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An automated platform that can synthesize a wide range of complex carbohydrates will greatly increase their accessibility and should facilitate progress in glycoscience. Here we report a fully automated process for enzyme-mediated oligosaccharide synthesis that can give easy access to different classes of complex glycans including poly- N -acetyllactosamine derivatives, human milk oligosaccharides, gangliosides and N -glycans. Our automated platform uses a catch and release approach in which glycosyltransferase-catalysed reactions are performed in solution and product purification is accomplished by solid phase extraction. We developed a sulfonate tag that can easily be installed and enables highly efficient solid phase extraction and product release using a single set of washing conditions, regardless of the complexity of the glycan. Using this custom-built synthesizer, as many as 15 reaction cycles can be performed in an automated fashion without a need for lyophilization or buffer exchange steps. An automated platform that can synthesize a wide range of complex glycans could greatly facilitate progress in glycoscience. Now, a fully automated process for enzyme-mediated oligosaccharide synthesis has been developed. This process uses glycosyltransferase-catalysed reactions performed in solution, with product purification being accomplished by solid phase extraction using a sulfonate tag.

Glycosyltransferases (GTs) play fundamental roles in nearly all cellular processes through the biosynthesis of complex carbohydrates and glycosylation of diverse protein and small molecule substrates. The extensive structural and functional diversification of GTs presents a major challenge in mapping the relationships connecting sequence, structure, fold and function using traditional bioinformatics approaches. Here, we present a convolutional neural network with attention (CNN-attention) based deep learning model that leverages simple secondary structure representations generated from primary sequences to provide GT fold prediction with high accuracy. The model learns distinguishing secondary structure features free of primary sequence alignment constraints and is highly interpretable. It delineates sequence and structural features characteristic of individual fold types, while classifying them into distinct clusters that group evolutionarily divergent families based on shared secondary structural features. We further extend our model to classify GT families of unknown folds and variants of known folds. By identifying families that are likely to adopt novel folds such as GT91, GT96 and GT97, our studies expand the GT fold landscape and prioritize targets for future structural studies. Glycosyltransferases (GT) are proteins that display extensive sequence and functional variation on a subset of 3D folds. Here, the authors use interpretable deep learning to predict 3D folds from sequence without the need for sequence alignment, which also enables the prediction of GTs with new folds.

Glycan microarrays provide a high-throughput means of profiling the interactions of glycan-binding proteins with their ligands. However, the construction of current glycan microarray platforms is time consuming and expensive. Here, we report a fast and cost-effective method for the assembly of cell-based glycan arrays to probe glycan–glycan-binding protein interactions directly on the cell surface. Chinese hamster ovary cell mutants with a narrow and relatively homogeneous repertoire of glycoforms serve as the foundation platforms to develop these arrays. Using recombinant glycosyltransferases, sialic acid, fucose, and analogs thereof are installed on cell-surface glycans to form cell-based arrays displaying diverse glycan epitopes that can be probed with glycan-binding proteins by flow cytometry. Using this platform, high-affinity glycan ligands are discovered for Siglec-15—a sialic acid-binding lectin involved in osteoclast differentiation. Incubating human osteoprogenitor cells with cells displaying a high-affinity Siglec-15 ligand impairs osteoclast differentiation, demonstrating the utility of this cell-based glycan array technology. Glycans, interaction platforms protruding from the surface of cells, are hard to study due to their diverse architecture. Here, the authors present a method to obtain cells carrying defined glycans, which can then be used to find proteins specifically recognizing these tags.

Heparan sulfate (HS) proteoglycans are extended (-GlcAβ1,4GlcNAcα1,4-) 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 β1,4GlcA 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. Heparan sulfate proteoglycans are extended disaccharide co-polymers containing decorations of sulfation and epimerization linked to cell surface and extracellular matrix proteins that contribute to cell signaling and tissue homeostasis. This structural and mutagenic study on the EXT1–2 complex explains molecular details of the backbone co-polymer synthesis.

Despite mammalian glycans typically having highly complex asymmetrical multiantennary architectures, chemical and chemoenzymatic synthesis has almost exclusively focused on the preparation of simpler symmetrical structures. This deficiency hampers investigations into the biology of glycan-binding proteins, which in turn complicates the biomedical use of this class of biomolecules. Herein, we describe an enzymatic strategy, using a limited number of human glycosyltransferases, to access a collection of 60 asymmetric, multiantennary human milk oligosaccharides (HMOs), which were used to develop a glycan microarray. Probing the array with several glycan-binding proteins uncovered that not only terminal glycoepitopes but also complex architectures of glycans can influence binding selectivity in unanticipated manners. N- and O-linked glycans express structural elements of HMOs, and thus, the reported synthetic principles will find broad applicability.

Contemporary chemoenzymatic approaches can provide highly complex multi-antennary N -linked glycans. These procedures are, however, very demanding and typically involve as many as 100 chemical steps to prepare advanced intermediates that can be diversified by glycosyltransferases in a branch-selective manner to give asymmetrical structures commonly found in nature. Only highly specialized laboratories can perform such syntheses, which greatly hampers progress in glycoscience. Here we describe a biomimetic approach in which a readily available bi-antennary glycopeptide can be converted in ten or fewer chemical and enzymatic steps into multi-antennary N -glycans that at each arm can be uniquely extended by glycosyltransferases to give access to highly complex asymmetrically branched N -glycans. A key feature of our approach is the installation of additional branching points using recombinant MGAT4 and MGAT5 in combination with unnatural sugar donors. At an appropriate point in the enzymatic synthesis, the unnatural monosaccharides can be converted into their natural counterpart, allowing each arm to be elaborated into a unique appendage. Preparation of well-defined N -glycans is very demanding, which hampers progress in glycoscience. Now, a biomimetic synthetic approach has been developed in which a readily available bi-antennary glycan can be converted in ten or fewer steps into multi-antennary N -glycans. This approach enables each arm to be uniquely extended by glycosyltransferases to give complex branched N -glycans.

Vertebrate glycoproteins and glycolipids are synthesized in complex biosynthetic pathways localized predominantly within membrane compartments of the secretory pathway. The enzymes that catalyze these reactions are exquisitely specific, yet few have been extensively characterized because of challenges associated with their recombinant expression as functional products. We used a modular approach to create an expression vector library encoding all known human glycosyltransferases, glycoside hydrolases, and sulfotransferases, as well as other glycan-modifying enzymes. We then expressed the enzymes as secreted catalytic domain fusion proteins in mammalian and insect cell hosts, purified and characterized a subset of the enzymes, and determined the structure of one enzyme, the sialyltransferase ST6GalNAcII. Many enzymes were produced at high yields and at similar levels in both hosts, but individual protein expression levels varied widely. This expression vector library will be a transformative resource for recombinant enzyme production, broadly enabling structure-function studies and expanding applications of these enzymes in glycochemistry and glycobiology.

Pectin is a vital component of the plant cell wall and provides the molecular glue that maintains cell-cell adhesion, among other functions. As the most complex wall polysaccharide, pectin is composed of several covalently linked domains, such as homogalacturonan (HG) and rhamnogalacturonan I (RG I). Pectin has widespread uses in the food industry and has emerging biomedical applications, but its synthesis remains poorly understood. For instance, the enzymes that catalyze RG I elongation remain unknown. Recently, a coexpression- and sequence-based MUCILAGE-RELATED (MUCI) reverse genetic screen uncovered hemicellulose biosynthetic enzymes in the Arabidopsis (Arabidopsis thaliana) seed coat. Here, we use an extension of this strategy to identify MUCI70 as the founding member of a glycosyltransferase family essential for the accumulation of seed mucilage, a gelatinous wall rich in unbranched RG I. Detailed biochemical and histological characterization of two muci70 mutants and two galacturonosyltransferase11 (gaut11) mutants identified MUCI70 and GAUT11 as required for two distinct RG I domains in seed mucilage. We demonstrate that, unlike MUCI70, GAUT11 catalyzes HG elongation in vitro and, thus, likely is required for the synthesis of an HG region important for RG I elongation. Analysis of a muci70 gaut11 double mutant confirmed that MUCI70 and GAUT11 are indispensable for the production and release of the bulk of mucilage RG I and for shaping the surface morphology of seeds. In addition, we uncover relationships between pectin and hemicelluloses and show that xylan is essential for the elongation of at least one RG I domain.

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. Access to glycoenzymes for basic and applied research is limited by difficulties with their recombinant expression. Here, the authors describe a universal strategy for converting membrane-bound glycosyltransferases into water-soluble biocatalysts, which are expressed at high levels with retention of activity.

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. Human milk oligosaccharides are fundamental to infant health. Here the authors deploy a multi-omics systems biology approach to elucidate their biosynthetic network, including the associated enzymes and likely structures of ambiguous oligosaccharides.