Biology subjects

Moremen, K. W.

Publications and source records attributed to Moremen, K. W..

18 recordsLinked to original sources

bioRxiv2026

Identification of glycosyltransferases that synthesize pectin heteroglycan block polymers and a structural motif required for their activity

Pectin is a family of plant cell wall complex polysaccharides and proteoglycans comprised of the glycan domains homogalacturonan (HG), rhamnogalacturonan I (RG-I), and the substituted HGs rhamnogalacturonan II (RG-II), xylogalacturonan and apiogalacturonan. The number, type, and arrangement of the pectic domains in different pectic polymers are unknown, as is the identity of the enzymes that link the domains together. Here we show that two Arabidopsis -1,4-galacturonosyltransferases, GAUT13 and GAUT14, in addition to previously reported HG acceptor-dependent and HG de novo synthesis activities, initiate and elongate HG on the non-reducing end of RG-I, identifying enzymes that synthesize pectic RG-I:HG heteroglycans. While other catalytic GAUTs did not initiate HG on RG-I acceptors, they were able to elongate HG once initiated on RG-I by GAUT13 and GAUT14. We show that the three different GAUT14 acceptors (UDP-GalA, HG, and RG-I) compete for the same binding pocket, and amino acids required for GAUT14 catalytic activity were identified using in vitro enzyme assays, computational modeling, and site-directed mutagenesis. The transfer of the GAUT14 hypervariable 1 (HV1) motif (RTFASKLQSRS) was sufficient and necessary to transfer RG-I:HG extension activity to GAUT10, which otherwise does not have this activity, representing a novel functional glycosyltransferase domain swapping strategy. The presence of all three catalytic activities in a GAUT13/14 homolog from the non-vascular seedless plant Physcomitrium established the early evolution of GAUT13/14 trifunctionality. The identification of GAUT13 and GAUT14 as RG-I:HG extension catalysts provides targets for probing in planta structure and function of RG-I:HG heteropolymers.

plant biology↗

bioRxiv2026

B3GNT7 regulates mucin glycosylation and protects against colitis and infection

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. SIGNIFICANCE STATEMENTUlcerative colitis is a chronic inflammatory bowel disease that affects 5 million people globally. The colonic mucus layer forms a protective barrier over colonic epithelial cells and is disrupted in ulcerative colitis. Mucus is composed of mucin proteins decorated by carbohydrates, called glycans. Glycans confer protective properties to the mucus barrier, and mucin glycans change in ulcerative colitis. B3GNT7 is an enzyme that elongates glycans and is downregulated in ulcerative colitis. In this study, we use in vitro and in vivo models to demonstrate that B3GNT7 regulates colonic mucus glycans and protects mice against colitis and infection. Our findings provide molecular insight into the contributions of B3GNT7-dependent glycans to colonic homeostasis.

biochemistry↗

bioRxiv2026

Glydentify: An explainable deep learning platform for glycosyltransferase donor substrate prediction

Glycosyltransferases (GTs) are a large family of enzymes that catalyze glycosidic linkages formation between chemically diverse donor and acceptor molecules to regulate diverse cellular processes across all domains of life. Despite their importance, the activated sugar donors (donor substrates) used by most GTs remain unidentified, limiting our understanding of GT functions. To address this challenge, we developed Glydentify, a deep learning framework that predicts donor usage across GT-A and GT-B fold glycosyltransferases. Trained on large-scale UniProt annotations, Glydentify integrates protein sequence embeddings learned from protein language models with chemical features derived from molecular encoders trained on extensive chemical datasets. The resulting models achieve high predictive performance, with precision-recall AUCs (PR-AUC) of 0.86 for GT-A and 0.91 for GT-B, surpassing general enzyme-substrate predictors while requiring minimal manual curation. We employed Glydentify to predict the donor specificity of uncharacterized plant GTs and experimentally tested the predictions using in vitro biochemical assays. Furthermore, we demonstrate that the model utilizes a combination of evolutionary, structural, and biochemical features to predict donor specificity through residue attention score analysis. Together, these results establish Glydentify as a robust, explainable framework for decoding donor-glycosyltransferase relationships and highlight its potential as a broadly applicable framework for modeling enzyme classes that act on chemically diverse substrates.

bioinformatics↗

bioRxiv2025

JAAG: a JSON input file Assembler for AlphaFold 3 with Glycan integration

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.

bioinformatics↗

bioRxiv2025

Biochemical and structural characterization of glycosyltransferase family 61 proteins reveal a key determinant of sugar donor specificity

Xylan, the most abundant non-cellulosic polymer in plant cell walls, is structurally diverse, especially in grasses where it is heavily substituted with arabinofuranose and further modified by various residues. Common substitutions across species include glucuronic and 4-O-methyl-glucuronic acid. Arabinose and xylose sidechains are synthesized by glycosyltransferase family 61 (GT61) proteins, many of which remain uncharacterized in plants, with limited structural and mechanistic understanding. In this study, we identified two novel GT61 enzymes in Sorghum bicolor, functioning as xylan arabinosyltransferase (SbXAT) and xylan xylosyltransferase (SbXXT). We resolved the crystal structure of SbXAT, which exhibits a GT-B fold with two Rossmann-like domains linked by a cleft that accommodates the catalytic site. Structural comparison with a predicted SbXXT model revealed a substrate-binding residue critical for sugar donor specificity, validated through site-directed mutagenesis and enzymatic assays. These findings enhance understanding of xylan biosynthesis and provide a foundation for engineering glycosyltransferases and predicting their functions.

biochemistry↗

bioRxiv2025

Enforced E-selectin ligand installation enhances homing and efficacy of adoptively transferred T cells

Adoptive T-cell transfer has revolutionized the treatment of hematological malignancies. However, this approach has had very limited success in treating solid tumors, largely due to inadequate infiltration of vascularly administered T cells at tumor sites. The shear-resistant interaction between endothelial E-selectin and its cognate ligand expressed on leukocytes, sialyl Lewis X (sLeX), is an essential prerequisite for extravasation of circulating leukocytes. Here, we report that enforced E-selectin ligand expression (enforced sLeX display) on antigen-specific T cells can be achieved by fucosylating cells via cell surface treatment with the human 1-3-fucosyltransferase, FUT6 ("exofucosylation"), or via Golgi-targeted FUT6 overexpression ("Golgi-fucosylation"). However, despite comparable E-selectin binding, only sLeX-modified T cells engendered by exofucosylation, not by Golgi-fucosylation, exhibited enhanced parenchymal infiltration of target malignant sites. This heightened homing yielded significantly improved therapeutic efficacy in various murine syngeneic and xenograft cancer models, including subcutaneous solid tumors, lymphoma and leukemia, as well as lung and bone marrow metastases. Therefore, exofucosylation represents a promising strategy to improve the efficacy of adoptive T-cell therapy, particularly in the treatment of solid tumors and metastatic disease.

cancer biology↗

bioRxiv2023

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 prepare well-defined KS oligosaccharides by exploiting known and newly discovered substrate specificities of relevant sulfotransferases. The premise of the approach is that recombinant GlcNAc-6-O-sulfotransferases (CHST2) only sulfates terminal GlcNAc moieties to give GlcNAc6S that can be galactosylated by B4GalT4. Furthermore, CHST1 can modify 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 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 presentation of epitopes as part of an O- or N-glycan determines binding properties.

biochemistry↗

bioRxiv2023

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.

plant biology↗

bioRxiv2022

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

N-glycosylation is an essential eukaryotic post-translational 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 influences glycan availability to enzymatic modification. We tested this hypothesis by expression of a panel of reporter N-linked glycoproteins in 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. By comparison, proteolytic digestion of the reporters prior to N-glycan processing eliminated differences in in vitro enzymatic modification. Comparison of in vitro rates of enzymatic modification with the glycan structures found on the mature reporters expressed in wild type cells correlate well with the enzymatic bottlenecks found in vitro. These data suggest that 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 site-specific microheterogeneity found on N-linked glycoproteins.

biochemistry↗

bioRxiv2021

Mapping the glycosyltransferase fold landscape using deep learning

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 learned distinguishing features free of primary sequence alignment constraints and, unlike other models, is highly interpretable and helped identify common secondary structural features shared by divergent families. The model delineated 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 identify targets for future structural studies and expand the GT fold landscape.

bioinformatics↗

bioRxiv2021

Cell Surface Glycan Engineering Reveals that Matriglycan Alone can Recapitulate Dystroglycan Binding and Function

-Dystroglycan (-DG) is uniquely modified on O-mannose sites by a repeating disaccharide (-Xyl1,3-GlcA{beta}1,3-)n termed matriglycan, which is a receptor for laminin-G domain-containing proteins and employed by old-world arenaviruses for infection. Using chemoenzymatically synthesized matriglycans printed as a microarray, we demonstrated length-dependent binding to Laminin, Lassa virus GP1, and the clinically-important antibody IIH6. Utilizing an enzymatic engineering approach, an N-linked glycoprotein was converted into a IIH6-positive Laminin-binding glycoprotein. Engineering of the surface of cells deficient for either -DG or O-mannosylation with matriglycans of sufficient length recovered infection with a Lassa-pseudovirus. Finally free matriglycan in a dose and length dependent manner inhibited viral infection of wildtype cells. These results indicate that matriglycan alone is necessary and sufficient for IIH6 staining, Laminin and LASV GP1 binding, and Lassa-pseudovirus infection and support a model in which it is a tunable receptor for which increasing chain length enhances ligand-binding capacity. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY Graphical Abstract C_FIG_DISPLAY

biochemistry↗

bioRxiv2020

Glycan remodeled erythrocytes facilitate antigenic characterizationof 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. We examined receptor specificities of A/H3N2 viruses, revealing that recent viruses compensated for decreased binding of the prototypic human receptor by recognizing 2,6-sialosides on extended LacNAc moieties. Erythrocyte glycomics showed an absence of extended glycans, providing a rationale for lack of agglutination by recent A/H3N2 viruses. A glycan remodeling approach installed functional receptors on erythrocytes, allowing antigenic characterization of recent A/H3N2 viruses and confirming the cocirculation of several antigenically different viruses in humans. Computational studies of HAs in complex with a sialoside having an extended LacNAc moiety revealed that mutations distal to the RBD reoriented the Y159 side chain, resulting in an extended receptor binding site.

biochemistry↗

bioRxiv2020

Modulate 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 epitopes overexpressed on tumor cells, inhibitory Siglecs on innate and adaptive immune cells modulate signaling cascades to restrain anti-tumor immune responses. However, the mechanisms underlying these processes are just starting to be elucidated. We discover 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 transfer of sialylated glycans from NK cells to target tumor cells and accelerated de novo synthesis of sialosides on the tumor cell themselves. The functionalization of NK cells with a high-affinity ligand of Siglec-7 leads to multifaceted consequences in modulating Siglec-7-regulated NK-activation. At high levels, the added Siglec-7 ligand suppresses NK-cytotoxicity through the recruitment of Siglec-7, whereas at low levels the same ligand triggers the release of Siglec-7 from the cell surface into the culture medium, preventing Siglec-7-mediated inhibition of NK cytotoxicity. These results suggest that glycan engineering of NK cells may provide a means to boost NK effector functions.

biochemistry↗

bioRxiv2020

Glycoengineering of NK cells with glycan ligands of CD22 and selectins for B-cell lymphoma therapy

CD22, a member of Siglec family of sialic acid binding proteins, has restricted expression on B cells. Antibody-based agents targeting CD22 or CD20 (Rituxan) on B lymphoma and leukemia cells exhibit clinical efficacy for treating these malignancies, but also attack normal B cells leading to immune deficiency. Here, we report a chemoenzymatic glycocalyx editing strategy to introduce high-affinity and specific CD22 ligands onto NK-92MI and cytokine-induced killer (CIK) cells to achieve tumor-specific CD22 targeting. These CD22-ligand modified cells exhibited significantly enhanced tumor cell binding and killing in vitro without harming healthy B cells. For effective lymphoma cell killing in vivo we further functionalized CD22 ligand-modified NK-92MI cells with the E-selectin ligand sialyl Lewis X to promote trafficking to bone marrow. The cells containing the ligands of both CD22 and selectins resulted in the efficient suppression of B lymphoma in a xenograft model. Our results suggest that NK cells modified with glycan ligands to CD22 and selectins promote both targeted killing of B lymphoma cells and improved trafficking to sites where the cancer cells reside, respectively.

bioengineering↗

bioRxiv2020

Structure of the Xylan O-Acetyltransferase AtXOAT1 Reveals Molecular Insight into Polysaccharide Acetylation in Plants

Acetylation of biomolecules is gaining increased attention due to both the abundance and importance of this modification across all kingdoms of life. Xylans are a major component of plant cell walls and are the third most abundant biopolymer in Nature. 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 proper wall architecture and normal plant development. Here, we describe the biochemical, structural, and mechanistic characterization of Arabidopsis thaliana xylan O-acetyltransferase 1 (AtXOAT1), a member of the plant-specific Trichome Birefrigence Like (TBL) family that catalyzes the 2-O-acetylation of xylan. A multipronged approach involving X-ray crystallography, biochemical analyses, mutagenesis, and molecular simulations show that XOAT1 catalyzes xylan acetylation through formation of an acyl-enzyme intermediate by a double displacement bi-bi mechanism involving a Ser-His-Asp catalytic triad and unconventionally employs an arginine residue in formation of an oxyanion hole.

biochemistry↗

bioRxiv2019

Deep evolutionary analysis reveals the design principles of fold A glycosyltransferases

Glycosyltransferases (GTs) are prevalent across the tree of life and regulate nearly all aspects of cellular functions by catalyzing synthesis of glycosidic linkages between diverse donor and acceptor substrates. Despite the availability of GT sequences from diverse organisms, the evolutionary basis for their complex and diverse modes of catalytic and regulatory functions remain enigmatic. Here, based on deep mining of over half a million GT-A fold sequences from diverse organisms, we define a minimal core component shared among functionally diverse enzymes. We find that variations in the common core and the emergence of hypervariable loops extending from the core contributed to the evolution of catalytic and functional diversity. We provide a phylogenetic framework relating diverse GT-A fold families for the first time and show that inverting and retaining mechanisms emerged multiple times independently during the course of evolution. We identify conserved modes of donor and acceptor recognition in evolutionarily divergent families and pinpoint the sequence and structural features for functional specialization. Using the evolutionary information encoded in primary sequences, we trained a machine learning classifier to predict donor specificity with nearly 88% accuracy and deployed it for the annotation of understudied GTs in five model organisms. Our studies provide an evolutionary framework for investigating the complex relationships connecting GT-A fold sequence, structure, function and regulation.

bioinformatics↗

bioRxiv2019

hFUT1-based live cell assay to profile α1-2-fucosides enhanced influenza A virus infection

Host cell-surface glycans play critical roles in influenza A virus (IAV) infection ranging from modulation of IAV attachment to membrane fusion and host tropism. Approaches for quick and sensitive profiling of the viral avidity towards a specific type of host-cell glycan can contribute to the understanding of tropism switching among different strains of IAV. In this study, we developed a method based on chemoenzymatic glycan engineering to investigate the possible involvement of 1-2-fucosides in IAV infections. Using a truncated human fucosyltransferase 1 (hFuT1), we were able to create 1-2-linked fucosides in situ on the host cell surface to assess their influence on the host cell binding to IAV hemagglutinin and the susceptibility of host cells toward IAV induced killing. We discovered that the newly added 1-2-fucosides on host cells enhanced the infection of several human pandemic IVA subtypes. These findings suggest that glycan epitopes other than sialic aicds should be taken into consideration for assessing the human pandemic risk of this viral pathogen.

biochemistry↗

bioRxiv2018

Refinement of highly flexible protein structures using simulation-guided spectroscopy

Highly flexible proteins present a special challenge for structure determination because they are multi-structured yet not disordered, and the resulting conformational ensembles are essential for understanding function. Determining such ensembles is difficult because many measurements that capture multiple conformational populations provide sparse data. A powerful opportunity exists to leverage molecular simulations for spectroscopic experiment selection. We have developed an information-theoretic approach to guide experiments by identifying which measurements best refine the underlying conformational ensemble. We have tested this approach on three flexible bacterial proteins. For proteins where a clear mechanistic hypothesis drives label selection, our approach systematically identifies labels that would test this hypothesis. Furthermore, when available data do not yield an obvious mechanistically-guided label selection strategy, our approach guides label selection and produces conformational refinement that significantly outperforms standard structure-guided approaches. Our information-theoretic approach to label selection thus offers a particular advantage when refining challenging, underdetermined protein conformational ensembles.

bioinformatics↗