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Sequential in vitro enzymatic N-glycoprotein modification reveals site-specific rates of glycoenzyme processing

Open AccessPublished:September 08, 2022DOI:https://doi.org/10.1016/j.jbc.2022.102474
      N-glycosylation is an essential eukaryotic posttranslational modification that affects various glycoprotein properties, including folding, solubility, protein–protein interactions, and half-life. N-glycans are processed in the secretory pathway to form varied ensembles of structures, and diversity at a single site on a glycoprotein is termed ‘microheterogeneity’. To understand the factors that influence glycan microheterogeneity, we hypothesized that local steric and electrostatic factors surrounding each site influence glycan availability for enzymatic modification. We tested this hypothesis via expression of reporter N-linked glycoproteins in N-acetylglucosaminyltransferase MGAT1-null HEK293 cells to produce immature Man5GlcNAc2 glycoforms (38 glycan sites total). These glycoproteins were then sequentially modified in vitro from high mannose to hybrid and on to biantennary, core-fucosylated, complex structures by a panel of N-glycosylation enzymes, and each reaction time course was quantified by LC-MS/MS. Substantial differences in rates of in vitro enzymatic modification were observed between glycan sites on the same protein, and differences in modification rates varied depending on the glycoenzyme being evaluated. In comparison, proteolytic digestion of the reporters prior to N-glycan processing eliminated differences in in vitro enzymatic modification. Furthermore, comparison of in vitro rates of enzymatic modification with the glycan structures found on the mature reporters expressed in WT cells correlated well with the enzymatic bottlenecks observed in vivo. These data suggest higher order local structures surrounding each glycosylation site contribute to the efficiency of modification both in vitro and in vivo to establish the spectrum of microheterogeneity in N-linked glycoproteins.

      Keywords

      Abbreviations:

      CID (collision-induced dissociation), ER (endoplasmic reticulum), MD (molecular dynamics), SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2)
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      There have been several approaches to studying N-glycan microheterogeneity. Early studies demonstrated that N-glycan microheterogeneity is reproducible on a site-by-site basis (
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      ). The involvement of the peptide–glycan interactions in affecting glycan conformation and thus potentially N-glycan processing, has also been supported by molecular dynamics (MD) simulations using yeast protein disulfide isomerase (PDI) as a model reporter glycoprotein (
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      ). A recent study by Mathew et al. studied early N-glycan processing steps through MD simulations and in vitro processing of N-glycans on the yeast protein disulfide isomerase, a similar approach as this study (
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      In this study, we report extensive site-specific in vitro N-glycan processing data for five multiply N-linked glycosylated proteins, with 38 different sites of N-glycosylation in total. By enriching all sites of all glycoproteins with a common Man5GlcNAc2 substrate and then monitoring N-glycan processing through time-course reactions, we were able to identify key bottlenecks that prevent specific sites on glycoproteins from being converted from high mannose to complex N-glycans. These bottlenecks appear to persist in vivo upon microheterogeneity analysis of each site of the reporter proteins when expressed in WT cells. Additionally, we found that removing the tertiary structure of the protein abolished all site specificity of N-glycan processing, highlighting the importance of protein tertiary structure in defining N-glycan microheterogeneity.

      Results

      Expression of reporter proteins in WT-HEK293F cells

      In order to probe individual steps of N-glycan processing, we first established a set of reporter proteins to be used as case studies (Table 1, Fig. S1). These proteins were selected based on their various applications in biology, virology, and use as therapeutics as well as their diversity in displayed glycans and the availability of quality crystal structures. CD16a (Fc ᵧ receptor IIIa) is an IgG receptor that is known to have differential affinities to antibodies depending on its glycan presentation, which impacts downstream signaling (
      • Patel K.R.
      • Roberts J.T.
      • Subedi G.P.
      • Barb A.W.
      Restricted processing of CD16a/Fc γ receptor IIIa N-glycans from primary human NK cells impacts structure and function.
      ,
      • Patel K.R.
      • Nott J.D.
      • Barb A.W.
      Primary human natural killer cells retain proinflammatory IgG1 at the cell surface and express CD16a glycoforms with donor-dependent variability.
      ,
      • Patel K.R.
      • Rodriguez Benavente M.C.
      • Lorenz W.W.
      • Mace E.M.
      • Barb A.W.
      Fc γ receptor IIIa/CD16a processing correlates with the expression of glycan-related genes in human natural killer cells.
      ). PDI is a resident ER glycoprotein that has been used as a model protein for studying N-glycan processing due to its ease of expression, analysis, and well-defined site-specific glycan heterogeneity (
      • Losfeld M.-E.
      • Scibona E.
      • Lin C.-W.
      • Villiger T.K.
      • Gauss R.
      • Morbidelli M.
      • et al.
      Influence of protein/glycan interaction on site-specific glycan heterogeneity.
      ,
      • Arigoni-Affolter I.
      • Scibona E.
      • Lin C.-W.
      • Brühlmann D.
      • Souquet J.
      • Broly H.
      • et al.
      Mechanistic reconstruction of glycoprotein secretion through monitoring of intracellular N-glycan processing.
      ,
      • Hang I.
      • Lin C.
      • Grant O.C.
      • Fleurkens S.
      • Villiger T.K.
      • Soos M.
      • et al.
      Analysis of site-specific N -glycan remodeling in the endoplasmic reticulum and the Golgi.
      ,
      • Weiß R.G.
      • Losfeld M.-E.
      • Aebi M.
      • Riniker S.
      N-glycosylation enhances conformational flexibility of protein disulfide isomerase revealed by microsecond molecular dynamics and Markov state modeling.
      ,
      • Mathew C.
      • Weiß R.G.
      • Giese C.
      • Lin C.
      • Losfeld M.-E.
      • Glockshuber R.
      • et al.
      Glycan–protein interactions determine kinetics of N -glycan remodeling.
      ). Etanercept is a bioengineered therapeutic fusion protein of a TNF⍺ receptor and an IgG1 Fc domain commonly used to help treat autoimmune disorders (
      • Liu L.
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      • et al.
      The impact of glycosylation on the pharmacokinetics of a TNFR2:fc fusion protein expressed in glycoengineered pichia pastoris.
      ). Erythropoietin is a therapeutic glycoprotein that stimulates red blood cell growth, and its glycosylation is known to impact its pharmacokinetics (
      • Čaval T.
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      Direct quality control of glycoengineered erythropoietin variants.
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      Glycoengineering: the effect of glycosylation on the properties of therapeutic proteins.
      ,
      • Banks D.D.
      The effect of glycosylation on the folding kinetics of erythropoietin.
      ). Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein is a highly glycosylated trimer that is responsible for the viral entry of the associated coronavirus SARS-CoV-2 via binding to the human receptor ACE2 (
      • Zhao P.
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      • Xiao T.
      • Rosenbalm K.E.
      • et al.
      Virus-receptor interactions of glycosylated SARS-CoV-2 spike and human ACE2 receptor.
      ,
      • Sztain T.
      • Ahn S.-H.
      • Bogetti A.T.
      • Casalino L.
      • Goldsmith J.A.
      • Seitz E.
      • et al.
      A glycan gate controls opening of the SARS-CoV-2 spike protein.
      ).
      Table 1Reporter proteins used as models for studying N-glycan processing
      Glycoprotein reporters
      ReporterUniprot IDAmino acidsGlycan sitesGlycan structuresPDB
      Etanercept TNFR-IgG Fc fusionP20333 + P018571–235 + 236–4673Varied per site3ALQ 3AVE
      ErythropoietinP0158828–1933Triantennary and tetra-antennary1EER
      SARS-CoV2 SpikeP0DTC21–120822Varied high man to complex6VSB
      CD16a Fc ɣ receptor IIIaP0863719–1925Mostly complex biantennary, triantennary, and tetra-antennary5BW7
      Pdi1p (yeast)P1796729–5225Complex, varied2B5E
      The reporter proteins were first transiently expressed in high yields in WT HEK293F cells and then harvested from supernatant and purified with Ni-NTA chromatography (Fig. S1, BF). Glycopeptide analysis using LC-MS/MS was performed on these purified proteins in order to determine their glycan occupancy and diversity when expressed in a “WT” background (Fig. 1). With LC-MS/MS techniques, we were able to obtain a detailed characterization of the N-glycan profile at each site on the reporter proteins, some of which contained dozens of different glycan moieties with a variety of terminal structures including sialylation, as well as core fucosylation (Fig. 2, AC).
      Figure thumbnail gr1
      Figure 1Graphical representation of approach. Reporter proteins were expressed in HEK293F WT and MGAT1-cells, analyzed via LC-MS/MS, and then processed by purified glycosyltransferases and hydrolases in vitro.
      Figure thumbnail gr2
      Figure 2Microheterogeneity at a single site on CD16a (Site 2, N063). Glycopeptide analysis with individual glycan types quantified via spectral count when expressed in a WT HEK293F cells or Lec1-HEK293F (MGAT1-) cells, divided by glycan class. A, WT-expressed CD16a high-mannose N-glycans. B, WT-expressed CD16a hybrid N-glycans. C, WT-expressed CD16a complex N-glycans. D, Lec1-expressed CD16a N-glycans (all). Colored bars denote glycan terminal features.
      All classes of glycans were observed at most sites (Figs. 3, S5–S42), with SARS-CoV-2 spike glycoprotein pictured separately due to its large number of sites (Fig. S2A). Of particular interest are the sites on reporter glycoproteins that greatly differ from other sites on the same protein: sequons 2 and 4 on CD16a (Fig. 3A) and sequon 4 on PDI (Fig. 3B) are predominantly less processed high mannose and hybrid structures, while the other sites of N-glycosylation on the same proteins are mostly highly processed complex structures. This is in contrast to etanercept (Fig. 3C) and erythropoietin (Fig. 3D), which have more homogenous N-glycan presentations. The SARS-CoV-2 spike glycoprotein had a diversity of N-glycan presentations on its 22 sites, with most sites enriched with complex N-glycans and certain sites mostly presenting high mannose N-glycans (Fig. S2). Interestingly, we noted that Man5GlcNAc2 was always the most abundant high mannose structure on 36 of our 38 sites with two exceptions being N0234 and N0717 of SARS-CoV-2 that both contain less than 15% complex structures (Figs. S2, S27, S35).
      Figure thumbnail gr3
      Figure 3Site occupancy of reporter proteins expressed in WT-HEK293F and Lec1-HEK293F cells. Relative proportion of glycan classes at each site on reporter proteins when expressed in a WT or Lec1 (MGAT1-) background. A, CD16a. B, PDI. C, etanercept. D, erythropoietin. Relative populations were ascertained with glycopeptide analysis and quantified with spectral counts.

      Expression of reporter proteins in Lec1-HEK293F cells

      Next, these same proteins were transiently overexpressed in HEK293S GnTI- (MGAT1 null) cells. The activity of MGAT1 is necessary for the formation of both hybrid and complex N-glycans, as the addition of GlcNAc to the nonreducing end α3-linked mannose is needed for further elaboration and capping by downstream enzymes. KO of MGAT1 substantially reduces the diversity of glycans at all sites of N-glycosylation and causes a significant enrichment of Man5GlcNAc2 structures N-glycans on expressed glycoproteins, as shown on sequon 2 of CD16a (Fig. 2D), as well as the other reporter sites (Figs. S5–S42). This is useful because it allows for the in vitro processing of all N-glycans on a glycoprotein to begin from a common substrate. This enrichment was successful for most sites on all reporter proteins (Figs. 3, S2B). However, there is an exception at Sequon 2 of etanercept, which contained a significant population of apparent hybrid N-glycans by an unknown processing event that we are currently exploring. These appear to be true hybrid structures with an attached GlcNAc on the ⍺-3-linked mannose based on MS2 fragmentation data (Fig. S16B).

      Conversion of high mannose glycans to hybrid glycans

      In order to probe the effects of tertiary structure on N-glycan processing, we first monitored the conversion of Man5GlcNAc2 N-glycans to GlcNAcMan5GlcNAc2 N-glycans via the addition of GlcNAc by the glycosyltransferase MGAT1 on intact reporter proteins expressed and purified from MGAT1-deficient cells. We did this through a series of time-course reactions using purified protein and MGAT1 in the presence of the nucleotide sugar donor UDP-GlcNAc followed by analysis and quantitation via LC-MS/MS (Fig. 4, example TIC can be found in Fig. S43). The ratio of enzymes to molarity of reporter N-glycan sites was kept constant so that we could compare the interprotein as well as intraprotein rates of N-glycan processing. We observed site-specific rates of GlcNAc addition across the range of our 38 sites of N-glycosylation. Generally, these rates corresponded well to the distributions of N-glycans that were found on the respective sites when the reporters were expressed in WT HEK293F cells (Figs. 3, S5–42). For example, sequons 2 and 4 on CD16a are both modified relatively slowly by MGAT1 (Fig. 4A) and are also enriched with high mannose structures when expressed in WT HEK293 cells (Fig. 3A). This also holds for sequon 4 of PDI (Fig. 3B), which is the only site on PDI that is modified slowly by MGAT1 (Fig. 4B). This pattern is also demonstrated on SARS-CoV-2 spike glycoprotein: we observed a broad distribution of MGAT1 activity rates across its 22 sites of N-glycosylation (Figs. 4E, S3), and the fastest and slowest sites are enriched in complex and high mannose N-glycans, respectively (Fig. 4F). This is best represented by sites N0234 and N0717 on Spike glycoprotein, which had slow transfer rates and primarily were occupied by high mannose N-glycans when expressed in a WT background (Figs. 4F, S2).
      Figure thumbnail gr4
      Figure 4Site-specific monitoring of MGAT1 activity. Time-course reaction of GlcNAc addition to reporter proteins with recombinant MGAT1. A, CD16a. B, Protein disulfide isomerase. C, Etanercept. D, Erythropoietin. E, SARS-CoV-2 spike glycoprotein. F, WT glycopeptide profiles for the sites on SARS-CoV-2 spike glycoprotein with the fastest (N1194) and slowest (N0234) rates of MGAT1 activity. Error bars and legend omitted for SARS-CoV-2 spike glycoprotein due to large number of sites. Reaction progress calculated as proportion of the sum of monoisotopic peak heights of product (Man5GlcNAc3) versus the sum of product and reactant (Man5GlcNAc2) peak heights. Experiments performed in triplicate, error bars represent SD. SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.
      In contrast to the demonstration of site specificity for PDI and CD16a, all sites on etanercept have relatively high levels of high mannose glycans (Fig. 3C), and all are processed slowly by MGAT1 (Fig. 4C). Additionally, erythropoietin’s three sites of N-glycosylation are all processed efficiently by MGAT1 (Fig. 4D) and when expressed in WT HEK293 cells mostly produce complex N-glycans (Fig. 3D).

      Conversion of hybrid glycans to complex glycans

      The conversion of hybrid N-glycans such as GlcNAcMan5GlcNAc2 to complex N-glycans requires the activity of two enzymes: the glycoside hydrolase MAN2A1 and the GlcNAc transferase MGAT2. In order to probe the site-specific rates of MAN2A1 activity, we first reacted the reporters expressed in the MGAT1-null cell line with an excess of MGAT1 to enrich GlcNAcMan5GlcNAc2 structures. After ensuring >80% conversion to product at each site, we then examined conversion of the GlcNAcMan5GlcNAc2 product to GlcNAcMan3GlcNAc2 following digestion with MAN2A1 (Fig. 5). Similar patterns of site-specific rates were seen as with MGAT1, with sequons 2 and 4 of CD16a (Fig. 5A) and sequon 4 of PDI (Fig. 5B) all having much lower levels of activity compared to the other sites on the same protein. Again, all sites on etanercept (Fig. 5C) were processed much more slowly than those on erythropoietin (Fig. 5D). Notably, no cleavage products were observed at sequon 5 (N0149) on SARS-CoV-2 spike glycoprotein (Fig. S3). Inspection of the GlcNAcMan4GlcNAc2 intermediate in MAN2A1 processing revealed that at this site, only one mannose was able to be removed (Fig. S4). Despite this in vitro observation, when expressed in a WT background, this site produces an abundance of complex-type N-glycans (Figs. S2, S25). This is in contrast to sites N0234 and N0717 on the spike glycoprotein, which exhibit both slow transfer rates and an enrichment of high mannose N-glycans (Figs. 5F, S2 and S3) but still form the GlcNAcMan3GlcNAc2 product.
      Figure thumbnail gr5
      Figure 5Site-specific monitoring of MAN2A1 activity. Time-course reaction of GlcNAc addition to reporter proteins with recombinant MGAT1. A, CD16a. B, Protein disulfide isomerase. C, etanercept. D, erythropoietin. E, SARS-CoV-2 spike glycoprotein. F, WT glycopeptide profiles for the sites on SARS-CoV-2 spike glycoprotein with the fastest (N0603) and slowest (N0747) rates of MAN2A1 activity for a site that was able to form product. Legend omitted for SARS-CoV-2 spike glycoprotein due to large number of sites. Reaction progress calculated as proportion of the sum of monoisotopic peak heights of product (Man3GlcNAc3) versus the sum of product and reactant (Man5GlcNAc3) peak heights. Experiments performed in triplicate; error bars represent SD. SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.
      The next step in the formation of complex glycans is the addition of a β-2-linked GlcNAc to the ⍺6-mannose of the GlcNAcMan3GlcNAc2 moiety. We reacted the reporter proteins expressed in the MGAT1-null cells with an excess of MGAT1, MAN2A1, and the UDP-GlcNAc donor in order to enrich the GlcNAcMan3GlcNAc2 substrate, then performed another set of time-course reactions with MGAT2. Sites which could not be efficiently converted to GlcNAcMan3GlcNAc2 structures by MGAT1 and MAN2A1 treatment (e.g., sequon 4 on PDI (Fig. 5B)) were excluded from further analyses. Similar patterns of modification were observed in the MGAT2 reactions as were seen in the MGAT1 and MAN2A1 experiments, with lower levels of activity observed at sequons 2 and 4 on CD16a compared to other sites on the same protein (Fig. 6A). Additionally, all sites on etanercept (Fig. 6C) were processed more slowly than those on erythropoietin (Fig. 6D). A broad range of processing rates was observed on SARS-CoV-2 spike glycoprotein (Fig. 6E). Sites that were modified fastest in our in vitro modification studies were also enriched in complex N-glycans when expressed in WT HEK293 cells, and the sites that were the slowest for in vitro modification corresponded to sites that were relatively enriched with high mannose N-glycans when generated in WT cells (Figs. 6F, S3).
      Figure thumbnail gr6
      Figure 6Site-specific monitoring of MGAT2 activity. Time-course reaction of GlcNAc addition to reporter proteins with recombinant MGAT1. A, CD16a. B, protein disulfide isomerase. C, etanercept. D, erythropoietin. E, SARS-CoV-2 spike glycoprotein. F, WT glycopeptide profiles for the sites on SARS-CoV-2 spike glycoprotein with the fastest (N0603) and slowest (N0717) rates of MGAT2 activity. Error bars and legend omitted for SARS-CoV-2 spike glycoprotein due to large number of sites. Asterisks on site legend indicate that not enough substrate was generated from previous N-glycan processing steps to monitor reaction progress. Reaction progress calculated as proportion of the sum of monoisotopic peak heights of product (Man3GlcNAc4) versus the sum of product and reactant (Man3GlcNAc3) peak heights. Experiments performed in triplicate; error bars represent SD. SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

      Core fucosylation of N-glycans by FUT8

      Following the aforementioned experiments, we wanted to see if similar patterns of site-specific N-glycan processing rates would apply to core fucosylation. Core fucosylation is the attachment of an ⍺1,6-linked fucose to the GlcNAc that is directly attached to the asparagine at the core of N-linked glycans, a reaction which is catalyzed by the fucosyltransferase FUT8. This reaction is generally specific to complex N-glycans (
      • García-García A.
      • Serna S.
      • Yang Z.
      • Delso I.
      • Taleb V.
      • Hicks T.
      • et al.
      FUT8-Directed core fucosylation of N-glycans is regulated by the glycan structure and protein environment.
      ), and thus, we generated GlcNAc2Man3GlcNAc2 glycans on our collection of reporter proteins by reacting with an excess of MGAT1, MAN2A1, and MGAT2 in the presence of the UDP-GlcNAc sugar donor. We then examined the rates of modification of the respective glycans with FUT8 (Fig. 7). Interestingly, at many sites we found substantial core fucosylation prior to in vitro processing despite the reporter proteins being expressed in an MGAT1-null cell line and thus lacking complex N-glycans (Fig. 7, A, B, D and E, S5–S42). Otherwise, we observed a diversity of fucosylation rates among the different sites. Sequon 2 on CD16a (Fig. 7A) and sequon 1 and 5 on PDI were markedly slow (Fig. 7B), as well as sequon 3 on etanercept (Fig. 7C). All sites on erythropoietin were fucosylated rapidly, which possibly reflects their high initial levels of fucosylation even before the FUT8 reaction (Fig. 7D). Most sites on SARS-CoV-2 spike glycoprotein were efficiently fucosylated (Fig. 7E), with a few exceptions (N0122, N0801, N1074, and N1098) (Fig. S3). Glycopeptide analysis data reflects that sites modified more rapidly by FUT8 in vitro had higher levels of fucosylation in vivo (Fig. 7F).
      Figure thumbnail gr7
      Figure 7Site-specific monitoring of FUT8 activity. Time-course reaction of fucose addition to reporter proteins with recombinant FUT8. A, CD16a. B, protein disulfide isomerase. C, etanercept. D, erythropoietin. E, SARS-CoV-2 spike glycoprotein. F, WT glycopeptide profiles for the sites on SARS-CoV-2 spike glycoprotein with the fastest (N0657) and slowest (N1098) rates of FUT8 activity. Error bars and legend omitted for SARS-CoV-2 spike glycoprotein due to large number of sites. Asterisks on site legend indicate that not enough substrate was generated from previous N-glycan processing steps to monitor reaction progress. Reaction progress calculated as proportion of the sum of monoisotopic peak heights of product (Man3GlcNAc4) versus the sum of product and reactant (Man3GlcNAc4Fuc1) peak heights. Experiments performed in triplicate; error bars represent SD. SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

      Impact of tertiary structure on site specificity

      In order to determine whether these site-specific differences in glycosyltransferase rates were due to tertiary structure, we repeated the transfer of GlcNAc onto PDI-Man5GlcNAc2 glycans using MGAT1 but first digested the protein with trypsin to cleave the fully folded protein substrate into glycopeptides. Without the reporter protein tertiary structure, all site specificity of GlcNAc transfer rate was lost and the overall rate of transfer was reduced (Fig. 8A compared to Fig. 3B). Since FUT8 activity requires access to the core GlcNAc linked to the Asn residue of the peptide backbone, we were curious to see if the glycoprotein being cleaved to glycopeptides would eliminate the site specificity observed on intact protein. Similar to MGAT1, all site-specific modification by FUT8 was lost following cleavage of CD16a to glycopeptides, with all sites (including the slow site 2 (Fig. 7A)) exhibiting similar rates of modification (Fig. 8B). CD16a was used instead of PDI for the FUT8 experiment to more fully illustrate that local tertiary structure was important for more than just one of our reporter proteins and because site 4 on PDI cannot adequately form complex N-glycans due to low MAN2A1 activity (Fig. 5B).
      Figure thumbnail gr8
      Figure 8Tertiary structure is responsible for site specificity of N-glycan processing rates. Time-course reaction of N-glycan processing on proteins digested into glycopeptides with proteases. A, reaction of MGAT1 with PDI glycopeptides. B, reaction of FUT8 with CD16a glycopeptides. Reaction progress calculated as proportion of the sum of monoisotopic peak heights of product versus the sum of product and reactant peak heights. Experiments performed in triplicate; error bars represent SD.

      Discussion

      While N-glycans are a crucial component in the production of membrane bound and secreted glycoproteins, the determinants that define the diversity of N-glycan structures at any given site are not well understood. Factors that have been suggested to influence N-glycan microheterogeneity include the expression of glycosyltransferases and glycoside hydrolases (
      • Narimatsu Y.
      • Büll C.
      • Chen Y.-H.
      • Wandall H.H.
      • Yang Z.
      • Clausen H.
      Genetic glycoengineering in mammalian cells.
      ,
      • Hirschberg K.
      • Lippincott-Schwartz J.
      Secretory pathway kinetics and in vivo analysis of protein traffic from the Golgi complex to the cell surface.
      ), the availability of nucleotide sugar donors (
      • Burleigh S.C.
      • van de Laar T.
      • Stroop C.J.M.
      • van Grunsven W.M.J.
      • O’Donoghue N.
      • Rudd P.M.
      • et al.
      Synergizing metabolic flux analysis and nucleotide sugar metabolism to understand the control of glycosylation of recombinant protein in CHO cells.
      ), secretory pathway trafficking (
      • Arigoni-Affolter I.
      • Scibona E.
      • Lin C.-W.
      • Brühlmann D.
      • Souquet J.
      • Broly H.
      • et al.
      Mechanistic reconstruction of glycoprotein secretion through monitoring of intracellular N-glycan processing.
      ,
      • Hang I.
      • Lin C.
      • Grant O.C.
      • Fleurkens S.
      • Villiger T.K.
      • Soos M.
      • et al.
      Analysis of site-specific N -glycan remodeling in the endoplasmic reticulum and the Golgi.
      ,
      • Hirschberg K.
      • Lippincott-Schwartz J.
      Secretory pathway kinetics and in vivo analysis of protein traffic from the Golgi complex to the cell surface.
      ), and the accessibility of the acceptor site (
      • Losfeld M.-E.
      • Scibona E.
      • Lin C.-W.
      • Villiger T.K.
      • Gauss R.
      • Morbidelli M.
      • et al.
      Influence of protein/glycan interaction on site-specific glycan heterogeneity.
      ,
      • Mathew C.
      • Weiß R.G.
      • Giese C.
      • Lin C.
      • Losfeld M.-E.
      • Glockshuber R.
      • et al.
      Glycan–protein interactions determine kinetics of N -glycan remodeling.
      ,
      • Yu X.
      • Baruah K.
      • Harvey D.J.
      • Vasiljevic S.
      • Alonzi D.S.
      • Song B.-D.
      • et al.
      Engineering hydrophobic protein-carbohydrate interactions to fine-tune monoclonal antibodies.
      ). The impact of enzyme availability in cells has mostly been probed through genetic engineering approaches (
      • Narimatsu Y.
      • Büll C.
      • Chen Y.-H.
      • Wandall H.H.
      • Yang Z.
      • Clausen H.
      Genetic glycoengineering in mammalian cells.
      ). However, availability of enzymes and sugar nucleotides cannot sufficiently explain site-specific differences on the same polypeptide nor can protein trafficking in the secretory pathway. Thus, we hypothesize that it is the impact of acceptor site accessibility that allows for site-specific differences on the same protein. This could occur by multiple mechanisms including the substrate glycan interacting with the substrate protein backbone at a specific site and thus hampering engagement of the glycoenzyme with the substrate glycan. An example of this is site 4 on PDI that was elegantly demonstrated by Aebi et al. (
      • Losfeld M.-E.
      • Scibona E.
      • Lin C.-W.
      • Villiger T.K.
      • Gauss R.
      • Morbidelli M.
      • et al.
      Influence of protein/glycan interaction on site-specific glycan heterogeneity.
      ). Additionally, the glycosylation on the Fc fragment of etanercept (site 3, N317) differs from what is normally seen on IgG as there is an abundance of high mannose glycans accompanying the expected biantennary complex structures (
      • Cobb B.A.
      The history of IgG glycosylation and where we are now.
      ), which may indicate altered accessibility to the N-glycan at that site in this non-naturally occurring fusion protein. Another possibility is local secondary and tertiary structure of the substrate protein at individual sites of modification and the glycoenzyme active site resulting in steric or electrostatic clashes that prohibit optimal binding for catalysis. Interestingly, our analysis revealed that proteolytically digesting substrate proteins before transfer reactions abolished site-specific rate differences (Fig. 8) and in the case of MGAT1 reduced the overall rate of reaction. This strongly supports higher order structure of the substrate playing an important role in transfer rates and in agreement with proposed mechanisms for microheterogeneity.
      MD studies and Markov state modeling by Mathew et al. demonstrated that the relative amount of time a glycan spends extended away from the protein and exposed to solvent correlates with site specificity of glycan-processing rates on the yeast model protein disulfide isomerase (
      • Mathew C.
      • Weiß R.G.
      • Giese C.
      • Lin C.
      • Losfeld M.-E.
      • Glockshuber R.
      • et al.
      Glycan–protein interactions determine kinetics of N -glycan remodeling.
      ). Additionally, they monitored in vitro N-glycan processing rates with ER mannosidases as well as MGAT1 and MAN2A1, and the results with overlapping enzymes in our present study agree well. Site 4 of PDI was identified as a “slow” site (Figs. 4B and 5B), and their Michaelis–Menten analysis of PDI processing kinetics are also consistent with our observations (Figs. 4B and 5B). Additionally, their studies on the kinetics of earlier glycan-processing steps involving ER mannosidase I and Golgi mannosidase IB show that this site specificity is conserved in earlier steps of N-glycan processing. However, their approach to reduce tertiary structure through reduction and alkylation prior to in vitro modification led to results contrasting with our glycopeptide experiments (Fig. 8), and they observed differences in modification rate at different sites. This may be due to some secondary structures of the protein not being completely disrupted without the use of protease digestion to cleave the model protein utilized or perhaps the denatured protein can still influence site kinetics.
      Generally, these results indicate that the tertiary structure specific to an acceptor site can be an important factor in defining the types of N-glycans seen at a given site. In particular, the efficiency (or lack thereof) of MGAT1 and MAN2A1 appears to be highly predictive of high mannose–type glycans at a sequon. In vivo, it is likely that MGAT1 is rate limiting as the most common high mannose structure at most sites when expressed in a WT background is the MGAT1 substrate Man5GlcNAc2 (Figs. S5–S42), and its activity is required for downstream processing by enzymes like MAN2A1. The rate-limiting role of MAN2A1 was also observed in our in vitro studies, particularly at sites that were also poorly modified with MGAT1. This may be partly due to lower activity of the recombinant enzyme employed in our in vitro studies; four times as much MAN2A1 had to be used in assays compared to the glycosyltransferases (1:250 enzyme:substrate molar ratio for MAN2A1 versus 1:1000 for glycosyltransferases). MAN2A1 processing has previously been identified as a potential bottleneck in N-glycan processing (
      • Mathew C.
      • Weiß R.G.
      • Giese C.
      • Lin C.
      • Losfeld M.-E.
      • Glockshuber R.
      • et al.
      Glycan–protein interactions determine kinetics of N -glycan remodeling.
      ). Potential steric barriers to MAN2A1 action are suggested by the structure of MAN2A1:substrate complex that demonstrates a significant portion of the total N-linked glycan must fit into the active site of the enzyme for efficient binding and catalysis (
      • Rose D.R.
      Structure, mechanism and inhibition of Golgi α-mannosidase II.
      ). If a site is not processed totally by MAN2A1, it may form hybrid structures but cannot form complex structures due to the necessity of mannose trimming on the ⍺6 branch of the trimannosyl N-glycan core. By contrast, the active site structure of MGAT1 involved in acceptor recognition has not yet been determined but likely also presents significant steric barriers for access to some poorly modified sites.
      While these studies provide a sound starting point for determining what structural features may be important in determining N-glycan destiny, much work remains. We purposely chose reporter proteins and processing enzymes with experimentally determined structures (Table 1), (
      • Sztain T.
      • Ahn S.-H.
      • Bogetti A.T.
      • Casalino L.
      • Goldsmith J.A.
      • Seitz E.
      • et al.
      A glycan gate controls opening of the SARS-CoV-2 spike protein.
      ,
      • Mukai Y.
      • Nakamura T.
      • Yoshikawa M.
      • Yoshioka Y.
      • Tsunoda S.
      • Nakagawa S.
      • et al.
      Solution of the structure of the TNF-TNFR2 complex.
      ,
      • Syed R.S.
      • Reid S.W.
      • Li C.
      • Cheetham J.C.
      • Aoki K.H.
      • Liu B.
      • et al.
      Efficiency of signalling through cytokine receptors depends critically on receptor orientation.
      ,
      • Isoda Y.
      • Yagi H.
      • Satoh T.
      • Shibata-Koyama M.
      • Masuda K.
      • Satoh M.
      • et al.
      Importance of the side chain at position 296 of antibody Fc in interactions with FcγRIIIa and other fcγ receptors.
      ,
      • Tian G.
      • Xiang S.
      • Noiva R.
      • Lennarz W.J.
      • Schindelin H.
      The crystal structure of yeast protein disulfide isomerase suggests cooperativity between its active sites.
      ). We are currently utilizing MD simulations of glycosylated reporter proteins and site-specific docking of specific glycan modified reporters with glycoenzymes to determine site-specific glycans interacting with the reporter protein as well as clashes between the reporter sites and the glycoenzymes. This will guide future work involving mutagenesis studies to influence the rate at which glycosyltransferases and glycosyl hydrolases are able to modify acceptor sites. There is evidence that this approach can indeed alter the distribution of N-glycans at a specific site, as evidenced through modification of a tyrosine residue proximal to sequon 4 on protein disulfide isomerase (
      • Losfeld M.-E.
      • Scibona E.
      • Lin C.-W.
      • Villiger T.K.
      • Gauss R.
      • Morbidelli M.
      • et al.
      Influence of protein/glycan interaction on site-specific glycan heterogeneity.
      ). Taking a systematic approach that involves site-specific rate monitoring coupled with modeling and mutagenesis should result in common rules that not only will allow prediction of microheterogeneity but will allow us to tune it.

      Experimental procedures

      Expression and purification of glycoprotein reporters and glycosylation enzymes for in vitro modification

      Expression constructs encoding the reporter proteins were generated with either NH2-terminal fusion tags (CD16a (low affinity immunoglobulin gamma Fc region receptor III-A, FCGR3A), UniProt P08637, residues 19 to 193; Erythropoietin (EPO), UniProt P01588, residues 28 to 193; Etanercept (TNF receptor-IgG1 fusion), GenBank AKX26891, residues 1–467) or C-terminal fusion tags (yeast PDI1 (protein disulfide-isomerase), UniProt P17967, residues 1–494; SARS-CoV-2 Spike glycoprotein, UniProt P0DTC2, residues 1–1208). The constructs employing N-terminal fusion sequences employed the pGEn2 expression vector while the PDI1 construct was generated in the PGEc2 vector as previously described (
      • Moremen K.W.
      • Ramiah A.
      • Stuart M.
      • Steel J.
      • Meng L.
      • Forouhar F.
      • et al.
      Expression system for structural and functional studies of human glycosylation enzymes.
      ). For the pGEn2 constructs, the fusion protein coding region was comprised of a 25 aa signal sequence, an His8 tag, AviTag, the “superfolder” GFP coding region, the 7 aa recognition sequence of the tobacco etch virus (TEV) protease followed by the catalytic domain region for reporter proteins (
      • Moremen K.W.
      • Ramiah A.
      • Stuart M.
      • Steel J.
      • Meng L.
      • Forouhar F.
      • et al.
      Expression system for structural and functional studies of human glycosylation enzymes.
      ). Constructs encoding MGAT1, MAN2A1, MGAT2, and FUT8 employed the pGEn2 vector and were expressed and purified as previously described (
      • Moremen K.W.
      • Ramiah A.
      • Stuart M.
      • Steel J.
      • Meng L.
      • Forouhar F.
      • et al.
      Expression system for structural and functional studies of human glycosylation enzymes.
      ). For the PDI1 construct, the pGEc2 vector was employed and encoded the segment of Saccromyces cerevisiae PDI1 indicated, followed by an SGSG tetrapeptide, the 7 aa TEV recognition sequence, the “superfolder” GFP coding region, and an His8 tag (
      • Moremen K.W.
      • Ramiah A.
      • Stuart M.
      • Steel J.
      • Meng L.
      • Forouhar F.
      • et al.
      Expression system for structural and functional studies of human glycosylation enzymes.
      ). For SARS-CoV-2 Spike, the construct contained an additional COOH-terminal trimerization sequence and His6 tag as previously described (
      • Xiao T.
      • Lu J.
      • Zhang J.
      • Johnson R.I.
      • McKay L.G.A.
      • Storm N.
      • et al.
      A trimeric human angiotensin-converting enzyme 2 as an anti-SARS-CoV-2 agent.
      ). The recombinant reporter proteins were expressed as a soluble secreted proteins by transient transfection of suspension culture HEK293F cells (FreeStyle 293-F cells, Thermo Fisher Scientific) for WT glycosylated structures and in HEK293S (GnTI-) cells (ATCC) to generate Man5GlcNAc2-Asn glycan structures (
      • Moremen K.W.
      • Ramiah A.
      • Stuart M.
      • Steel J.
      • Meng L.
      • Forouhar F.
      • et al.
      Expression system for structural and functional studies of human glycosylation enzymes.
      ,
      • Meng L.
      • Forouhar F.
      • Thieker D.
      • Gao Z.
      • Ramiah A.
      • Moniz H.
      • et al.
      Enzymatic basis for N-glycan sialylation: Structure of rat α2,6-sialyltransferase (ST6GAL1) reveals conserved and unique features for glycan sialylation.
      ). Cultures were maintained at 0.5 to 3.0 × 106 cells/ml in a humidified CO2 platform shaker incubator at 37 °C with 50% humidity. Transient transfection was performed using expression medium comprised of a 9:1 ratio of Freestyle293 expression medium (Thermo Fisher Scientific) and EX-Cell expression medium including Glutmax (Sigma–Aldrich). Transfection was initiated by the addition of plasmid DNA and PEI as transfection reagent (linear 25 kDa PEI, Polysciences, Inc). Twenty-four hours post-transfection, the cell cultures were diluted with an equal volume of fresh media supplemented with valproic acid (2.2 mM final concentration) and protein production was continued for an additional 5 days at 37 °C (3). The cell cultures were harvested, clarified by sequential centrifugation at 1200 rpm for 10 min and 3500 rpm for 15 min at 4 °C, and passed through a 0.8 μM filter (Millipore). The protein preparation was adjusted to contain 20 mM Hepes, 20 mM imidazole, 300 mM NaCl, pH 7.5, and subjected to Ni-NTA Superflow (Qiagen) chromatography using a column preequilibrated with 20 mM Hepes, 300 mM NaCl, 20 mM imidazole, pH 7.5 (Buffer I). Following loading of the sample, the column was washed with 3 column volumes of Buffer I followed by 3 column volumes of Buffer I containing 50 mM imidazole and eluted with Buffer I containing 300 mM imidazole at pH 7.0. The protein was concentrated to approximately 3 mg/ml using an ultrafiltration pressure cell (Millipore) with a 10 kDa molecular mass cutoff membrane and buffer exchanged with 20 mM Hepes, 100 mM NaCl, pH 7.0, 0.05% sodium azide, and 10% glycerol.

      In vitro N-Glycan processing

      For the time-course reactions, purified reporter proteins generated in HEK293S (GnTI-) cells were used. Reactions were performed at 37 °C in 1.5 ml Eppendorf tubes in a reaction volume of 150 μl, with 20 mM Hepes (VWR) pH 7.5, and 300 mM NaCl (Fisher). For glycosyltransferases, the corresponding nucleotide sugar, UDP-GlcNAc (Sigma) for MGAT1 and MGAT2 and GDP-Fucose (CarboSynth) for FUT8 was kept in excess at 1 mM. MGAT1 and MGAT2 reactions were supplemented with 1 mM MnCl2 (Sigma). The concentration of total N-glycans for each reaction was kept at 5 μM; for example, for a reporter protein with 5 sites of N-glycosylation, the concentration of the protein would be 1.25 μM. For MGAT1, MGAT2, and FUT8 reactions, a 1:1000 enzyme-to-glycan ratio was used with the concentration of respective enzyme at 5 nM; for MAN2A1 reactions, a 1:250 enzyme-to-glycan ratio was used with the concentration of MAN2A1 at 20 nM. Prior to adding enzymes, time-course reaction vessels were equilibrated at 37 °C for 15 min. At each time point, 20 μl of samples were taken and reactions were deactivated by heating at 95 °C for 5 min. The samples were then digested by proteases and processed for LC-MS/MS analysis. In order to prepare substrate reporter proteins for N-glycan processing steps downstream of MGAT1 (e.g., MAN2A1, MGAT2, FUT8), reporters were reacted with the appropriate combination of enzymes and sugar nucleotides at a 1:100 enzyme:glycan ratio for 3 h. Reporters were confirmed to have been >80% converted to desired product by LC-MS/MS, detailed later.

      Enzymatic digestion of PDI1, etanercept, EPO, CD16a, and SARS-CoV-2 spike from WT and HEK293S (GnTI-) cells

      All proteins were reduced by incubating with 10 mM of DTT (Sigma) at 56 °C and alkylated by 27.5 mM of iodoacetamide (Sigma) at room temperature in dark. For the intact glycopeptide analysis, aliquots of PDI1 proteins were digested respectively using trypsin (Promega), a combination of trypsin and Glu-C (Promega), or a combination of trypsin and AspN (Promega); aliquots of etanercept proteins were digested respectively using trypsin (Promega) or AspN (Promega); aliquots of EPO proteins were digested respectively using a combination of trypsin and Glu-C (Promega) or Glu-C (Promega); aliquots of CD16a proteins were digested respectively using chymotrypsin (Athens Research and Technology), AspN (Promega), or a combination of chymotrypsin (Athens Research and Technology) and Glu-C (Promega); aliquots of S proteins were digested respectively using alpha lytic protease (New England BioLabs), chymotrypsin (Athens Research and Technology), a combination of trypsin and Glu-C (Promega), or a combination of Glu-C and AspN (Promega). For the analysis of deglycosylated glycopeptides, aliquots of PDI1 proteins were digested respectively using trypsin (Promega) or a combination of trypsin and Glu-C (Promega); aliquots of etanercept proteins were digested respectively using trypsin (Promega) or AspN (Promega); aliquots of EPO proteins were digested respectively using a combination of trypsin and Glu-C (Promega) or Glu-C (Promega); aliquots of CD16a proteins were digested respectively using chymotrypsin (Athens Research and Technology) or AspN (Promega); aliquots of S proteins were digested respectively using chymotrypsin (Athens Research and Technology), a combination of trypsin and Glu-C (Promega), or AspN (Promega). Following digestion, the proteins were deglycosylated by Endo-H (Promega) followed by PNGaseF (Promega) treatment in the presence of 18O water (Cambridge Isotope Laboratories).

      LC-MS/MS analysis of glycopeptides of PDI1, etanercept, EPO, CD16a, and SARS-CoV-2 spike from WT and HEK293S (GnTI-) cells

      The resulting peptides from respective enzymatic digestion of each protein were separated on an Acclaim PepMap RSLC C18 column (75 μm × 15 cm) and eluted into the nanoelectrospray ion source of an Orbitrap Fusion Lumos Tribrid or an Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher Scientific) at a flow rate of 200 nl/min. The elution gradient for PDI1, etanercept, EPO, and CD16a proteins consists of 1% to 40% acetonitrile in 0.1% formic acid over 220 min followed by 10 min of 80% acetonitrile in 0.1% formic acid. The elution gradient for S protein consists of 1% to 40% acetonitrile in 0.1% formic acid over 370 min followed by 10 min of 80% acetonitrile in 0.1% formic acid. The spray voltage was set to 2.2 kV and the temperature of the heated capillary was set to 275 °C. For the intact glycopeptide analysis, full MS scans were acquired from m/z 200 to 2000 at 60k resolution, and MS/MS scans following higher energy collisional dissociation with stepped collision energy (15%, 25%, 35%) were collected in the orbitrap at 15k resolution. For the deglycosylated glycopeptide analysis, full MS scans were acquired from m/z 200 to 2000 at 60k resolution, and MS/MS scans following collision-induced dissociation (CID) at 38% collision energy were collected in the ion trap.
      For time-course reactions, a shorter LC gradient was used, and digests of the same reporters were combined prior to analysis for higher throughput. The elution gradient used for PDI, etanercept, EPO, and CD16a proteins was 1% to 80% acetonitrile in 0.1% formic acid over 60 min followed by 5 min of 80% acetonitrile in 0.1% formic acid. The peptides were eluted into the source of an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific). The spray voltage was set to 2.25 kV and the temperature of the heated capillary was set to 280 °C. Full MS scans were acquired from m/z 300 to 2000 at 60k resolution, and MS/MS scans following CID at 38% collision energy were collected in the ion trap. The elution gradient used for SARS-CoV-2 spike glycoprotein was 1% to 80% acetonitrile in 0.1% formic acid over 300 min followed by 10 min of 80% acetonitrile in 0.1% formic acid. The peptides were eluted into the source of an Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher Scientific). The spray voltage was set to 2.25 kV and the temperature of the heated capillary was set to 275 °C. Full MS scans were acquired from m/z 300 to 1900 at 60k resolution, and MS/MS scans following CID at 38% collision energy were collected in the ion trap.

      MS data analysis

      For the intact glycopeptide analysis, the raw spectra were analyzed using pGlyco3 (
      • Zeng W.-F.
      • Cao W.-Q.
      • Liu M.-Q.
      • He S.-M.
      • Yang P.-Y.
      Precise, fast and comprehensive analysis of intact glycopeptides and modified glycans with pGlyco3.
      ) for database searches with mass tolerance set as 20 ppm for both precursors and fragments. The database search output was filtered to reach a 1% false discovery rate for glycans and 10% for peptides. The filtered result was further validated by manual examination of the raw spectra. For isobaric glycan compositions, fragments in the MS/MS spectra were evaluated to provide the most probable topologies. Quantitation was performed by calculating spectral counts for each glycan composition at each site. Any N-linked glycan compositions identified by only one spectra were removed from the quantitation. For the deglycosylated glycopeptide analysis, the spectra were analyzed using SEQUEST (Proteome Discoverer 1.4 and 2.5, Thermo Fisher Scientific) with mass tolerance set as 20 ppm for precursors and 0.5 Da for fragments. The search output from Proteome Discoverer 1.4 was filtered using ProteoIQ (v2.7, Premier Biosoft) to reach a 1% false discovery rate at protein level and 10% at peptide level. The search output from Proteome Discoverer 2.5 was filtered within the program to reach a 1% false discovery rate at protein level and 10% at peptide level. Occupancy of each N-linked glycosylation site was calculated using spectral counts assigned to the 18O-Asp-containing (PNGaseF-cleaved) and/or HexNAc-modified (EndoH-cleaved) peptides and their unmodified counterparts.
      For time-course reactions, quantitation was performed through manual inspection of MS1 spectra using Thermo Freestyle 1.7 (Thermo Fischer Scientific). The intensities of monoisotopic peak heights for all observable charge states for reactants and products were determined and then summed and averaged in triplicate to determine percent conversion to product over time. Plots generated using RStudio (1.4.1717).

      Data availability

      All data generated or analyzed during this study are included in this article and supporting information files. The glycopeptide analysis MS data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD032149. MS data for time-course reactions available upon request.

      Supporting information

      Conflict of interest

      The authors declare that they have no conflicts of interest with the contents of this article.

      Acknowledgments

      We thank Dr Henrik Clausen for providing cell lines for this project.

      Author contributions

      T. M. A., K. W. M., and L. W. conceptualization; T. M. A. and P. Z. methodology; T. M. A. and P. Z. validation; T. M. A. and P. Z. formal analysis; T. M. A., D. C., and P. Z. investigation; D. C. resources; P. Z. data curation; T. M. A. writing–original draft; P. Z., D. C., K. W. M., and L. W. writing–review & editing; T. M. A., D. C., and P. Z. visualization; L. W. supervision; L. W. project administration; K. W. M. and L. W. funding acquisition.

      Funding and additional information

      This work was supported in part by National Institutes of Health Grant 5R01GM130915 from NIGMS (to K. W. M. and L. W.) and the Glycoscience Training Program ( 5T32GM107004 ) from NIGMS. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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