructs were used as templates to clone T2 constructs into pSBtet-GH (Kowarz et al., 2015) with a C-terminal VSV-G tag, using the primers T2_F3 and T2_R4 with an SfiI cloning strategy.
Full length GalNAc-T1 (NCBI GenBankÒ accession number NM_020474) was used as a gBlock (IDT) with overhangs and NotI/ BamHI restriction sites, and amplified with the primers T1_F1 and T1_R1 prior to cloning into pFLAG-CMV-5.1 using a NotI/BamHI strategy. Site-directed mutagenesis (I238A/L295A) was performed at this step according to a published procedure (Choi et al., 2019). These constructs were used as templates to clone T1 constructs into pSBtet-GH with a C-terminal VSV-G tag, using the primers T1_R1 and T1_R2 with an SfiI cloning strategy.
Crystals were grown using sitting drop vapor diffusion at room temperature. Drops were set-up using a Douglas Oryx8 Nanodrop Dispensing Robot (Douglas Instruments Ltd, Berkshire, United Kingdom).
Crystals of BH-T2 complexed with 1 and Mn 2+ were obtained through soaking of EA2 and 1 into co-crystals of BH-T2 with UDP and Mn 2+ . EA2 peptide electron density was too weak to properly discern in crystal structures. Initial drops were created using 0.15 mL of 5 mM MnCl 2 , 5 mM UDP, 10 mg/mL T2 BH protein in 25 mM Tris-HCl (pH 8.0), 0.5 mM EDTA, and 1 mM TCEP and 0.15 mL precipitant solution (11%-23% PEG8000, 0.1 mM HEPES, pH 7.5-8.5) against 80 mL of precipitant solution. Crystals were grown for 18 days before soaking and diffraction. Crystals were soaked in a solution with EA2 by adding 0.45 mL of a 50 mM EA2 solution in water to the original drop for 30 minutes to 2 hours. Crystals were harvested and soaking was performed with a solution of 20 mM 1 with 16% ethylene glycol, 11%-23% PEG8000, 0.1 mM HEPES, 5 mM MnCl 2 , pH 7.5-8.5 for 1-2 hours.
Data was processed using XDS (Kabsch, 2010), and scaled with SCALA (Evans, 2006) and other programs implemented with the CCP4 software (Winn et al., 2011). Crystal structures were determined using molecular replacement with Phaser (McCoy et al., 2007), using published structures (PDB: 2FFU for PDB: 67EI and PDB: 4D0T for PDB: 6NQT) as the templates Lira-Navarrete et al., 2014). The initial model was improved through multiple cycles of manual modeling in Coot (Emsley et al., 2010), and refinement using REFMAC5 (Murshudov et al., 1997). The final structural model was validated using MOLPROBITY (Chen et al., 2010). UDP-GalNAc analog conformation was validated using Privateer (Agirre et al., 2015). Images were prepared using Pymol 2.0.0 (Schrö dinger LLC, New York, USA). Electron density was rendered at 1 s and carved at 1.6 Å . Structure statistics are given in Table S1.
Screening peptide substrate selectivities of GalNAc-Ts using SAMDI-MS SAMDI-MS screenings were performed similarly to previously reported (Kightlinger et al., 2018). Briefly, 50 mM solutions of 361 peptides of the general formula AX -1 TX +1 APRC, where X -1 and X +1 are 19 natural amino acids except for Cys, were screened individually with 25 nM purified soluble FLAG-tagged WT-GalNAc-T1 or 50 nM purified soluble FLAG-tagged WT-GalNAc-T2 and 0.25 mM UDP-GalNAc in SAMDI buffer (50 mM Tris-HCl pH 7.4, 50 mM NaCl, 5 mM MnCl 2 ), at 37 C for 3 h (Choi et al., 2019). For BH-GalNAc-Ts, the same peptide array was screened with 30 nM purified soluble FLAG-tagged BH-GalNAc-T1 and 0.25 mM compound 1 in SAMDI buffer at 37 C for 21 h, or with 175 nM soluble FLAG-tagged BH-GalNAc-T2 and 0.25 mM compound 1 in SAMDI buffer at 37 C for 3 h (Choi et al., 2019). After the reaction of each 10 mL volume, 5 mM EDTA were added for quenching and 2.5 mL TCEP reducing gel (Thermo Fisher) were added for reduction incubation for 1 h at 37 C. Reduced mixture (2 mL) was added to the gold islands of a 384 SAMDI plate, prepared as described before (Kightlinger et al., 2018). The SAMDI plate was incubated at room temperature for 30 min, and washed with water, ethanol and water, nitrogen blow dried, and treated with 10 mg/mL 2 0 ,4',6'-trihydroxyacetophenone monohydrate (Sigma Aldrich) matrix in acetonitrile. The SAMDI plates were analyzed with a Sciex MALDI-TOF/TOF 5800 (Applied Biosystems, Foster City, USA), equipped with the software Explorer 4.1.0 to analyze data. Percent intensity of glycosylated peptides related to the sum of unreacted and glycosylated peptide was recorded. Preferred X -1 amino acids, combined with all X +1 amino acids, were chosen to repeat the experiment for BH-GalNAc-Ts.
Cloning and site-directed mutagenesis of AGX1 AGX1 F383G in pIRES-puro3 was used as a template to generate AGX1 constructs (Yu et al., 2012). Site-directed mutagenesis was performed using the following primers (positions 381 and 383 underlined): 383G_F and 383G_R for F381G; 381G/383G_F and 381G/383G_R for F381G/F383G. A C-terminal FLAG-tag was then introduced into these constructs using a site-directed mutagenesis strategy with FLAG introduction primer_F and _R. These constructs were then used to produce other AGX1 constructs. Primer pairs (positions 381 and 383 underlined) were: 381A_F/_R and for F381A; F383A_F/_R and for F383A; 381A/383GA_F/_R and for F381A/F383A; WT_F/_R for WT.
Cloning of AGX1 into pSBtet and pSBbi plasmids WT-AGX1 or mut-AGX1 were cloned into the backbone of pSBtet-GH containing WT-T1, BH-T1, WT-T2, or BH-T2 by replacing the eGFP coding sequence. C-terminally FLAG-tagged AGX1 sequences were amplified from the corresponding pIRES-puro3 constructs (see above) using the primers AGX1 subcloning_F/_R, purified by gel extraction, and subjected to an overlap extension PCR with equimolar amounts (10 fmol each) of the gBlocks (IDT) AGX1_gBlock_5 0 and AGX1_gBlock_3 0 using 10 mM dNTPs and Q5 polymerase. Briefly, a 50 mL reaction was run for 15 cycles using the following protocol: 95 C (2 min), 95 C (30 s), 64 C (30 s), 72 C (30 s -return to step 2), 72 C (5 min), then treated with the primers (500 nmol) AGX1 extension_F and AGX1 extension_R and continued for another 18 cycles with an annealing temperature of 55 C. The resulting PCR product was used to replace the eGFP cloning sequence of pSBtet-GH containing T1 or T2 using an XcmI/PasI strategy to digest the vector and In-Fusion HD Cloning Kit to insert AGX1 sequences. AGX1-containing pSBtet vectors are named pSBtet-WT-hAGX1 or pSBtet-mut-hAGX1. For constitutive expression of GalNAc-Ts, eGFP coding sequence was accordingly replaced with AGX1 in pSBbi-GH by GeneArt (Thermo).
HEK293T cells were seeded at 1.2 million cells per well of a 6-well plate. After 24 h, cells were transfected with 1.5 mg sgRNA-coding vector and a third generation lentiviral packaging mix (0.1 mg GAG/POL, 0.1 mg REV, and 0.2 mg VSV-G (Dull et al., 1998), using TransIT-293 (Mirus Bio LLC, Madison, USA). After 16 h, supernatant was aspirated and replaced with fresh growth medium. After another 30 h, supernatant was collected, and filtered by 0.45 mm syringe filtration. Virus-containing supernatant was stored at À80 C. ll Article For lentiviral infection, 200,000 K-562-spCas9 cells were seeded in 1 mL medium in a 24-well plate. After 24 h, 300 mL virus-containing supernatant and 8 mL polybrene (Sigma Aldrich) were added. After another 24 h, cells were harvested and treated with fresh growth medium. After another 24 h, cells were harvested and treated with growth medium containing 1.25 mg/mL puromycin. Selection was carried out for 96 h, and cells were expanded in puromycin-free growth medium. The knockout cells were fed 100 uM of GalNAc for 2 days before FACS sorting. After 48 h, the cells were stained with 5 mg/mL biotinylated Vicia villosa lectin (Vector Labs, Burlingame, USA) and 4 mg/mL of AlexaFluor488 conjugated streptavidin (Thermo Fisher) on ice for 30 min. The cells were washed twice with PBS + 0.5% BSA and viable cells were sorted based on AlexaFluor488 fluorescence intensity and low SYTOX Red (Thermo) staining according to a published procedure (Gaziel-Sovran et al., 2011). UDP-GalNAc was absent in these cells unless GalNAc was supplemented ( Figure S3C). Both sgRNAs had the same effect, and only sgRNA_GALE2 was used for further experiments.
HEK293T cells were transfected with pIRES-puro3 plasmids containing AGX1 constructs using TransIT-293 (Mirus Bio LLC, Madison, USA) according to the manufacturer's instructions and 37.5 mg DNA per 15 cm dish or 15 mg DNA per 10 cm dish. After 24 h, medium was aspirated, and cells were either treated with fresh growth medium and compounds for analysis of nucleotide-sugar biosynthesis (see below), or growth medium containing 5 mg/mL puromycin (Sigma Aldrich) for selection of stable cells for two weeks.
K-562 cells were transfected with pSBtet-based plasmids using Lipofectamine LTX (Thermo Fisher) according to the manufacturer's specifications, with 200000 cells in 1 mL growth medium, 1.25 mg pSBtet and 62.5 ng pCMV(CAT)T7-SB100 plasmid DNA. After 24 h, cells were harvested and selected in growth medium containing 150 mg/mL hygromycin B (Thermo Fisher) for 7-10 days to obtain stable cells.
Membrane protein glycosylation was performed on a membrane fraction from untransfected or stable HepG2 cells. Cells were seeded in a 15 cm dish, grown to 50% confluency and treated with doxycycline (5 mg/mL final concentration). After 24 h, doxycycline treatment was repeated. After another 24 h, cells were washed with PBS (10 mL) and scraped in PBS (5 mL). Cells were harvested by centrifugation (300 g, 5 min, 4 C), washed once with ice-cold PBS (2 mL) and frozen at À80 C. Cells were thawed on ice and fractionated using the Subcellular Fractionation Kit for Cultured Cells (Thermo Fisher) according to the manufacturer's instructions, with 0.4 mL of both Cytoplasmic Extraction Buffer and Membrane Extraction Buffer. Protein concentration was measured by BCA (Thermo Fisher), and fractions were frozen after addition of 80% (v/v) glycerol (10% final concentration).
In vitro glycosylation reactions were performed using 10 mg membrane protein of stably transfected HepG2 cell lines in 20 mL reaction volume containing 62.5 mM Tris-HCl pH 7.4, 150 mM NaCl, 10 mM MnCl 2 , 250 mM UDP-GalNAc analog and 500 mM UDP-GalNAc at 37 C for 12 h. Reactions were heat-inactivated at 95 C for 20 s and cooled to 4 C. Then, alkyne-containing reaction mixtures were sequentially treated with equal volumes (1.25 mL each) of 2 mM biotin-PEG 4 -azide (Thermo Fisher), 2 mM BTTAA (Click Chemistry Tools, Scottsdale, USA), 20 mM CuSO 4 and 100 mM sodium ascorbate (final concentrations 100 mM biotin probe, 100 mM BTTAA, 1 mM CuSO 4 and 5 mM sodium ascorbate). Click reactions were carried out at room temperature for 2 h and quenched by addition of 50 mM EDTA. Reaction mixtures were then subjected to SDS-PAGE and blotted on nitrocellulose membranes. The total protein amount was assessed using the REVERT protein staining kit (LI-COR Biosciences, Lincoln, USA), and biotinylation was detected using IRDye 800CW Streptavidin (LI-COR Biosciences) according to the manufacturer's instructions.
In vitro glycosylations were replicated using a membrane protein fraction from untransfected HepG2 cells that was depleted for internal GalNAc-T activity by three heat (90 C)-cool (4 C) cycles of 30 s each. Soluble FLAG-tagged  were added at a final concentration of 20 nM (T1) or 10 nM (T2), along with bumped UDP-GalNAc analog (250 mM) and UDP-GalNAc (500 mM). Glycosylation, click reaction and Streptavidin detection were performed as described above.

Fluorescence microscopy
Fluorescence microscopy was performed with stably transfected HepG2 cells. Cells were Dox-induced (T1-transfected cells: 2 mg/mL; T2-transfected cells: 0.2 mg/mL) for 24 h, then trypsinated and transferred in growth medium to a 24 well plate containing circular 12 mm coverslips (Electron Microscopy Services, Hatfield, USA) pre-treated with human fibronectin (5 mg/mL in PBS, Merck & Co., Kenilworth, USA) for 20 min and washed with PBS. Cells were treated with Dox again and incubated for another 24 h. Supernatant was aspirated, cells were washed in PBS with 100 mg/L Ca 2+ and 100 mg/mL Mg 2+ (DPBS), and fixed with 4% (v/v) paraformaldehyde (Sigma Aldrich) in PBS for 10 min in the dark. Cells were washed with 50 mM ammonium chloride in DPBS and DPBS with 2% (w/v) BSA (Incubation Buffer). Cells were then permeabilized with 0.5% (v/v) Tween-20 in incubation buffer for 10 min in the dark, and subsequently treated with antibody solutions in the same buffer: mouse anti-VSV-G-tag, (1:200), and rabbit anti-Giantin (1:500, 9B6, abcam). Cells were incubated for 1 h, washed twice with Incubation Buffer with 0.1% Tween-20, and incubated with secondary antibodies in Incubation Buffer containing 0.5% (v/v) Tween-20: Donkey anti-mouse Alexa Fluor 568 (1:200, abcam) and donkey anti-rabbit Alexa Fluor 647 (1:200, abcam). Cells were incubated for 1 h in the dark, washed twice with Incubation Buffer with 0.1% (v/v) tween-20, and mounted on glass slides using ProLong Diamond Antifade mounting solution (Thermo Fisher). Cells were imaged using a Nikon A1R+ Resonant Scanning Confocal Microscope.

Analysis of nucleotide-sugar biosynthesis by High Performance Anion Exchange Chromatography
Cells expressing AGX1-FLAG (transient or stably transfected HEK293T in 20 mL growth medium in a 15 cm dish or 5 million stable K-562 in 4 mL growth medium) were treated with caged GalNAc-1-phosphate analogs (100 mM final concentration from 10 mM or 100 mM stock solutions in DMSO; all samples of an experiment contained equal amounts of DMSO) or DMSO vehicle. After 7 h, K-562 cells were harvested (500 g, 5 min, 4 C) and the supernatant was aspirated. HEK293T cells were washed once on the plate with cold PBS (8 mL), scraped in cold 1 mM EDTA in PBS (8 mL), transferred to a conical tube and harvested (300 g, 5 min, 4 C). Cell pellets were resuspended in PBS (1 mL), 0.8 mL of that suspension was transferred to O-ring tubes (1.5 mL, Thermo Fisher) and harvested. Zirconia/silica beads (0.1 mm, BioSpec, Bertlesville, USA) were added at a similar volume as the cell pellet, followed by 1:1 acetonitrile/water (1 mL). Cells were lysed with a bead beater (FastPrep-24, MP Biomedicals, Santa Ana, USA) for 30 s at 6 m/s, and the mixture was left at 4 C for 10 min. Samples were centrifuged (14000 g, 10 min, 4 C), and the resulting supernatant was transferred to a new 1.5 mL tube. The solvent was removed by speed vac. The residue was resuspended in a solution of 15 mM ADP-a-Dglucose (Sigma Aldrich) in LCMS-grade water (Thermo Fisher, 0.2-0.4 mL). The solution was membrane-filtered (30 min, 14000 g) using a 3 kDa cut-off filter (Centricon, Merck) and analyzed by high performance anion exchange chromatography. The residual cell suspension in PBS (0.2 mL) was harvested, and the pellet was resuspended in M-PER lysis buffer (Thermo Fisher) containing cOmplete protease inhibitor (0.2 mL). The solution was incubated for 10 min at room temperature and centrifuged (14000 g, 10 min, 4 C). The protein concentration of the supernatant was measured by BCA, and samples were used for analysis of protein expression.
For in-gel fluorescence, cells were resuspended in Cell Buffer (35 mL), treated with a solution of 50 mM CuSO 4 , 300 mM BTTAA (Click Chemistry Tools, Scottsdale, USA), 2.5 mM sodium ascorbate, 2.5 mM aminoguanidinium chloride and 50 mM CF680 picolyl azide in cell buffer (35 mL), and incubated for 7 min at room temperature on an orbital shaker. The reaction was quenched by addition of 3 mM bathocuproinedisulfonic acid in PBS (35 mL). Cells were harvested, washed twice with Cell Buffer and once with PBS, and resuspended in ice-cold Lysis Buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% (v/v) Triton X-100, 0.5% (v/v) sodium deoxycholate, 0.1% (w/v) SDS, 1 mM MgCl 2 , and 100 mU/mL benzonase (Merck) containing cOmplete protease inhibitors, 0.1 mL). Cells were lysed for 20 min at 4 C on an orbital shaker and centrifuged (1500 g, 20 min, 4 C). The supernatant was transferred to a new plate and ll Article protein concentration was measured by BCA. For enzymatic digest, equal amounts of protein (typically 15 mg) were diluted to 40 mL with Lysis Buffer (PNGase F digest) or PBS (StcE digest), treated with PNGase F (2 U, Promega, Madison, USA) or StcE (50 nM) and incubated for 12 h at 37 C (Malaker et al., 2019). The reaction was quenched by heating to 95 C for 10 s with subsequent cooling at 4 C. Loading buffer (a 1:1:1:0.5 (v/v/v/v) mixture of 1 M Tris-HCl pH 7.0, 10% (w/v) SDS, 80% (v/v) glycerol and 1 M DTT) was added, samples were heated at 95 C for one minute, run on a 10% Criterion TM gel (Bio-Rad, Hercules, USA) for SDS-PAGE, and imaged on an Odyssey CLx (LI-COR Biosciences, Lincoln, USA). Total protein content was assessed by Coomassie staining using Acquastain (Bulldog Bio, Portsmouth, USA). Another aliquot of each sample was used for protein expression control by western blot, using antibodies against VSV-G-tag, GAPDH (ab128915, abcam) or FLAG tag (mouse anti-FLAG M2, Sigma Aldrich) and secondary antibodies IRDyeÒ 800CW Donkey anti-mouse IgG amd IRDyeÒ 680RD Donkey anti-rabbit IgG. Background and contrast for gel and blot images were linearly and uniformly adjusted using either the LI-COR software or Photoshop 2020 (Adobe, San Jose, USA).
For flow cytometry, cells were resuspended in cell buffer (50 mL) and treated with a solution of 50 mM CuSO 4 , 300 mM BTTAA, 2.5 mM sodium ascorbate, 2.5 mM aminoguanidinium chloride and 50 mM MB488 picolyl azide (Click Chemistry Tools) in Cell Buffer (50 mL), and incubated for 5 min at room temperature on an orbital shaker. The reaction was quenched by addition of 3 mM bathocuproinedisulfonic acid in PBS (50 mL). Cells were harvested, washed twice with cell buffer and once with PBS, Cells were then fixed with 0.5% (v/v) paraformaldehyde in PBS (100 mL) for 10 min at room temperature in the dark, harvested and washed with cell buffer once. Cells were permeabilized with 0.5% (v/v) Tween-20 in cell buffer (100 mL) for 10 min at room temperature. Cells were harvested, treated with mouse anti-VSV-G-tag (1:200)  Solvents and reagents were of commercial grade. Anhydrous solvents were obtained from a Dry Solvent System. Moisture-sensitive reactions were carried out in heat-dried glassware and under a nitrogen atmosphere. Thin layer chromatography was performed on Kieselgel 60 F254 glass plates pre-coated with silica gel (0.25 mm thickness). Spots were developed with sugar stain (0.1% (v/v) 3-methoxyphenol, 2.5% (v/v) sulfuric acid in EtOH) or ceric ammonium molybdate stain (5% (w/v) ammonium molybdate, 1% (w/v) cerium(II) sulfate and 10% (v/v) sulfuric acid in water) dipping solutions. Flash chromatography was carried out on Fluka Kieselgel 60 (230-400 mesh). Solvents were removed under reduced pressure using a rotary evaporator and high vacuum (1 mbar). 1 H, 13 C and 2D NMR spectra were measured with a Varian AS400 spectrometer or a Varian AS600 spectrometer at 25 C. Chemical shifts (s) are reported in parts per million (ppm) relative to the respective residual solvent peaks (CDCl 3 : s 7.26 in 1 H and 77.16 in 13 C NMR; acetone-D 6 : s 2.05 in 1 H and 29.84 in 13 C NMR). Two-dimensional NMR experiments (HH-COSY, CH-HSQC) were performed to assign peaks in 1 H spectra. The following abbreviations are used to indicate peak multiplicities: s singlet; d doublet; dd doublet of doublets; dt doublet of triplets; m multiplet. Coupling constants (J) are reported in Hertz (Hz). High resolution mass spectrometry by electrospray ionization (ESI-HRMS) was performed at Stanford University Mass Spectrometry, with a micrOTOF-Q II hybrid quadrupole time-of-flight mass spectrometer (Bruker) equipped with an Agilent 1260 UPLC.

3-Hydroxypropyl (6-azidomethyl)nicotinate (SI-7)
To a stirred solution of ester SI-5 (58 mg, 0.30 mmol) in 5:2 MeOH/water (1.5 mL) was added at room temperature triethylamine (0.3 mL). The reaction was warmed to 50 C and stirred for 20 h at that temperature, when the starting material was converted to a lower-running spot (TLC CH 2 Cl 2 /MeOH 10:1). The solvents were evaporated, and the residue was co-evaporated with toluene (3x5 mL) to give the intermediate triethylammonium salt as a yellow oil.

QUANTIFICATION AND STATISTICAL ANALYSIS
The number of replicates for each experiment is specified in Figure legends. Flow cytometry experiments were run in three independent replicates on three different days. Statistical analysis was performed by two-tailed ratio paired t test, and P values are indicated in figures.
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