Reading and Writing the Human Glycocode.

The complex carbohydrate structures decorating human proteins and lipids, also called glycans, are abundantly present at cell surfaces and in the secretome. Glycosylation is vital for biological processes including cell-cell recognition, immune responses, and signaling pathways. Therefore, the structural and functional characterization of the human glycome is gaining more and more interest in basic biochemistry research and in the context of developing new therapies, diagnostic tools, and biotechnology applications. For glycomics to reach its full potential in these fields, it is critical to appreciate the specific factors defining the function of the human glycome. Here, we review the glycosyltransferases (the writers) that form the glycome and the glycan-binding proteins (the readers) with an essential role in decoding glycan functions. While abundantly present throughout different cells and tissues, the function of specific glycosylation features is highly dependent on their context. In this review, we highlight the relevance of studying the glycome in the context of specific carrier proteins, cell types, and subcellular locations. With this, we hope to contribute to a richer understanding of the glycome and a more systematic approach to identifying the roles of glycosylation in human physiology.

Mucins and the Microbiome.

Generating the barriers that protect our inner surfaces from bacteria and other challenges requires large glycoproteins called mucins. These come in two types, gel-forming and transmembrane, all characterized by large, highly O-glycosylated mucin domains that are diversely decorated by Golgi glycosyltransferases to become extended rodlike structures. The general functions of mucins on internal epithelial surfaces are to wash away microorganisms and, even more importantly, to build protective barriers. The latter function is most evident in the large intestine, where the inner mucus layer separates the numerous commensal bacteria from the epithelial cells. The host's conversion of MUC2 to the outer mucus layer allows bacteria to degrade the mucin glycans and recover the energy content that is then shared with the host. The molecular nature of the mucins is complex, and how they construct the extracellular complex glycocalyx and mucus is poorly understood and a future biochemical challenge.

Biosynthesis and Export of Bacterial Glycolipids.

Complex carbohydrates are essential for many biological processes, from protein quality control to cell recognition, energy storage, and cell wall formation. Many of these processes are performed in topologically extracellular compartments or on the cell surface; hence, diverse secretion systems evolved to transport the hydrophilic molecules to their sites of action. Polyprenyl lipids serve as ubiquitous anchors and facilitators of these transport processes. Here, we summarize and compare bacterial biosynthesis pathways relying on the recognition and transport of lipid-linked complex carbohydrates. In particular, we compare transporters implicated in O antigen and capsular polysaccharide biosyntheses with those facilitating teichoic acid and N-linked glycan transport. Further, we discuss recent insights into the generation, recognition, and recycling of polyprenyl lipids.

Cryo-EM Structures and Regulation of Arabinofuranosyltransferase AftD from Mycobacteria.

Mycobacterium tuberculosis causes tuberculosis, a disease that kills over 1 million people each year. Its cell envelope is a common antibiotic target and has a unique structure due, in part, to two lipidated polysaccharides-arabinogalactan and lipoarabinomannan. Arabinofuranosyltransferase D (AftD) is an essential enzyme involved in assembling these glycolipids. We present the 2.9-Å resolution structure of M. abscessus AftD, determined by single-particle cryo-electron microscopy. AftD has a conserved GT-C glycosyltransferase fold and three carbohydrate-binding modules. Glycan array analysis shows that AftD binds complex arabinose glycans. Additionally, AftD is non-covalently complexed with an acyl carrier protein (ACP). 3.4- and 3.5-Å structures of a mutant with impaired ACP binding reveal a conformational change, suggesting that ACP may regulate AftD function. Mutagenesis experiments using a conditional knockout constructed in M. smegmatis confirm the essentiality of the putative active site and the ACP binding for AftD function.

Stereoisomer-specific reprogramming of a bacterial flagellin sialyltransferase.

Glycosylation of surface structures diversifies cells chemically and physically. Nucleotide-activated sialic acids commonly serve as glycosyl donors, particularly pseudaminic acid (Pse) and its stereoisomer legionaminic acid (Leg), which decorate eubacterial and archaeal surface layers or protein appendages. FlmG, a recently identified protein sialyltransferase, O-glycosylates flagellins, the subunits of the flagellar filament. We show that flagellin glycosylation and motility in Caulobacter crescentus and Brevundimonas subvibrioides is conferred by functionally insulated Pse and Leg biosynthesis pathways, respectively, and by specialized FlmG orthologs. We established a genetic glyco-profiling platform for the classification of Pse or Leg biosynthesis pathways, discovered a signature determinant of eubacterial and archaeal Leg biosynthesis, and validated it by reconstitution experiments in a heterologous host. Finally, by rewiring FlmG glycosylation using chimeras, we defined two modular determinants that govern flagellin glycosyltransferase specificity: a glycosyltransferase domain that either donates Leg or Pse and a specialized flagellin-binding domain that identifies the acceptor.

An Atlas of Human Glycosylation Pathways Enables Display of the Human Glycome by Gene Engineered Cells.

The structural diversity of glycans on cells-the glycome-is vast and complex to decipher. Glycan arrays display oligosaccharides and are used to report glycan hapten binding epitopes. Glycan arrays are limited resources and present saccharides without the context of other glycans and glycoconjugates. We used maps of glycosylation pathways to generate a library of isogenic HEK293 cells with combinatorially engineered glycosylation capacities designed to display and dissect the genetic, biosynthetic, and structural basis for glycan binding in a natural context. The cell-based glycan array is self-renewable and reports glycosyltransferase genes required (or blocking) for interactions through logical sequential biosynthetic steps, which is predictive of structural glycan features involved and provides instructions for synthesis, recombinant production, and genetic dissection strategies. Broad utility of the cell-based glycan array is demonstrated, and we uncover higher order binding of microbial adhesins to clustered patches of O-glycans organized by their presentation on proteins.

UDP-glycosyltransferases act as key determinants of host plant range in generalist and specialist Spodoptera species.

Phytophagous insects have evolved sophisticated detoxification systems to overcome the antiherbivore chemical defenses produced by many plants. However, how these biotransformation systems differ in generalist and specialist insect species and their role in determining insect host plant range remains an open question. Here, we show that UDP-glucosyltransferases (UGTs) play a key role in determining the host range of insect species within the Spodoptera genus. Comparative genomic analyses of Spodoptera species that differ in host plant breadth identified a relatively conserved number of UGT genes in generalist species but high levels of UGT gene pseudogenization in the specialist Spodoptera picta. CRISPR-Cas9 knockouts of the three main UGT gene clusters of Spodoptera frugiperda revealed that UGT33 genes play an important role in allowing this species to utilize the poaceous plants maize, wheat, and rice, while UGT40 genes facilitate utilization of cotton. Further functional analyses in vivo and in vitro identified the UGT SfUGT33F32 as the key mechanism that allows generalist S. frugiperda to detoxify the benzoxazinoid DIMBOA (2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one), a potent insecticidal phytotoxin produced by poaceous plants. However, while this detoxification capacity is conserved in several generalist Spodoptera species, Spodoptera picta, which specializes on Crinum plants, is unable to detoxify DIMBOA due to a nonfunctionalizing mutation in SpUGT33F34. Collectively, these findings provide insight into the role of insect UGTs in host plant adaptation, the mechanistic basis of evolutionary transitions between generalism and specialism and offer molecular targets for controlling a group of notorious insect pests.

Three-component systems represent a common pathway for extracytoplasmic addition of pentofuranose sugars into bacterial glycans.

Cell surface glycans are major drivers of antigenic diversity in bacteria. The biochemistry and molecular biology underpinning their synthesis are important in understanding host-pathogen interactions and for vaccine development with emerging chemoenzymatic and glycoengineering approaches. Structural diversity in glycostructures arises from the action of glycosyltransferases (GTs) that use an immense catalog of activated sugar donors to build the repeating unit and modifying enzymes that add further heterogeneity. Classical Leloir GTs incorporate α- or ß-linked sugars by inverting or retaining mechanisms, depending on the nucleotide sugar donor. In contrast, the mechanism of known ribofuranosyltransferases is confined to ß-linkages, so the existence of α-linked ribofuranose in some glycans dictates an alternative strategy. Here, we use Citrobacter youngae O1 and O2 lipopolysaccharide O antigens as prototypes to describe a widespread, versatile pathway for incorporating side-chain α-linked pentofuranoses by extracytoplasmic postpolymerization glycosylation. The pathway requires a polyprenyl phosphoribose synthase to generate a lipid-linked donor, a MATE-family flippase to transport the donor to the periplasm, and a GT-C type GT (founding the GT136 family) that performs the final glycosylation reaction. The characterized system shares similarities, but also fundamental differences, with both cell wall arabinan biosynthesis in mycobacteria, and periplasmic glucosylation of O antigens first discovered in Salmonella and Shigella. The participation of auxiliary epimerases allows the diversification of incorporated pentofuranoses. The results offer insight into a broad concept in microbial glycobiology and provide prototype systems and bioinformatic guides that facilitate discovery of further examples from diverse species, some in currently unknown glycans.

The exostosin glycosyltransferase 1/STAT3 axis is a driver of breast cancer aggressiveness.

The epithelial-mesenchymal transition (EMT) program is crucial for transforming carcinoma cells into a partially mesenchymal state, enhancing their chemoresistance, migration, and metastasis. This shift in cell state is tightly regulated by cellular mechanisms that are not yet fully characterized. One intriguing EMT aspect is the rewiring of the proteoglycan landscape, particularly the induction of heparan sulfate proteoglycan (HSPG) biosynthesis. This proteoglycan functions as a co-receptor that accelerates cancer-associated signaling pathways through its negatively-charged residues. However, the precise mechanisms through which EMT governs HSPG biosynthesis and its role in cancer cell plasticity remain elusive. Here, we identified exostosin glycosyltransferase 1 (EXT1), a central enzyme in HSPG biosynthesis, to be selectively upregulated in aggressive tumor subtypes and cancer cell lines, and to function as a key player in breast cancer aggressiveness. Notably, ectopic expression of EXT1 in epithelial cells is sufficient to induce HSPG levels and the expression of known mesenchymal markers, subsequently enhancing EMT features, including cell migration, invasion, and tumor formation. Additionally, EXT1 loss in MDA-MB-231 cells inhibits their aggressiveness-associated traits such as migration, chemoresistance, tumor formation, and metastasis. Our findings reveal that EXT1, through its role in HSPG biosynthesis, governs signal transducer and activator of transcription 3 (STAT3) signaling, a known regulator of cancer cell aggressiveness. Collectively, we present the EXT1/HSPG/STAT3 axis as a central regulator of cancer cell plasticity that directly links proteoglycan synthesis to oncogenic signaling pathways.

Generation of a bloodstream form Trypanosoma brucei double glycosyltransferase null mutant competent in receptor-mediated endocytosis of transferrin.

The bloodstream form of Trypanosoma brucei expresses large poly-N-acetyllactosamine (pNAL) chains on complex N-glycans of a subset of glycoproteins. It has been hypothesised that pNAL may be required for receptor-mediated endocytosis. African trypanosomes contain a unique family of glycosyltransferases, the GT67 family. Two of these, TbGT10 and TbGT8, have been shown to be involved in pNAL biosynthesis in bloodstream form Trypanosoma brucei, raising the possibility that deleting both enzymes simultaneously might abolish pNAL biosynthesis and provide clues to pNAL function and/or essentiality. In this paper, we describe the creation of a TbGT10 null mutant containing a single TbGT8 allele that can be excised upon the addition of rapamycin and, from that, a TbGT10 and TbGT8 double null mutant. These mutants were analysed by lectin blotting, glycopeptide methylation linkage analysis and flow cytometry. The data show that the mutants are defective, but not abrogated, in pNAL synthesis, suggesting that other GT67 family members can compensate to some degree for loss of TbGT10 and TbGT8. Despite there being residual pNAL synthesis in these mutants, certain glycoproteins appear to be particularly affected. These include the lysosomal CBP1B serine carboxypeptidase, cell surface ESAG2 and the ESAG6 subunit of the essential parasite transferrin receptor (TfR). The pNAL deficient TfR in the mutants continued to function normally with respect to protein stability, transferrin binding, receptor mediated endocytosis of transferrin and subcellular localisation. Further the pNAL deficient mutants were as viable as wild type parasites in vitro and in in vivo mouse infection experiments. Although we were able to reproduce the inhibition of transferrin uptake with high concentrations of pNAL structural analogues (N-acetylchito-oligosaccharides), this effect disappeared at lower concentrations that still inhibited tomato lectin uptake, i.e., at concentrations able to outcompete lectin-pNAL binding. Based on these findings, we recommend revision of the pNAL-dependent receptor mediated endocytosis hypothesis.

Coupling delay controls synchronized oscillation in the segmentation clock.

Individual cellular activities fluctuate but are constantly coordinated at the population level via cell-cell coupling. A notable example is the somite segmentation clock, in which the expression of clock genes (such as Hes7) oscillates in synchrony between the cells that comprise the presomitic mesoderm (PSM)1,2. This synchronization depends on the Notch signalling pathway; inhibiting this pathway desynchronizes oscillations, leading to somite fusion3-7. However, how Notch signalling regulates the synchronicity of HES7 oscillations is unknown. Here we establish a live-imaging system using a new fluorescent reporter (Achilles), which we fuse with HES7 to monitor synchronous oscillations in HES7 expression in the mouse PSM at a single-cell resolution. Wild-type cells can rapidly correct for phase fluctuations in HES7 oscillations, whereas the absence of the Notch modulator gene lunatic fringe (Lfng) leads to a loss of synchrony between PSM cells. Furthermore, HES7 oscillations are severely dampened in individual cells of Lfng-null PSM. However, when Lfng-null PSM cells were completely dissociated, the amplitude and periodicity of HES7 oscillations were almost normal, which suggests that LFNG is involved mostly in cell-cell coupling. Mixed cultures of control and Lfng-null PSM cells, and an optogenetic Notch signalling reporter assay, revealed that LFNG delays the signal-sending process of intercellular Notch signalling transmission. These results-together with mathematical modelling-raised the possibility that Lfng-null PSM cells shorten the coupling delay, thereby approaching a condition known as the oscillation or amplitude death of coupled oscillators8. Indeed, a small compound that lengthens the coupling delay partially rescues the amplitude and synchrony of HES7 oscillations in Lfng-null PSM cells. Our study reveals a delay control mechanism of the oscillatory networks involved in somite segmentation, and indicates that intercellular coupling with the correct delay is essential for synchronized oscillation.

Structure and mechanism of the ER-based glucosyltransferase ALG6.

In eukaryotic protein N-glycosylation, a series of glycosyltransferases catalyse the biosynthesis of a dolichylpyrophosphate-linked oligosaccharide before its transfer onto acceptor proteins1. The final seven steps occur in the lumen of the endoplasmic reticulum (ER) and require dolichylphosphate-activated mannose and glucose as donor substrates2. The responsible enzymes-ALG3, ALG9, ALG12, ALG6, ALG8 and ALG10-are glycosyltransferases of the C-superfamily (GT-Cs), which are loosely defined as containing membrane-spanning helices and processing an isoprenoid-linked carbohydrate donor substrate3,4. Here we present the cryo-electron microscopy structure of yeast ALG6 at 3.0 Å resolution, which reveals a previously undescribed transmembrane protein fold. Comparison with reported GT-C structures suggests that GT-C enzymes contain a modular architecture with a conserved module and a variable module, each with distinct functional roles. We used synthetic analogues of dolichylphosphate-linked and dolichylpyrophosphate-linked sugars and enzymatic glycan extension to generate donor and acceptor substrates using purified enzymes of the ALG pathway to recapitulate the activity of ALG6 in vitro. A second cryo-electron microscopy structure of ALG6 bound to an analogue of dolichylphosphate-glucose at 3.9 Å resolution revealed the active site of the enzyme. Functional analysis of ALG6 variants identified a catalytic aspartate residue that probably acts as a general base. This residue is conserved in the GT-C superfamily. Our results define the architecture of ER-luminal GT-C enzymes and provide a structural basis for understanding their catalytic mechanisms.

Recapitulating the human segmentation clock with pluripotent stem cells.

Pluripotent stem cells are increasingly used to model different aspects of embryogenesis and organ formation1. Despite recent advances in in vitro induction of major mesodermal lineages and cell types2,3, experimental model systems that can recapitulate more complex features of human mesoderm development and patterning are largely missing. Here we used induced pluripotent stem cells for the stepwise in vitro induction of presomitic mesoderm and its derivatives to model distinct aspects of human somitogenesis. We focused initially on modelling the human segmentation clock, a major biological concept believed to underlie the rhythmic and controlled emergence of somites, which give rise to the segmental pattern of the vertebrate axial skeleton. We observed oscillatory expression of core segmentation clock genes, including HES7 and DKK1, determined the period of the human segmentation clock to be around five hours, and demonstrated the presence of dynamic travelling-wave-like gene expression in in vitro-induced human presomitic mesoderm. Furthermore, we identified and compared oscillatory genes in human and mouse presomitic mesoderm derived from pluripotent stem cells, which revealed species-specific and shared molecular components and pathways associated with the putative mouse and human segmentation clocks. Using CRISPR-Cas9-based genome editing technology, we then targeted genes for which mutations in patients with segmentation defects of the vertebrae, such as spondylocostal dysostosis, have been reported (HES7, LFNG, DLL3 and MESP2). Subsequent analysis of patient-like and patient-derived induced pluripotent stem cells revealed gene-specific alterations in oscillation, synchronization or differentiation properties. Our findings provide insights into the human segmentation clock as well as diseases associated with human axial skeletogenesis.

Global View of Domain-Specific O-Linked Mannose Glycosylation in Glycoengineered Cells.

Protein O-linked mannose (O-Man) glycosylation is an evolutionary conserved posttranslational modification that fulfills important biological roles during embryonic development. Three nonredundant enzyme families, POMT1/POMT2, TMTC1-4, and TMEM260, selectively coordinate the initiation of protein O-Man glycosylation on distinct classes of transmembrane proteins, including α-dystroglycan, cadherins, and plexin receptors. However, a systematic investigation of their substrate specificities is lacking, in part due to the ubiquitous expression of O-Man glycosyltransferases in cells, which precludes analysis of pathway-specific O-Man glycosylation on a proteome-wide scale. Here, we apply a targeted workflow for membrane glycoproteomics across five human cell lines to extensively map O-Man substrates and genetically deconstruct O-Man initiation by individual and combinatorial knockout of O-Man glycosyltransferase genes. We established a human cell library for the analysis of substrate specificities of individual O-Man initiation pathways by quantitative glycoproteomics. Our results identify 180 O-Man glycoproteins, demonstrate new protein targets for the POMT1/POMT2 pathway, and show that TMTC1-4 and TMEM260 pathways widely target distinct Ig-like protein domains of plasma membrane proteins involved in cell-cell and cell-extracellular matrix interactions. The identification of O-Man on Ig-like folds adds further knowledge on the emerging concept of domain-specific O-Man glycosylation which opens for functional studies of O-Man-glycosylated adhesion molecules and receptors.

"Mix and match" auto-assembly of glycosyltransferase domains delivers biocatalysts with improved substrate promiscuity.

Glycosyltransferases (GT) catalyze the glycosylation of bioactive natural products, including peptides and proteins, flavonoids, and sterols, and have been extensively used as biocatalysts to generate glycosides. However, the often narrow substrate specificity of wild-type GTs requires engineering strategies to expand it. The GT-B structural family is constituted by GTs that share a highly conserved tertiary structure in which the sugar donor and acceptor substrates bind in dedicated domains. Here, we have used this selective binding feature to design an engineering process to generate chimeric glycosyltransferases that combine auto-assembled domains from two different GT-B enzymes. Our approach enabled the generation of a stable dimer with broader substrate promiscuity than the parent enzymes that were related to relaxed interactions between domains in the dimeric GT-B. Our findings provide a basis for the development of a novel class of heterodimeric GTs with improved substrate promiscuity for applications in biotechnology and natural product synthesis.

Structural and biochemical basis for regiospecificity of the flavonoid glycosyltransferase UGT95A1.

Glycosylation is a predominant strategy plants use to fine-tune the properties of small molecule metabolites to affect their bioactivity, transport, and storage. It is also important in biotechnology and medicine as many glycosides are utilized in human health. Small molecule glycosylation is largely carried out by family 1 glycosyltransferases. Here, we report a structural and biochemical investigation of UGT95A1, a family 1 GT enzyme from Pilosella officinarum that exhibits a strong, unusual regiospecificity for the 3'-O position of flavonoid acceptor substrate luteolin. We obtained an apo crystal structure to help drive the analyses of a series of binding site mutants, revealing that while most residues are tolerant to mutations, key residues M145 and D464 are important for overall glycosylation activity. Interestingly, E347 is crucial for maintaining the strong preference for 3'-O glycosylation, while R462 can be mutated to increase regioselectivity. The structural determinants of regioselectivity were further confirmed in homologous enzymes. Our study also suggests that the enzyme contains large, highly dynamic, disordered regions. We showed that while most disordered regions of the protein have little to no implication in catalysis, the disordered regions conserved among investigated homologs are important to both the overall efficiency and regiospecificity of the enzyme. This report represents a comprehensive in-depth analysis of a family 1 GT enzyme with a unique substrate regiospecificity and may provide a basis for enzyme functional prediction and engineering.

Tyrosine-modifying glycosylation by Yersinia effectors.

Mono-O-glycosylation of target proteins by bacterial toxins or effector proteins is a well-known mechanism by which bacteria interfere with essential functions of host cells. The respective glycosyltransferases are important virulence factors such as the Clostridioides difficile toxins A and B. Here, we describe two glycosyltransferases of Yersinia species that have a high sequence identity: YeGT from the zoonotic pathogen Yersinia enterocolitica and YkGT from the murine pathogen Yersinia kristensenii. We show that both modify Rho family proteins by attachment of GlcNAc at tyrosine residues (Tyr-34 in RhoA). Notably, the enzymes differed in their target protein specificity. While YeGT modified RhoA, B, and C, YkGT possessed a broader substrate spectrum and glycosylated not only Rho but also Rac and Cdc42 subfamily proteins. Mutagenesis studies indicated that residue 177 is important for this broader target spectrum. We determined the crystal structure of YeGT shortened by 16 residues N terminally (sYeGT) in the ligand-free state and bound to UDP, the product of substrate hydrolysis. The structure assigns sYeGT to the GT-A family. It shares high structural similarity to glycosyltransferase domains from toxins. We also demonstrated that the 16 most N-terminal residues of YeGT and YkGT are important for the mediated translocation into the host cell using the pore-forming protective antigen of anthrax toxin. Mediated introduction into HeLa cells or ectopic expression of YeGT and YkGT caused morphological changes and redistribution of the actin cytoskeleton. The data suggest that YeGT and YkGT are likely bacterial effectors belonging to the family of tyrosine glycosylating bacterial glycosyltransferases.

Identification of glycosyltransferases mediating 2-O-arabinopyranosyl and 2-O-galactosyl substitutions of glucuronosyl side chains of xylan.

Xylan is one of the major hemicelluloses in plant cell walls and its xylosyl backbone is often decorated at O-2 with glucuronic acid (GlcA) and/or methylglucuronic acid (MeGlcA) residues. The GlcA/MeGlcA side chains may be further substituted with 2-O-arabinopyranose (Arap) or 2-O-galactopyranose (Gal) residues in some plant species, but the enzymes responsible for these substitutions remain unknown. During our endeavor to investigate the enzymatic activities of Arabidopsis MUR3-clade members of the GT47 glycosyltransferase family, we found that one of them was able to transfer Arap from UDP-Arap onto O-2 of GlcA side chains of xylan, and thus it was named xylan 2-O-arabinopyranosyltransferase 1 (AtXAPT1). The function of AtXAPT1 was verified in planta by its T-DNA knockout mutation showing a loss of the Arap substitution on xylan GlcA side chains. Further biochemical characterization of XAPT close homologs from other plant species demonstrated that while the poplar ones had the same catalytic activity as AtXAPT1, those from Eucalyptus, lemon-scented gum, sea apple, 'Ohi'a lehua, duckweed and purple yam were capable of catalyzing both 2-O-Arap and 2-O-Gal substitutions of xylan GlcA side chains albeit with differential activities. Sequential reactions with XAPTs and glucuronoxylan methyltransferase 3 (GXM3) showed that XAPTs acted poorly on MeGlcA side chains, whereas GXM3 could efficiently methylate arabinosylated or galactosylated GlcA side chains of xylan. Furthermore, molecular docking and site-directed mutagenesis analyses of Eucalyptus XAPT1 revealed critical roles of several amino acid residues at the putative active site in its activity. Together, these findings establish that XAPTs residing in the MUR3 clade of family GT47 are responsible for 2-O-arabinopyranosylation and 2-O-galactosylation of GlcA side chains of xylan.

Four glycosyltransferase genes are responsible for synthesis and accumulation of different flavonol glycosides in apple tissues.

Flavonols are widely synthesized throughout the plant kingdom, playing essential roles in plant physiology and providing unique health benefits for humans. Their glycosylation plays significant role in improving their stability and solubility, thus their accumulation and function. However, the genes encoding the enzymes catalyze this glycosylation remain largely unknown in apple. This study utilized a combination of methods to identify genes encoding such enzymes. Initially, candidate genes were selected based on their potential to encode UDP-dependent glycosyltransferases (UGTs) and their expression patterns in response to light induction. Subsequently, through testing the in vitro enzyme activity of the proteins produced in Escherichia coli cells, four candidates were confirmed to encode a flavonol 3-O-galactosyltransferase (UGT78T6), flavonol 3-O-glucosyltransferase (UGT78S1), flavonol 3-O-xylosyltransferase/arabinosyltransferase (UGT78T5), and flavonol 3-O-rhamnosyltransferase (UGT76AE22), respectively. Further validation of these genes' functions was conducted by modulating their expression levels in stably transformed apple plants. As anticipated, a positive correlation was observed between the expression levels of these genes and the content of specific flavonol glycosides corresponding to each gene. Moreover, overexpression of a flavonol synthase gene, MdFLS, resulted in increased flavonol glycoside content in apple roots and leaves. These findings provide valuable insights for breeding programs aimed at enriching apple flesh with flavonols and for identifying flavonol 3-O-glycosyltransferases of other plant species.

Specific protein interactions between rice members of the GT43 and GT47 families form various central cores of putative xylan synthase complexes.

Members of the glycosyltransferase (GT)43 and GT47 families have been associated with heteroxylan synthesis in both dicots and monocots and are thought to assemble into central cores of putative xylan synthase complexes (XSCs). Currently, it is unknown whether protein-protein interactions within these central cores are specific, how many such complexes exist, and whether these complexes are functionally redundant. Here, we used gene association network and co-expression approaches in rice to identify four OsGT43s and four OsGT47s that assemble into different GT43/GT47 complexes. Using two independent methods, we showed that (i) these GTs assemble into at least six unique complexes through specific protein-protein interactions and (ii) the proteins interact directly in vitro. Confocal microscopy showed that, when alone, all OsGT43s were retained in the endoplasmic reticulum (ER), while all OsGT47s were localized in the Golgi. co-expression of OsGT43s and OsGT47s displayed complexes that form in the ER but accumulate in Golgi. ER-to-Golgi trafficking appears to require interactions between OsGT43s and OsGT47s. Comparison of the central cores of the three putative rice OsXSCs to wheat, asparagus, and Arabidopsis XSCs, showed great variation in GT43/GT47 combinations, which makes the identification of orthologous central cores between grasses and dicots challenging. However, the emerging picture is that all central cores from these species seem to have at least one member of the IRX10/IRX10-L clade in the GT47 family in common, suggesting greater functional importance for this family in xylan synthesis. Our findings provide a new framework for future investigation of heteroxylan biosynthesis and function in monocots.