Kelley W. Moremen

Heparan sulfate (HS) proteoglycans are essential regulators of cell signaling, development, and extracellular matrix organization. Central to the HS biosynthesis pathway is the exostosin family of enzymes that control commitment to HS assembly and catalyze formation of the polymer backbone in the Golgi apparatus. Recent structural studies have substantially revised our understanding by demonstrating that the HS co-polymerase is a tightly organized EXT1-EXT2 heterodimer, wherein the GT-B domain of EXT1 and the GT-A domain of EXT2 provide the β1,4-GlcA and α1,4-GlcNAc transferase activities, respectively. Parallel work on EXTL3 clarified how selective recognition of the linker-region glycopeptide commits to HS extension, outcompeting the default chondroitin sulfate pathway. In contrast, EXTL2 is best viewed as a regulatory GlcNAc transferase that can cap or divert linker intermediates rather than as a polymerase, whereas EXTL1 remains the least defined family member despite evidence for GlcNAc transferase activity. Genetic studies continue to reveal their importance in human disease, including hereditary multiple exostoses and EXTL3-associated immunodeficiency. Despite this progress, major questions remain regarding chain length control, enzyme coordination, and therapeutic targeting. This short review integrates recent structural, biochemical, and genetic advances to provide an updated narrative of how mammalian EXT proteins govern HS biosynthesis.

Heparan sulfate proteoglycans (HSPGs) are essential cell surface and extracellular matrix glycoconjugates that mediate diverse biological processes through interactions between their heparan sulfate (HS) chains and extracellular ligands. While HS sulfation patterning is known to dictate ligand specificity, how cells control HS assembly to regulate these interactions remains incompletely understood. To systematically identify genetic modifiers of HS-protein interactions, we performed genome-wide CRISPR activation (CRISPRa) screens in HEK293T cells using binding of antithrombin (AT), which selectively recognizes 3- O -sulfated HS motifs, or the N -sulfation-specific antibody 10E4 as functional readouts. Strikingly, the screens revealed proteoglycan core proteins as key modulators of HS function. In particular, syndecan-1 (SDC1) emerged as a preferential enhancer of AT binding compared to other syndecan family members. Targeted upregulation of syndecan family members increased total HS levels, but only SDC1 enhanced AT binding. Structural and enzymatic analyses demonstrated that SDC1-associated HS chains contain elevated 6- O -sulfation and serve as superior substrates for 3- O -sulfotransferases relative to SDC2-associated HS chains. Additionally, SDC1 exhibited slower cell surface recovery, which was blocked by cycloheximide treatment, consistent with extended trafficking and biosynthetic processing. Overall, these findings indicate that proteoglycan core protein identity influences HS sulfation patterning and ligand-binding specificity and trafficking kinetics may contribute to core protein-dependent regulation of HS modification.

Pectins are a major class of plant cell wall polysaccharide crucial for plant survival and adaptation. Despite their importance, the biosynthesis of pectin remains poorly understood due to its exceptional structural and functional diversity. Functional interrogation of pectin biosynthetic enzymes in planta is often limited by severe developmental or difficult to interpret carbohydrate phenotypes resulting from genetic disruption. Using a direct biochemical approach, we report the identification and functional characterization of a Galactan Initiation (GalTi) enzyme from the aquatic monocot Spirodela polyrhiza, a member of CAZy glycosyltransferase family 47, that initiates galactan side‑chain biosynthesis on rhamnogalacturonan‑I. We further show that orthologs from Arabidopsis thaliana exhibit identical catalytic activity, revealing conservation of this biosynthetic initiation mechanism across monocots and dicots. Comprehensive carbohydrate structural analyses demonstrate that GalTi enzymes specifically catalyze the transfer of single β‑D‑galactopyranosyl residues to the O‑4 position of backbone rhamnose residues, establishing their direct role in galactan side‑chain biosynthesis. Co-synthesis experiments show 3-O-acetylation of backbone rhamnose residues inhibits SpGalTi‑mediated galactosylation, suggesting a potential regulatory mechanism by which plants use to modulate side‑chain patterning in vivo. Together, these findings provide a new framework for RG‑I galactan assembly, advancing our mechanistic understanding of plant cell wall biosynthesis.

Chondroitin sulfate (CS) proteoglycans are extended (-GlcAβ1,3GalNAcβ1,4-)_n co-polymers attached to cell surface and extracellular matrix core proteins that are further modified by extensive sulfation and epimerization. Four homologous proteins contribute to CS backbone synthesis (CHPF1, CHPF2, CHSY1, and CHSY3) and prior data suggests assembly of the proteins into heterocomplexes is required for function. Here we show by sequence alignment and structural modeling that all CHSYs and CHPFs contain an N-terminal CAZy GT31-like domain and a C-terminal GT7-like domain separated by a cystatin-like linker domain. Co-expression of one CHPF and one CHSY is required to form a soluble, functional heterodimeric CS synthase and structural modeling indicates all four potential CHSY-CHPF combinations can form equivalent heterodimeric complexes. Cryo-EM studies on CHSY3-CHPF1 confirm the structure, interface, and active site features predicted by the structural models. Enzymatic analyses of catalytic mutants demonstrate that only the glycosyltransferase domains in the CHSYs are responsible for polymer synthesis: the GT31 domain transfers β1,3-GlcA while the GT7 domain transfers β1,4-GalNAc. The corresponding CHPF domains do not contribute to polymer synthesis but stabilize the corresponding CHSY functional domains. Additional mutagenesis and modeling suggest that the bridging cystatin-like domains may contribute to efficient polymer synthesis.

Mucus covers and protects colonic epithelial cells. Mucus is mainly composed of heavily O-glycosylated proteins called mucins, and disruption of normal mucin glycosylation occurs in ulcerative colitis (UC). Mucin-2 (MUC2) is the major colonic mucin, and MUC2 O-glycans are often extended with sulfated polyLacNAc, also known as keratan sulfate (KS). The GlcNAc residues in KS are added by B3GNT family members. B3GNT7 is highly expressed in the colon, and B3GNT7 expression is dramatically reduced in UC. However, the function of B3GNT7 in colonic physiology is unexplored. Here we show that B3gnt7 is a key player in colonic physiology through its function in controlling the structure of mucus glycans. We found that B3GNT7 prefers to extend a sulfated acceptor substrate and is required for production of polyLacNAc-modified mucus in a human goblet cell model. In vivo, B3GNT7 regulates Muc2, Muc13, and Muc17 O-glycosylation. Intestinal B3GNT7 deficiency increases susceptibility to colitis and enteric infection in mice, showing that B3GNT7-dependent glycosylation confers protective properties to colonic mucus. Taken together, these results demonstrate that B3GNT7 has a function distinct from other B3GNT family members and is critical for maintaining colonic homeostasis.

SUMMARY: Glycosyltransferases (GTs) are the primary enzymes responsible for the biosynthesis of the complex polysaccharides in plant cell walls. Given the important role of GTs in plants, it is necessary to undertake their functional characterization to better understand plant cell wall synthesis pathways to develop improved feedstocks for efficient conversion into fuels and products to support the emerging bioeconomy. The GT47 family in plants represents a unique target for characterization due to the substantial diversity of donor and acceptor substrates observed within a single family. Here, we have carried out the biochemical characterization of MUR3 and XLT2 orthologs from the aquatic monocot Spirodela polyrhiza . Our findings support existing genetic and phylogenetic data classifying these enzymes as regio‐specific galactosyltransferases involved in xyloglucan (XyG) sidechain biosynthesis. In addition, we have identified novel characteristics for both enzymes, such as in vitro arabinopyranosyltransferase activity and distinctiveness in xyloglucan reducing end specificity.

Glycosyltransferases (GTs) are the primary enzymes responsible for the biosynthesis of the complex polysaccharides in plant cell walls. Given the important role of GTs in plants, it is necessary to undertake their functional characterization to better understand plant cell wall synthesis pathways to develop improved feedstocks for efficient conversion into fuels and products to support the emerging bioeconomy. The GT47 family in plants represents a unique target for characterization due to the substantial diversity of donor and acceptor substrates observed within a single family. Here, we have carried out the biochemical characterization of MUR3 and XLT2 orthologs from the aquatic monocot Spirodela polyrhiza. Our findings support existing genetic and phylogenetic data classifying these enzymes as regio-specific galactosyltransferases involved in xyloglucan (XyG) sidechain biosynthesis. In addition, we have identified novel characteristics for both enzymes, such as in vitro arabinopyranosyltransferase activity and distinctiveness in xyloglucan reducing end specificity.

Standalone AlphaFold 3 (AF3) models proteins, post-translational modifications, and ligands (including glycans) from a single JSON input file. Plausible glycan stereochemistry is more consistently achieved when monosaccharides are specified as Chemical Component Dictionary (CCD) entries and connected using bondedAtomPairs (BAP) syntax. Although this approach preserves glycan stereochemistry, assembling JSON input files manually is challenging due to the diversity of monosaccharides, linkages, branching, and structural complexity. To simplify this process, we developed JAAG (JSON input file Assembler for AlphaFold 3 with Glycan integration (https://biofgreat.org/JAAG and https://github.com/chinchc/JAAG), a web-based graphical interface that streamlines AF3 JSON file creation, reduces errors, and facilitates modeling of glycans and glycan-macromolecule interactions.

Standalone AlphaFold 3 (AF3) models proteins, post-translational modifications, and ligands (including glycans) from a single JSON input file. Plausible glycan stereochemistry is more consistently achieved when monosaccharides are specified as Chemical Component Dictionary (CCD) entries and connected using bondedAtomPairs (BAP) syntax. Although this approach preserves glycan stereochemistry, assembling JSON input files manually is challenging due to the diversity of monosaccharides, linkages, branching, and structural complexity. To simplify this process, we developed JAAG (JSON input file Assembler for AlphaFold 3 with Glycan integration ( https://biofgreat.org/JAAG and https://github.com/chinchc/JAAG )), a web-based graphical interface that streamlines AF3 JSON file creation, reduces errors, and facilitates modeling of glycans and glycan-macromolecule interactions.

Attachment of human influenza virus to the surface of airway epithelial cells is mediated by the hemagglutinin (HA) that binds to Neu5Acα2-6Gal-terminated glycans referred to as human-type receptors. Numerous reports suggest that current H1N1 and H3N2 influenza viruses that annually infect humans have evolved to preferentially recognize only a subset of human-type receptors. Based on glycomics analysis of nasal and tracheal human epithelial cells, we have chemoenzymatically synthesized a library of N-linked glycans that represent the diversity of structures that are candidate receptors. Using this library, we investigated the specificity of the HAs of H1N1 viruses stemming from two pandemics in 1918 and 2009. We find that H1N1 viruses from both pandemics have maintained a remarkably similar receptor specificity, with a strong preference for α2-6 sialylated bi-antennary N-linked glycans extended with two or more Galβ1-4GlcNAc (LacNAc) repeats on at least one antenna.