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.

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.

Site-to-site cross-talk in OST-B glycosylation of hCEACAM1-IgV.

N-glycosylation is a common posttranslational modification of secreted proteins in eukaryotes. This modification targets asparagine residues within the consensus sequence, N-X-S/T. While this sequence is required for glycosylation, the initial transfer of a high-mannose glycan by oligosaccharyl transferases A or B (OST-A or OST-B) can lead to incomplete occupancy at a given site. Factors that determine the extent of transfer are not well understood, and understanding them may provide insight into the function of these important enzymes. Here, we use mass spectrometry (MS) to simultaneously measure relative occupancies for three N-glycosylation sites on the N-terminal IgV domain of the recombinant glycoprotein, hCEACAM1. We demonstrate that addition is primarily by the OST-B enzyme and propose a kinetic model of OST-B N-glycosylation. Fitting the kinetic model to the MS data yields distinct rates for glycan addition at most sites and suggests a largely stochastic initial order of glycan addition. The model also suggests that glycosylation at one site influences the efficiency of subsequent modifications at the other sites, and glycosylation at the central or N-terminal site leads to dead-end products that seldom lead to full glycosylation of all three sites. Only one path of progressive glycosylation, one initiated by glycosylation at the C-terminal site, can efficiently lead to full occupancy for all three sites. Thus, the hCEACAM1 domain provides an effective model system to study site-specific recognition of glycosylation sequons by OST-B and suggests that the order and efficiency of posttranslational glycosylation is influenced by steric cross-talk between adjoining acceptor sites.

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.

Vertebrate protein glycosylation: diversity, synthesis and function.

Protein glycosylation is a ubiquitous post-translational modification found in all domains of life. Despite their significant complexity in animal systems, glycan structures have crucial biological and physiological roles, from contributions in protein folding and quality control to involvement in a large number of biological recognition events. As a result, they impart an additional level of 'information content' to underlying polypeptide structures. Improvements in analytical methodologies for dissecting glycan structural diversity, along with recent developments in biochemical and genetic approaches for studying glycan biosynthesis and catabolism, have provided a greater understanding of the biological contributions of these complex structures in vertebrates.

Solid-Phase-Supported Chemoenzymatic Synthesis and Analysis of Chondroitin Sulfate Proteoglycan Glycopeptides.

Proteoglycans (PGs), consisting of glycosaminoglycans (GAGs) linked with the core protein through a tetrasaccharide linkage region, play roles in many important biological events. The chemical synthesis of PG glycopeptides is extremely challenging. In this work, the enzymes required for synthesis of chondroitin sulfate (CS) PG (CSPG) have been expressed and the suitable sequence of enzymatic reactions has been established. To expedite CSPG synthesis, the peptide acceptor was immobilized on solid phase and the glycan units were directly installed enzymatically onto the peptide. Subsequent enzymatic chain elongation and sulfation led to the successful synthesis of CSPG glycopeptides. The CS dodecasaccharide glycopeptide was the longest homogeneous CS glycopeptide synthesized to date. The enzymatic synthesis was much more efficient than the chemical synthesis of the corresponding CS glycopeptides, which could reduce the total number of synthetic steps by 80 %. The structures of the CS glycopeptides were confirmed by mass spectrometry analysis and NMR studies. In addition, the interactions between the CS glycopeptides and cathepsin G were studied. The sulfation of glycan chain was found to be important for binding with cathepsin G. This efficient chemoenzymatic strategy opens new avenues to investigate the structures and functions of PGs.

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.

Modularity of the hydrophobic core and evolution of functional diversity in fold A glycosyltransferases.

Hydrophobic cores are fundamental structural properties of proteins typically associated with protein folding and stability; however, how the hydrophobic core shapes protein evolution and function is poorly understood. Here, we investigated the role of conserved hydrophobic cores in fold-A glycosyltransferases (GT-As), a large superfamily of enzymes that catalyze formation of glycosidic linkages between diverse donor and acceptor substrates through distinct catalytic mechanisms (inverting versus retaining). Using hidden Markov models and protein structural alignments, we identify similarities in the phosphate-binding cassette (PBC) of GT-As and unrelated nucleotide-binding proteins, such as UDP-sugar pyrophosphorylases. We demonstrate that GT-As have diverged from other nucleotide-binding proteins through structural elaboration of the PBC and its unique hydrophobic tethering to the F-helix, which harbors the catalytic base (xED-Asp). While the hydrophobic tethering is conserved across diverse GT-A fold enzymes, some families, such as B3GNT2, display variations in tethering interactions and core packing. We evaluated the structural and functional impact of these core variations through experimental mutational analysis and molecular dynamics simulations and find that some of the core mutations (T336I in B3GNT2) increase catalytic efficiency by modulating the conformational occupancy of the catalytic base between "D-in" and acceptor-accessible "D-out" conformation. Taken together, our studies support a model of evolution in which the GT-A core evolved progressively through elaboration upon an ancient PBC found in diverse nucleotide-binding proteins, and malleability of this core provided the structural framework for evolving new catalytic and substrate-binding functions in extant GT-A fold enzymes.

Harnessing galactose oxidase in the development of a chemoenzymatic platform for glycoconjugate vaccine design.

In the preparation of commercial conjugate vaccines, capsular polysaccharides (CPSs) must undergo chemical modification to generate the reactive groups necessary for covalent attachment to a protein carrier. One of the most common approaches employed for this derivatization is sodium periodate (NaIO4) oxidation of vicinal diols found within CPS structures. This procedure is largely random and structurally damaging, potentially resulting in significant changes in the CPS structure and therefore its antigenicity. Additionally, periodate activation of CPS often gives rise to heterogeneous conjugate vaccine products with variable efficacy. Here, we explore the use of an alternative agent, galactose oxidase (GOase) isolated from Fusarium sp. in a chemoenzymatic approach to generate a conjugate vaccine against Streptococcus pneumoniae. Using a colorimetric assay and NMR spectroscopy, we found that GOase generated aldehyde motifs on the CPS of S. pneumoniae serotype 14 (Pn14p) in a site-specific and reversible fashion. Direct comparison of Pn14p derivatized by either GOase or NaIO4 illustrates the functionally deleterious role chemical oxidation can have on CPS structures. Immunization with the conjugate synthesized using GOase provided a markedly improved humoral response over the traditional periodate-oxidized group. Further, functional protection was validated in vitro by measure of opsonophagocytic killing and in vivo through a lethality challenge in mice. Overall, this work introduces a strategy for glycoconjugate development that overcomes limitations previously known to play a role in the current approach of vaccine design.

Fringe GlcNAc-transferases differentially extend O-fucose on endogenous NOTCH1 in mouse activated T cells.

NOTCH1 is a transmembrane receptor that initiates a cell-cell signaling pathway controlling various cell fate specifications in metazoans. The addition of O-fucose by protein O-fucosyltransferase 1 (POFUT1) to epidermal growth factor-like (EGF) repeats in the NOTCH1 extracellular domain is essential for NOTCH1 function, and modification of O-fucose with GlcNAc by the Fringe family of glycosyltransferases modulates Notch activity. Prior cell-based studies showed that POFUT1 modifies EGF repeats containing the appropriate consensus sequence at high stoichiometry, while Fringe GlcNAc-transferases (LFNG, MFNG, and RFNG) modify O-fucose on only a subset of NOTCH1 EGF repeats. Previous in vivo studies showed that each FNG affects naïve T cell development. To examine Fringe modifications of NOTCH1 at a physiological level, we used mass spectral glycoproteomic methods to analyze O-fucose glycans of endogenous NOTCH1 from activated T cells obtained from mice lacking all Fringe enzymes or expressing only a single FNG. While most O-fucose sites were modified at high stoichiometry, only EGF6, EGF16, EGF26, and EGF27 were extended in WT T cells. Additionally, cell-based assays of NOTCH1 lacking fucose at each of those O-fucose sites revealed small but significant effects of LFNG on Notch-Delta binding in the EGF16 and EGF27 mutants. Finally, in activated T cells expressing only LFNG, MFNG, or RFNG alone, the extension of O-fucose with GlcNAc in the same EGF repeats was diminished, consistent with cooperative interactions when all three Fringes were present. The combined data open the door for the analysis of O-glycans on endogenous NOTCH1 derived from different cell types.

Multiple Arabidopsis galacturonosyltransferases synthesize polymeric homogalacturonan by oligosaccharide acceptor-dependent or de novo synthesis.

Homogalacturonan (HG), the most abundant pectic glycan, functions as a cell wall structural and signaling molecule essential for plant growth, development and response to pathogens. HG exists as a component of pectic homoglycans, heteroglycans and glycoconjugates. HG is synthesized by members of the GALACTURONOSYLTRANSFERASE (GAUT) family. UDP-GalA-dependent homogalacturonan:galacturonosyltransferase (HG:GalAT) activity has previously been demonstrated for GAUTs 1, 4 and 11, as well as the GAUT1:GAUT7 complex. Here, we show that GAUTs 10, 13 and 14 are also HG:GalATs and that GAUTs 1, 10, 11, 13, 14 and 1:7 synthesize polymeric HG in vitro. Comparison of the in vitro HG:GalAT specific activities of the heterologously-expressed proteins demonstrates GAUTs 10 and 11 with the lowest, GAUT1 and GAUT13 with moderate, and GAUT14 and the GAUT1:GAUT7 complex with the highest HG:GalAT activity. GAUT13 and GAUT14 are also shown to de novo synthesize (initiate) HG synthesis in the absence of exogenous HG acceptors, an activity previously demonstrated for GAUT1:GAUT7. The rate of de novo HG synthesis by GAUT13 and GAUT14 is similar to their acceptor dependent HG synthesis, in contrast to GAUT1:GAUT7 for which de novo synthesis occurred at much lower rates than acceptor-dependent synthesis. The results suggest a unique role for de novo HG synthesis by GAUTs 13 and 14. The reducing end of GAUT13-de novo-synthesized HG has covalently attached UDP, indicating that UDP-GalA serves as both a donor and acceptor substrate during de novo HG synthesis. The functional significance of unique GAUT HG:GalAT catalytic properties in the synthesis of different pectin glycan or glycoconjugate structures is discussed.

Molecular Mechanism of Polysaccharide Acetylation by the Arabidopsis Xylan O-acetyltransferase XOAT1.

Xylans are a major component of plant cell walls. O-Acetyl moieties are the dominant backbone substituents of glucuronoxylan in dicots and play a major role in the polymer-polymer interactions that are crucial for wall architecture and normal plant development. Here, we describe the biochemical, structural, and mechanistic characterization of Arabidopsis (Arabidopsis thaliana) xylan O-acetyltransferase 1 (XOAT1), a member of the plant-specific Trichome Birefringence Like (TBL) family. Detailed characterization of XOAT1-catalyzed reactions by real-time NMR confirms that it exclusively catalyzes the 2-O-acetylation of xylan, followed by nonenzymatic acetyl migration to the O-3 position, resulting in products that are monoacetylated at both O-2 and O-3 positions. In addition, we report the crystal structure of the catalytic domain of XOAT1, which adopts a unique conformation that bears some similarities to the α/ß/α topology of members of the GDSL-like lipase/acylhydrolase family. Finally, we use a combination of biochemical analyses, mutagenesis, and molecular simulations to show that XOAT1 catalyzes xylan acetylation through formation of an acyl-enzyme intermediate, Ac-Ser-216, by a double displacement bi-bi mechanism involving a Ser-His-Asp catalytic triad and unconventionally uses an Arg residue in the formation of an oxyanion hole.

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.

Influence of Side Chain Conformation on the Activity of Glycosidase Inhibitors.

Substrate side chain conformation impacts reactivity during glycosylation and glycoside hydrolysis and is restricted by many glycosidases and glycosyltransferases during catalysis. We show that the side chains of gluco and manno iminosugars can be restricted to predominant conformations by strategic installation of a methyl group. Glycosidase inhibition studies reveal that iminosugars with the gauche,gauche side chain conformations are 6- to 10-fold more potent than isosteric compounds with the gauche,trans conformation; a manno-configured iminosugar with the gauche,gauche conformation is a 27-fold better inhibitor than 1-deoxymannojirimycin. The results are discussed in terms of the energetic benefits of preorganization, particularly when in synergy with favorable hydrophobic interactions. The demonstration that inhibitor side chain preorganization can favorably impact glycosidase inhibition paves the way for improved inhibitor design through conformational preorganization.

Comparison of human poly-N-acetyl-lactosamine synthase structure with GT-A fold glycosyltransferases supports a modular assembly of catalytic subsites.

Poly-N-acetyl-lactosamine (poly-LacNAc) structures are composed of repeating [-Galß(1,4)-GlcNAcß(1,3)-]n glycan extensions. They are found on both N- and O-glycoproteins and glycolipids and play an important role in development, immune function, and human disease. The majority of mammalian poly-LacNAc is synthesized by the alternating iterative action of ß1,3-N-acetylglucosaminyltransferase 2 (B3GNT2) and ß1,4-galactosyltransferases. B3GNT2 is in the largest mammalian glycosyltransferase family, GT31, but little is known about the structure, substrate recognition, or catalysis by family members. Here we report the structures of human B3GNT2 in complex with UDP:Mg2+ and in complex with both UDP:Mg2+ and a glycan acceptor, lacto-N-neotetraose. The B3GNT2 structure conserves the GT-A fold and the DxD motif that coordinates a Mg2+ ion for binding the UDP-GlcNAc sugar donor. The acceptor complex shows interactions with only the terminal Galß(1,4)-GlcNAcß(1,3)- disaccharide unit, which likely explains the specificity for both N- and O-glycan acceptors. Modeling of the UDP-GlcNAc donor supports a direct displacement inverting catalytic mechanism. Comparative structural analysis indicates that nucleotide sugar donors for GT-A fold glycosyltransferases bind in similar positions and conformations without conserving interacting residues, even for enzymes that use the same donor substrate. In contrast, the B3GNT2 acceptor binding site is consistent with prior models suggesting that the evolution of acceptor specificity involves loops inserted into the stable GT-A fold. These observations support the hypothesis that GT-A fold glycosyltransferases employ coevolving donor, acceptor, and catalytic subsite modules as templates to achieve the complex diversity of glycan linkages in biological systems.

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.

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.

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.

A Clickable Bioorthogonal Sydnone-Aglycone for the Facile Preparation of a Core 1 O-Glycan-Array.

Protein-O-glycosylation has been shown to be essential for many biological processes. However, determining the exact relationship between O-glycan structures and their biological activity remains challenging. Here we report that, unlike azides, sydnones can be incorporated as an aglycon into core 1 O-glycans early-on in their synthesis since it is compatible with carbohydrate chemistry and enzymatic glycosylations, allowing us to generate a small library of sydnone-containing core 1 O-glycans by chemoenzymatic synthesis. The sydnone-aglycon was then employed for the facile preparation of an O-glycan array, via bioorthogonal strain-promoted sydnone-alkyne cycloaddition click reaction, and in turn was utilized for the high-throughput screening of O-glycan-lectin interactions. This sydnone-aglycon, particularly adapted for O-glycomics, is a valuable chemical tool that complements the limited technologies available for investigating O-glycan structure-activity relationships.