Integrated chemoenzymatic synthesis of a comprehensive sulfated ganglioside glycan library to decipher functional sulfoglycomics and sialoglycomics.

Ganglioside glycans are ubiquitous and complex biomolecules that are involved in a wide range of biological functions and disease processes. Variations in sialylation and sulfation render the structural complexity and diversity of ganglioside glycans, and influence protein-carbohydrate interactions. Structural and functional insights into the biological roles of these glycans are impeded due to the limited accessibility of well-defined structures. Here we report an integrated chemoenzymatic strategy for expeditious and systematic synthesis of a comprehensive 65-membered ganglioside glycan library covering all possible patterns of sulfation and sialylation. This strategy relies on the streamlined modular assembly of three common sialylated precursors by highly stereoselective iterative sialylation, modular site-specific sulfation through flexible orthogonal protecting-group manipulations and enzymatic-catalysed diversification using three sialyltransferase modules and a galactosidase module. These diverse ganglioside glycans enable exploration into their structure-function relationships using high-throughput glycan microarray technology, which reveals that different patterns of sulfation and sialylation on these glycans mediate their unique binding specificities.

A Biomimetic Synthetic Strategy Can Provide Keratan Sulfate I and II Oligosaccharides with Diverse Fucosylation and Sulfation Patterns.

Keratan sulfate (KS) is a proteoglycan that is widely expressed in the extracellular matrix of various tissue types, where it performs multiple biological functions. KS is the least understood proteoglycan, which in part is due to a lack of panels of well-defined KS oligosaccharides that are needed for structure-binding studies, as analytical standards, to examine substrate specificities of keratinases, and for drug development. Here, we report a biomimetic approach that makes it possible to install, in a regioselective manner, sulfates and fucosides on oligo-N-acetyllactosamine (LacNAc) chains to provide any structural element of KS by using specific enzyme modules. It is based on the observation that α1,3-fucosides, α2,6-sialosides and C-6 sulfation of galactose (Gal6S) are mutually exclusive and cannot occur on the same LacNAc moiety. As a result, the pattern of sulfation on galactosides can be controlled by installing α1,3-fucosides or α2,6-sialosides to temporarily block certain LacNAc moieties from sulfation by keratan sulfate galactose 6-sulfotransferase (CHST1). The patterns of α1,3-fucosylation and α2,6-sialylation can be controlled by exploiting the mutual exclusivity of these modifications, which in turn controls the sites of sulfation by CHST1. Late-stage treatment with a fucosidase or sialidase to remove blocking fucosides or sialosides provides selectively sulfated KS oligosaccharides. These treatments also unmasked specific galactosides for further modification by CHST1. To showcase the potential of the enzymatic strategy, we have prepared a range of poly-LacNAc derivatives having different patterns of fucosylation and sulfation and several N-glycans decorated by specific arrangements of sulfates.

Glycosyltransferase 8 domain-containing protein 1 (GLT8D1) is a UDP-dependent galactosyltransferase.

Glycosyltransferases (GTs) are enzymes that catalyze the formation of glycosidic bonds and hundreds of GTs have been identified so far in humans. Glycosyltransferase 8 domain-containing protein 1 (GLT8D1) has been associated with central nervous system diseases and cancer. However, evidence on its enzymatic properties, including its substrates, has been scarcely described. In this paper, we have produced and purified recombinant secretory GLT8D1. The enzyme was found to be N-glycosylated. Differential scanning fluorimetry was employed to analyze the stabilization of GLT8D1 by Mn2+ and nucleotides, revealing UDP as the most stabilizing nucleotide scaffold. GLT8D1 displayed glycosyltransferase activity from UDP-galactose onto N-acetylgalactosamine but with a low efficiency. Modeling of the structure revealed similarities with other GT-A fold enzymes in CAZy family GT8 and glycosyltransferases in other families with galactosyl-, glucosyl-, and xylosyltransferase activities, each with retaining catalytic mechanisms. Our study provides novel structural and functional insights into the properties of GLT8D1 with implications in pathological processes.

Exploiting Substrate Specificities of 6-O-Sulfotransferases to Enzymatically Synthesize Keratan Sulfate Oligosaccharides.

Keratan sulfate (KS) is a glycosaminoglycan that is widely expressed in the extracellular matrix of various tissue types, where it is involved in many biological processes. Herein, we describe a chemo-enzymatic approach to preparing well-defined KS oligosaccharides by exploiting the known and newly discovered substrate specificities of relevant sulfotransferases. The premise of the approach is that recombinant GlcNAc-6-O-sulfotransferases (CHST2) only sulfate terminal GlcNAc moieties to give GlcNAc6S that can be galactosylated by B4GalT4. Furthermore, CHST1 can modify the internal galactosides of a poly-LacNAc chain; however, it was found that a GlcNAc6S residue greatly increases the reactivity of CHST1 of a neighboring and internal galactoside. The presence of a 2,3-linked sialoside further modulates the site of modification by CHST1, and a galactoside flanked by 2,3-Neu5Ac and GlcNAc6S is preferentially sulfated over the other Gal residues. The substrate specificities of CHST1 and 2 were exploited to prepare a panel of KS oligosaccharides, including selectively sulfated N-glycans. The compounds and several other reference derivatives were used to construct a microarray that was probed for binding by several plant lectins, Siglec proteins, and hemagglutinins of influenza viruses. It was found that not only the sulfation pattern but also the presentation of epitopes as part of an O- or N-glycan determines binding properties.

The Plasminogen-Apple-Nematode (PAN) domain suppresses JA/ET defense pathways in plants.

Suppression of immune response is a phenomenon that enables biological processes such as gamete fertilization, cell growth, cell proliferation, endophyte recruitment, parasitism, and pathogenesis. Here, we show for the first time that the Plasminogen-Apple-Nematode (PAN) domain present in G-type lectin receptor-like kinases is essential for immunosuppression in plants. Defense pathways involving jasmonic acid and ethylene are critical for plant immunity against microbes, necrotrophic pathogens, parasites, and insects. Using two Salix purpurea G-type lectin receptor kinases, we demonstrated that intact PAN domains suppress jasmonic acid and ethylene signaling in Arabidopsis and tobacco. Variants of the same receptors with mutated residues in this domain could trigger induction of both defense pathways. Assessment of signaling processes revealed significant differences between receptors with intact and mutated PAN domain in MAPK phosphorylation, global transcriptional reprogramming, induction of downstream signaling components, hormone biosynthesis and resistance to Botrytis cinerea . Further, we demonstrated that the domain is required for oligomerization, ubiquitination, and proteolytic degradation of these receptors. These processes were completely disrupted when conserved residues in the domain were mutated. Additionally, we have tested the hypothesis in recently characterized Arabidopsis mutant which has predicted PAN domain and negatively regulates plant immunity against root nematodes. ern1.1 mutant complemented with mutated PAN shows triggered immune response with elevated WRKY33 expression, hyperphosphorylation of MAPK and resistant to necrotrophic fungus Botrytis cinerea . Collectively, our results suggest that ubiquitination and proteolytic degradation mediated by the PAN domain plays a role in receptor turn-over to suppress jasmonic acid and ethylene defense signaling in plants.

Structural basis for Lewis antigen synthesis by the α1,3-fucosyltransferase FUT9.

Mammalian cell surface and secreted glycoproteins exhibit remarkable glycan structural diversity that contributes to numerous physiological and pathogenic interactions. Terminal glycan structures include Lewis antigens synthesized by a collection of α1,3/4-fucosyltransferases (CAZy GT10 family). At present, the only available crystallographic structure of a GT10 member is that of the Helicobacter pylori α1,3-fucosyltransferase, but mammalian GT10 fucosyltransferases are distinct in sequence and substrate specificity compared with the bacterial enzyme. Here, we determined crystal structures of human FUT9, an α1,3-fucosyltransferase that generates Lewisx and Lewisy antigens, in complex with GDP, acceptor glycans, and as a FUT9-donor analog-acceptor Michaelis complex. The structures reveal substrate specificity determinants and allow prediction of a catalytic model supported by kinetic analyses of numerous active site mutants. Comparisons with other GT10 fucosyltransferases and GT-B fold glycosyltransferases provide evidence for modular evolution of donor- and acceptor-binding sites and specificity for Lewis antigen synthesis among mammalian GT10 fucosyltransferases.

Structural and biochemical insight into a modular ß-1,4-galactan synthase in plants.

Rhamnogalacturonan I (RGI) is a structurally complex pectic polysaccharide with a backbone of alternating rhamnose and galacturonic acid residues substituted with arabinan and galactan side chains. Galactan synthase 1 (GalS1) transfers galactose and arabinose to either extend or cap the ß-1,4-galactan side chains of RGI, respectively. Here we report the structure of GalS1 from Populus trichocarpa, showing a modular protein consisting of an N-terminal domain that represents the founding member of a new family of carbohydrate-binding module, CBM95, and a C-terminal glycosyltransferase family 92 (GT92) catalytic domain that adopts a GT-A fold. GalS1 exists as a dimer in vitro, with stem domains interacting across the chains in a 'handshake' orientation that is essential for maintaining stability and activity. In addition to understanding the enzymatic mechanism of GalS1, we gained insight into the donor and acceptor substrate binding sites using deep evolutionary analysis, molecular simulations and biochemical studies. Combining all the results, a mechanism for GalS1 catalysis and a new model for pectic galactan side-chain addition are proposed.

Structural basis for heparan sulfate co-polymerase action by the EXT1-2 complex.

Heparan sulfate (HS) proteoglycans are extended (-GlcAß1,4GlcNAcα1,4-)n co-polymers containing decorations of sulfation and epimerization that are linked to cell surface and extracellular matrix proteins. In mammals, HS repeat units are extended by an obligate heterocomplex of two exostosin family members, EXT1 and EXT2, where each protein monomer contains distinct GT47 (GT-B fold) and GT64 (GT-A fold) glycosyltransferase domains. In this study, we generated human EXT1-EXT2 (EXT1-2) as a functional heterocomplex and determined its structure in the presence of bound donor and acceptor substrates. Structural data and enzyme activity of catalytic site mutants demonstrate that only two of the four glycosyltransferase domains are major contributors to co-polymer syntheses: the EXT1 GT-B fold ß1,4GlcA transferase domain and the EXT2 GT-A fold α1,4GlcNAc transferase domain. The two catalytic sites are over 90 Å apart, indicating that HS is synthesized by a dissociative process that involves a single catalytic site on each monomer.

Chemoenzymatic Tagging of Tn/TF/STF Antigens in Living Systems.

Truncated mucin-type O-glycans, such as Tn-associated antigens, are aberrantly expressed biomarkers of cancer, but remain challenging to target. Reactive antibodies to these antigens either lack high-affinity or are prone to antigen escape. Here, we have developed a robust chemoenzymatic strategy for the global labeling of Tn-associated antigens, i.e. Tn (GalNAcα-O-Ser/Thr), Thomsen-Friedenreich (Galß1-3GalNAcα-O-Ser/Thr, TF) and STF (Neu5Acα2-3Galß1-3GalNAcα-O-Ser/Thr, STF) antigens, in human whole blood with high efficiency and selectivity. This method relies on the use of the O-glycan sialyltransferase ST6GalNAc1 to transfer a sialic acid-functionalized adaptor to the GalNAc residue of these antigens. By tagging, the adaptor functionalized antigens can be easily targeted by customized strategies such as, but not limited to, chimeric antigen receptor T-Cells (CAR-T). We expect this tagging system to find broad applications in cancer diagnostics and targeting in combination with established strategies.

A universal glycoenzyme biosynthesis pipeline that enables efficient cell-free remodeling of glycans.

The ability to reconstitute natural glycosylation pathways or prototype entirely new ones from scratch is hampered by the limited availability of functional glycoenzymes, many of which are membrane proteins that fail to express in heterologous hosts. Here, we describe a strategy for topologically converting membrane-bound glycosyltransferases (GTs) into water soluble biocatalysts, which are expressed at high levels in the cytoplasm of living cells with retention of biological activity. We demonstrate the universality of the approach through facile production of 98 difficult-to-express GTs, predominantly of human origin, across several commonly used expression platforms. Using a subset of these water-soluble enzymes, we perform structural remodeling of both free and protein-linked glycans including those found on the monoclonal antibody therapeutic trastuzumab. Overall, our strategy for rationally redesigning GTs provides an effective and versatile biosynthetic route to large quantities of diverse, enzymatically active GTs, which should find use in structure-function studies as well as in biochemical and biomedical applications involving complex glycomolecules.

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

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.

Site-selective sulfation of N-glycans by human GlcNAc-6-O-sulfotransferase 1 (CHST2) and chemoenzymatic synthesis of sulfated antibody glycoforms.

Sulfation is a common modification of glycans and glycoproteins. Sulfated N-glycans have been identified in various glycoproteins and implicated for biological functions, but in vitro synthesis of structurally well-defined full length sulfated N-glycans remains to be described. We report here the first in vitro enzymatic sulfation of biantennary complex type N-glycans by recombinant human CHST2 (GlcNAc-6-O-sulfotransferase 1, GlcNAc6ST-1). We found that the sulfotransferase showed high antennary preference and could selectively sulfate the GlcNAc moiety located on the Manα1,3Man arm of the biantennary N-glycan. The glycan chain was further elongated by bacterial ß1,4 galactosyltransferase from Neiserria meningitidis and human ß1,4 galactosyltransferase IV（B4GALT4）, which led to the formation of different sulfated N-glycans. Using rituximab as a model IgG antibody, we further demonstrated that the sulfated N-glycans could be efficiently transferred to an intact antibody by using a chemoenzymatic Fc glycan remodeling method, providing homogeneous sulfated glycoforms of antibodies. Preliminary binding analysis indicated that sulfation did not affect the apparent affinity of the antibody for FcγIIIa receptor.

Extracellular sialyltransferase st6gal1 in breast tumor cell growth and invasiveness.

The sialyltransferase ST6GAL1 that adds α2-6 linked sialic acids to N-glycans of cell surface and secreted glycoproteins is prominently associated with many human cancers. Tumor-native ST6GAL1 promotes tumor cell behaviors such as invasion and resistance to cell stress and chemo- and radio-treatments. Canonically, ST6GAL1 resides in the intracellular secretory apparatus and glycosylates nascent glycoproteins in biosynthetic transit. However, ST6GAL1 is also released into the extracellular milieu and extracellularly remodels cell surface and secreted glycans. The impact of this non-canonical extrinsic mechanism of ST6GAL1 on tumor cell pathobiology is not known. We hypothesize that ST6GAL1 action is the combined effect of natively expressed sialyltransferase acting cell-autonomously within the ER-Golgi complex and sialyltransferase from extracellular origins acting extrinsically to remodel cell-surface glycans. We found that shRNA knockdown of intrinsic ST6GAL1 expression resulted in decreased ST6GAL1 cargo in the exosome-like vesicles as well as decreased breast tumor cell growth and invasive behavior in 3D in vitro cultures. Extracellular ST6GAL1, present in cancer exosomes or the freely soluble recombinant sialyltransferase, compensates for insufficient intrinsic ST6GAL1 by boosting cancer cell proliferation and increasing invasiveness. Moreover, we present evidence supporting the existence novel but yet uncharacterized cofactors in the exosome-like particles that potently amplify extrinsic ST6GAL1 action, highlighting a previously unknown mechanism linking this enzyme and cancer pathobiology. Our data indicate that extracellular ST6GAL1 from remote sources can compensate for cellular ST6GAL1-mediated aggressive tumor cell proliferation and invasive behavior and has great clinical potential for extracellular ST6GAL1 as these molecules are in the extracellular space should be easily accessible targets.

Extracellular ST6GAL1 regulates monocyte-macrophage development and survival.

Interaction of immune cells with the systemic environment is necessary for the coordinated development and execution of immune responses. Monocyte-macrophage lineage cells reside at the junction of innate and adaptive immunity. Previously we reported that the sialyltransferase ST6GAL1 in the extracellular milieu modulates B cell development and IgG production, granulocyte production, and attenuates acute airway inflammation to bacterial challenge in mouse models. Here, we report that extracellular ST6GAL1 also elicits profound responses in monocyte-macrophage lineage cells. We show that recombinant ST6GAL1 adheres to subsets of thioglycolate-elicited inflammatory cells in the mouse peritoneum and to cultured human monocyte THP-1 cells. Exposure of the inflammatory cells to recombinant ST6GAL1 elicited wholesale changes in the gene expression profile of primary mouse myeloid cells; most notable was the striking up-regulation of monocyte-macrophage and monocyte-derived dendritic cell development pathway signature genes and transcription factors PU.1, NFκB and their target genes, driving increased monocyte-macrophage population and survival ex vivo. In the cultured human monocyte cells, the essential cell surface receptor of the monocyte-macrophage lineage, the M-CSF receptor (M-CSF-R, Csfr1) was a target of extracellular ST6GAL1 catalytic activity. Extracellular ST6GAL1 activated the M-CSF-R and initiated intracellular signaling events, namely, the nuclear translocation of NFκB subunit p65, and phosphorylation of ERK 1/2 and AKT. The findings implicate extracellular ST6GAL1 in monocyte development by a mechanism initiated at the cell surface and support an emerging paradigm of an extracellular glycan-modifying enzyme as a central regulator coordinating immune hematopoietic cell development and function.

Integrated Chemoenzymatic Approach to Streamline the Assembly of Complex Glycopeptides in the Liquid Phase.

Glycosylation of proteins is a complicated post-translational modification. Despite the significant progress in glycoproteomics, accurate functions of glycoproteins are still ambiguous owing to the difficulty in obtaining homogeneous glycopeptides or glycoproteins. Here, we describe a streamlined chemoenzymatic method to prepare complex glycopeptides by integrating hydrophobic tag-supported chemical synthesis and enzymatic glycosylations. The hydrophobic tag is utilized to facilitate peptide chain elongation in the liquid phase and expeditious product separation. After removal of the tag, a series of glycans are installed on the peptides via efficient glycosyltransferase-catalyzed reactions. The general applicability and robustness of this approach are exemplified by efficient preparation of 16 well-defined SARS-CoV-2 O-glycopeptides, 4 complex MUC1 glycopeptides, and a 31-mer glycosylated glucagon-like peptide-1. Our developed approach will open up a new range of easy access to various complex glycopeptides of biological importance.

Human Intelectin-1 Promotes Cellular Attachment and Neutrophil Killing of Streptococcus pneumoniae in a Serotype-Dependent Manner.

Human intelectin-1 (hIntL-1) is a secreted glycoprotein capable of binding exocyclic 1,2-diols within surface glycans of human pathogens such as Streptococcus pneumoniae, Vibrio cholerae, and Helicobacter pylori. For the latter, lectin binding was shown to cause bacterial agglutination and increased phagocytosis, suggesting a role for hIntL-1 in pathogen surveillance. In this study, we investigated the interactions between hIntL-1 and S. pneumoniae, the leading cause of bacterial pneumonia. We show that hIntL-1 also agglutinates S. pneumoniae serotype 43, which displays an exocyclic 1,2-diol moiety in its capsular polysaccharide but is unable to kill in a complement-dependent manner or to promote bacterial killing by peripheral blood mononuclear cells. In contrast, hIntL-1 not only significantly increases serotype-specific S. pneumoniae killing by neutrophils but also enhances the attachment of these bacteria to A549 lung epithelial cells. Taken together, our results suggest that hIntL-1 participates in host surveillance through microbe sequestration and enhanced targeting to neutrophils.

Elucidating Human Milk Oligosaccharide biosynthetic genes through network-based multi-omics integration.

Human Milk Oligosaccharides (HMOs) are abundant carbohydrates fundamental to infant health and development. Although these oligosaccharides were discovered more than half a century ago, their biosynthesis in the mammary gland remains largely uncharacterized. Here, we use a systems biology framework that integrates glycan and RNA expression data to construct an HMO biosynthetic network and predict glycosyltransferases involved. To accomplish this, we construct models describing the most likely pathways for the synthesis of the oligosaccharides accounting for >95% of the HMO content in human milk. Through our models, we propose candidate genes for elongation, branching, fucosylation, and sialylation of HMOs. Our model aggregation approach recovers 2 of 2 previously known gene-enzyme relations and 2 of 3 empirically confirmed gene-enzyme relations. The top genes we propose for the remaining 5 linkage reactions are consistent with previously published literature. These results provide the molecular basis of HMO biosynthesis necessary to guide progress in HMO research and application with the goal of understanding and improving infant health and development.

Modulation of Siglec-7 Signaling Via In Situ-Created High-Affinity cis-Ligands.

Sialic acid-binding immunoglobulin-like lectins, also known as Siglecs, have recently been designated as glyco-immune checkpoints. Through their interactions with sialylated glycan ligands overexpressed on tumor cells, inhibitory Siglecs on innate and adaptive immune cells modulate signaling cascades to restrain anti-tumor immune responses. However, the elucidation of the mechanisms underlying these processes is just beginning. We find that when human natural killer (NK) cells attack tumor cells, glycan remodeling occurs on the target cells at the immunological synapse. This remodeling occurs through both the transfer of sialylated glycans from NK cells to target tumor cells and the accumulation of de novo synthesized sialosides on the tumor cells. The functionalization of NK cells with a high-affinity ligand of Siglec-7 leads to multifaceted consequences in modulating a Siglec-7-regulated NK-activation. At high levels of ligand, an enzymatically added Siglec-7 ligand suppresses NK cytotoxicity through the recruitment of Siglec-7 to an immune synapse, whereas at low levels of ligand an enzymatically added Siglec-7 ligand triggers the release of Siglec-7 from the cell surface into the culture medium, preventing a Siglec-7-mediated inhibition of NK cytotoxicity. These results suggest that a glycan engineering of NK cells may provide a means to boost NK effector functions for related applications.

Glycan remodeled erythrocytes facilitate antigenic characterization of recent A/H3N2 influenza viruses.

During circulation in humans and natural selection to escape antibody recognition for decades, A/H3N2 influenza viruses emerged with altered receptor specificities. These viruses lost the ability to agglutinate erythrocytes critical for antigenic characterization and give low yields and acquire adaptive mutations when cultured in eggs and cells, contributing to recent vaccine challenges. Examination of receptor specificities of A/H3N2 viruses reveals that recent viruses compensated for decreased binding of the prototypic human receptor by recognizing α2,6-sialosides on extended LacNAc moieties. Erythrocyte glycomics shows an absence of extended glycans providing a rationale for lack of agglutination by recent A/H3N2 viruses. A glycan remodeling approach installing functional receptors on erythrocytes, allows antigenic characterization of recent A/H3N2 viruses confirming the cocirculation of antigenically different viruses in humans. Computational analysis of HAs in complex with sialosides having extended LacNAc moieties reveals that mutations distal to the RBD reoriented the Y159 side chain resulting in an extended receptor binding site.

Variable posttranslational modifications of severe acute respiratory syndrome coronavirus 2 nucleocapsid protein.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), started in 2019 in China and quickly spread into a global pandemic. Nucleocapsid protein (N protein) is highly conserved and is the most abundant protein in coronaviruses and is thus a potential target for both vaccine and point-of-care diagnostics. N Protein has been suggested in the literature as having posttranslational modifications (PTMs), and accurately defining these PTMs is critical for its potential use in medicine. Reports of phosphorylation of N protein have failed to provide detailed site-specific information. We have performed comprehensive glycomics, glycoproteomics and proteomics experiments on two different N protein preparations. Both were expressed in HEK293 cells; one was in-house expressed and purified without a signal peptide (SP) sequence, and the other was commercially produced with a SP channeling it through the secretory pathway. Our results show completely different PTMs on the two N protein preparations. The commercial product contained extensive N- and O-linked glycosylation as well as O-phosphorylation on site Thr393. Conversely, the native N Protein model had O-phosphorylation at Ser176 and no glycosylation, highlighting the importance of knowing the provenance of any commercial protein to be used for scientific or clinical studies. Recent studies have indicated that N protein can serve as an important diagnostic marker for COVID-19 and as a major immunogen by priming protective immune responses. Thus, detailed structural characterization of N protein may provide useful insights for understanding the roles of PTMs on viral pathogenesis, vaccine design and development of point-of-care diagnostics.