Kelley Moremen

Followers

Following

Co-authors

Public Views

Interests

Uploads

Papers by Kelley Moremen

Transcriptional Regulation of the Protocadherin β Cluster during Her-2 Protein-induced Mammary Tumorigenesis Results from Altered N-Glycan Branching

Journal of Biological Chemistry, Jul 1, 2012

Background: Her-2-induced mammary tumor onset is significantly delayed in GnT-V knock-out mice. R... more Background: Her-2-induced mammary tumor onset is significantly delayed in GnT-V knock-out mice. Results: The gene expression of the Pcdh␤ cluster is up-regulated in her-2-induced tumors with GnT-V deletion. Conclusion: Up-regulation of the Pcdh␤ cluster is one of the mechanisms for the reduced her-2-mediated tumorigenesis resulting from GnT-V deletion. Significance: Our findings shed new light on the molecular mechanisms of the effects of GnT-V on mammary tumorigenesis. GnT-V knockout, resulting in a significant delay in the onset of her-2-induced mammary tumors. To identify the genes that contributed to this GnT-V regulation of early events in tumorigenesis, microarray analysis was performed using her-2 induced mammary tumors from wild-type and GnT-V-null mice. We found that 142 genes were aberrantly expressed (>2.0-fold) with 64 genes up-regulated and 78 genes down-regulated after deletion of GnT-V. Among differentially expressed genes, the expression of a subgroup of the cadherin superfamily, the protocadherin ␤ (Pcdh␤) cluster, was up-regulated in GnT-V-null tumors. Altered expression of the Pcdh␤ cluster in GnT-V-null tumors was not due to changes in promoter methylation; instead, impaired her-2-mediated signaling pathways were implicated at least in part resulting from reduced microRNA-21 expression. Overexpression of Pcdh␤ genes inhibited tumor cell growth, decreased the proportion of tumor-initiating cells, and decreased tumor formation in vivo, demonstrating that expression of the Pcdh␤ gene cluster can serve as an inhibitor of the transformed phenotype. Our results suggest the up-regulation of the Pcdh␤ gene cluster as a mechanism for reduced her-2-mediated tumorigenesis resulting from GnT-V deletion. Grants U01CA128454 (to M. P.) and P41RR018502 (to M. P. and K. M.).

Download

Molecular Mechanism of Polysaccharide Acetylation by the Arabidopsis Xylan <i>O</i>-acetyltransferase XOAT1

The Plant Cell, Apr 30, 2020

Xylans are a major component of plant cell walls. O-Acetyl moieties are the dominant backbone sub... more 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-Oacetylation 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 a/b/a 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.

Download

O-Linked N-Acetylglucosamine (O-GlcNAc) Expression Levels Epigenetically Regulate Colon Cancer Tumorigenesis by Affecting the Cancer Stem Cell Compartment via Modulating Expression of Transcriptional Factor MYBL1

Journal of Biological Chemistry, Mar 1, 2017

transferase (OGT) knockdown grew significantly slower than those formed from control cells, indic... more transferase (OGT) knockdown grew significantly slower than those formed from control cells, indicating a reduced proliferation of tumor cells due to inhibition of OGT expression. Significant reduction of the CCSC population was observed in the tumor cells after OGT knockdown, whereas tumor cells treated with the O-GlcNAcase inhibitor showed an increased CCSC population, indicating that O-GlcNAc levels regulated the CCSC compartment. When grown in suspension, tumor cells with OGT knockdown showed a reduced ability to form tumorspheres, indicating a reduced self-renewal of CCSC due to reduced levels of O-GlcNAc. ChIP-sequencing experiments using an anti-O-GlcNAc antibody revealed significant chromatin enrichment of O-GlcNAcmodified proteins at the promoter of the transcription factor MYBL1, which was also characterized by the presence of H3K27me3. RNA-sequencing analysis showed an increased expression of MYBL1 in tumor cells with OGT knockdown. Forced overexpression of MYBL1 led to a reduced population of CCSC and tumor growth in vivo, similar to the effects of OGT silencing. Moreover, two CpG islands near the transcription start site of MYBL1 were identified, and O-GlcNAc levels regulated their methylation status. These results strongly argue that O-GlcNAc epigenetically regulates MYBL1, functioning similarly to H3K27me3. The aberrant CCSC compartment observed after modulating O-GlcNAc levels is therefore likely to result, at least in part, from the epigenetic regulation of MYBL1 expression by O-GlcNAc, thereby significantly affecting tumor progression.

Download

Family 47 α‐Mannosidases in N‐Glycan Processing

Methods in Enzymology, 2006

α-Mannosidases in eukaryotic cells are involved in both glycan biosynthetic reactions and glycan ... more α-Mannosidases in eukaryotic cells are involved in both glycan biosynthetic reactions and glycan catabolism. Two broad families of enzymes have been identified that cleave terminal mannose linkages from Asn-linked oligosaccharides , including the Class 1 mannosidases (CAZy GH family 47 ) of the early secretory pathway involved in the processing of N-glycans and quality control and the Class 2 mannosidases (CAZy family GH38 ) involved in glycoprotein biosynthesis or catabolism. Within the Class 1 family of α-mannosidases, three subfamilies of enzymes have been identified . The endoplasmic reticulum (ER) α1,2-mannosidase I (ERManI) subfamily acts to cleave a single residue from Asn-linked glycans in the ER. The Golgi α-mannosidase I (GolgiManI) subfamily has at least three members in mammalian systems involved in glycan maturation in the Golgi complex to form the Man 5 GlcNAc 2 processing intermediate. The third subfamily ofGH47 proteins comprises the ER degradation, enhancing α-mannosidase-like proteins (EDEM proteins) . These proteins have been proposed to accelerate the degradation of misfolded proteins in the lumen of the ER by a lectin function that leads to retrotranslocation to the cytosol and proteasomal degradation. Recent studies have also indicated that ERManI acts as a timer for initiation of glycoprotein degradation via the ubiquitin-proteasome pathway . This article discusses methods for analysis of the GH47 α-mannosidases, including expression, purification, activity assays, generation of point mutants, and binding studies by surface plasmon resonance.

Download

Vertebrate protein glycosylation: diversity, synthesis and function

Nature Reviews Molecular Cell Biology, Jun 22, 2012

Protein glycosylation is a ubiquitous post-translational modification found in all domains of lif... more 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.

Download

Human <i>N</i> -acetylglucosaminyltransferase II substrate recognition uses a modular architecture that includes a convergent exosite

Proceedings of the National Academy of Sciences of the United States of America, Apr 16, 2018

Data deposition: The atomic coordinates and structure factors have been deposited in the Protein ... more

Download

Downstream Products are Potent Inhibitors of the Heparan Sulfate 2-O-Sulfotransferase

Scientific Reports, Aug 7, 2018

Heparan Sulfate (HS) is a cell signaling molecule linked to pathological processes ranging from c... more Heparan Sulfate (HS) is a cell signaling molecule linked to pathological processes ranging from cancer to viral entry, yet fundamental aspects of its biosynthesis remain incompletely understood. Here, the binding preferences of the uronyl 2-O-sulfotransferase (HS2ST) are examined with variablysulfated hexasaccharides. Surprisingly, heavily sulfated oligosaccharides formed by later-acting sulfotransferases bind more tightly to HS2ST than those corresponding to its natural substrate or product. Inhibition assays also indicate that the IC 50 values correlate simply with degree of oligosaccharide sulfation. Structural analysis predicts a mode of inhibition in which 6-O-sulfate groups located on glucosamine residues present in highly-sulfated oligosaccharides occupy the canonical binding site of the nucleotide cofactor. The unexpected finding that oligosaccharides associated with later stages in HS biosynthesis inhibit HS2ST indicates that the enzyme must be separated temporally and/or spatially from downstream products during biosynthesis in vivo, and highlights a challenge for the enzymatic synthesis of lengthy HS chains in vitro. Glycosaminoglycans (GAGs) are a group of linear polysaccharides that consist of alternating hexosamine and hexuronic acid units. With the exception of hyaluronan, GAGs are variably sulfated, and are categorized into heparan sulfate (HS), chondroitin sulfate, and dermatan sulfate based on the composition and linkage of the monosaccharide residues 1 . HS has received considerable attention due to the broad functionality it displays in key cellular signaling events that are crucial for proper development 2 , such as vasculogenesis 3 , axon guidance 4 , and cellular differentiation 5 . The critical role for HS within animals, as well as its presence on cell surfaces, has provided pathogenic organisms with an accessible route for attachment and entry into the cell. Families of viruses including Herpesviridae 6 , Flaviviridae 7 , and Filoviridae 8 each exploit HS for cell adhesion. HS has also been implicated in various forms of cancer 9 and may either promote or inhibit metastasis depending on the sulfation pattern along the carbohydrate chain 10 . Efforts to characterize HS-protein interactions indicate that some binding modes recognize specific sulfation patterns, while others are non-specific and appear to be dependent primarily on negative charge density . The prototypical example of highly specific HS binding is that between antithrombin and heparin (a more highly sulfated form of HS bearing antithrombin binding sites) whose affinity increases more than 1000-fold upon 3-O-sulfation of a key hexosamine residue . There is growing interest in the therapeutic potential of HS mimetics due to the diverse functionality and possibility for highly specific interactions . Biosynthesis of HS begins with the assembly of a core tetrasaccharide to specific serine residues of proteins 20 . Two enzymes then add alternating N-acetyl glucosamine (GlcNAc) and glucuronic acid (GlcA) residues until the chain reaches between 20 and 150 disaccharide units in length 21 . Assembly of the HS chain continues via a set of enzymes that modify the repeating disaccharide unit, including concomitant N-deacetylation and N-sulfation of GlcNAc units 22 , epimerization of GlcA to iduronic acid (IdoA), 2-O-sulfation of uronic acids and 6-O-and 3-O-sulfation of hexosamine units (Fig. ). A sequential biosynthetic pathway was originally proposed based on label-chase studies of HS in mouse mastocytoma microsomal fractions . In general, the enzymatic

Download

Modulation of the NOTCH1 Pathway by LUNATIC FRINGE Is Dominant over That of MANIC or RADICAL FRINGE

Molecules, Sep 30, 2021

Fringes are glycosyltransferases that transfer a GlcNAc to O-fucose residues on Epidermal Growth ... more Fringes are glycosyltransferases that transfer a GlcNAc to O-fucose residues on Epidermal Growth Factor-like (EGF) repeats. Three Fringes exist in mammals: LUNATIC FRINGE (LFNG), MANIC FRINGE (MFNG), and RADICAL FRINGE (RFNG). Fringe modification of O-fucose on EGF repeats in the NOTCH1 (N1) extracellular domain modulates the activation of N1 signaling. Not all O-fucose residues of N1 are modified by all Fringes; some are modified by one or two Fringes and others not modified at all. The distinct effects on N1 activity depend on which Fringe is expressed in a cell. However, little data is available on the effect that more than one Fringe has on the modification of O-fucose residues and the resulting downstream consequence on Notch activation. Using mass spectral glycoproteomic site mapping and cell-based N1 signaling assays, we compared the effect of co-expression of N1 with one or more Fringes on modification of O-fucose and activation of N1 in three cell lines. Individual expression of each Fringe with N1 in the three cell lines revealed differences in modulation of the Notch pathway dependent on the presence of endogenous Fringes. Despite these cell-based differences, co-expression of several Fringes with N1 demonstrated a dominant effect of LFNG over MFNG or RFNG. MFNG and RFNG appeared to be co-dominant but strongly dependent on the ligands used to activate N1 and on the endogenous expression of Fringes. These results show a hierarchy of Fringe activity and indicate that the effect of MFNG and/or RFNG could be small in the presence of LFNG.

Download

O-fucosylation of thrombospondin type 1 repeats is essential for ECM remodeling and signaling during bone development

Matrix Biology, Mar 1, 2022

Many extracellular matrix (ECM) associated proteins that influence ECM properties have Thrombospo... more Many extracellular matrix (ECM) associated proteins that influence ECM properties have Thrombospondin type 1 repeats (TSRs) which are modified with O-linked fucose. The O-fucose is added in the endoplasmic reticulum to folded TSRs by the enzyme Protein O-fucosyltransferase-2 (POFUT2) and is proposed to promote efficient trafficking of substrates. The importance of this modification for function of TSR-proteins is underscored by the early embryonic lethality of mouse embryos lacking Pofut2. To overcome early lethality and investigate the impact of the Pofut2 knockout on the secretion of POFUT2 substrates and on extracellular matrix properties in vivo, we deleted Pofut2 in the developing limb mesenchyme using Prrx1-Cre recombinase. Loss of Pofut2 in the limb mesenchyme caused significant shortening of the limbs, long bones and tendons and stiff joint resembling the musculoskeletal dysplasias in human and in mice with mutations in ADAMTS or ADAMTSL proteins. Limb shortening was evident at embryonic day 14.5 where loss of O-fucosylation led to an accumulation of fibrillin 2 (FBN2), decreased BMP and IHH signaling, and increased TGF-β signaling. Consistent with these changes we saw a decrease in the size of the hypertrophic zone with lower levels of Collagen-X. Unexpectedly, we observed minimal effects of the Pofut2 knockout on secretion of two POFUT2 substrates, CCN2 or ADAMTS17, in the developing bone. In contrast, CCN2 and two other POFUT2 substrates important for bone development, ADAMTS6 and 10, showed a decrease in secretion from POFUT2-null HEK293T cells in vitro. These combined results suggest that the impact of the Pofut2 mutation is cell-type specific. In addition, these observations raise the possibility that the O-fucose modification on TSRs extends beyond promoting efficient trafficking of POFUT2 substrates and has the potential to influence their function in the extracellular environment.

The glycan receptors of Helicobacter pylori : decoding the pathways underlying gastric glycophenotype modulation

Glycobiology, 2016

"The deleterious effect of AB5 toxins on Campylobacter jejuni strains that mimic GM1 ganglioside:... more

Download

A universal glycoenzyme biosynthesis pipeline that enables efficient cell-free remodeling of glycans

Nature Communications

The ability to reconstitute natural glycosylation pathways or prototype entirely new ones from sc... more 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 quantit...

Download

AtFUT4 and AtFUT6 Are Arabinofuranose-Specific Fucosyltransferases

Frontiers in Plant Science

The bulk of plant biomass is comprised of plant cell walls, which are complex polymeric networks,... more The bulk of plant biomass is comprised of plant cell walls, which are complex polymeric networks, composed of diverse polysaccharides, proteins, polyphenolics, and hydroxyproline-rich glycoproteins (HRGPs). Glycosyltransferases (GTs) work together to synthesize the saccharide components of the plant cell wall. The Arabidopsis thaliana fucosyltransferases (FUTs), AtFUT4, and AtFUT6, are members of the plant-specific GT family 37 (GT37). AtFUT4 and AtFUT6 transfer fucose (Fuc) onto arabinose (Ara) residues of arabinogalactan (AG) proteins (AGPs) and have been postulated to be non-redundant AGP-specific FUTs. AtFUT4 and AtFUT6 were recombinantly expressed in mammalian HEK293 cells and purified for biochemical analysis. We report an updated understanding on the specificities of AtFUT4 and AtFUT6 that are involved in the synthesis of wall localized AGPs. Our findings suggest that they are selective enzymes that can utilize various arabinogalactan (AG)-like and non-AG-like oligosaccharide...

Download

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

Journal of Biological Chemistry

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 g... more 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...

Structural and Biochemical Insight into a Modular β-1,4-Galactan Synthase in Plants

Rhamnogalacturonan I (RGI) is a structurally complex pectic polysaccharide with a backbone of alt... more Rhamnogalacturonan I (RGI) is a structurally complex pectic polysaccharide with a backbone of alternating rhamnose and galacturonic acid residues substituted with arabinan and galactan side chains. Galactan synthase 1 (GalS1), transfers galactose and arabinose to either extend or cap the β-1,4 galactan side chains of RGI, respectively. Here we report the structure of GalS1 from Populus trichocarpa, showing a modular protein consisting of an N-terminal domain that represents the founding member of a new family of carbohydrate-binding module, CBMXX (number denoted as ‘XX” assigned upon publication), and a C-terminal glycosyltransferase family 92 (GT92) catalytic domain that adopts a GT-A fold. GalS1 exists as a dimer in vitro, with stem domains interacting across the chains in a 'handshake' orientation that is essential for maintaining stability and activity. In addition to understanding the enzymatic mechanism of GalS1, we gained insight into the donor and acceptor substrate ...

Download

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

Journal of Biological Chemistry

Extracellular ST6GAL1 regulates monocyte–macrophage development and survival

Glycobiology

Interaction of immune cells with the systemic environment is necessary for the coordinated develo... more 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 ...

Crystal structures of β-1,4-N-acetylglucosaminyltransferase 2: structural basis for inherited muscular dystrophies

Acta Crystallographica Section D Structural Biology, 2021

The canonical O-mannosylation pathway in humans is essential for the functional glycosylation of ... more The canonical O-mannosylation pathway in humans is essential for the functional glycosylation of α-dystroglycan. Disruption of this post-translational modification pathway leads to congenital muscular dystrophies. The first committed step in the construction of a functional matriglycan structure involves the post-translational modification of α-dystroglycan. This is essential for binding extracellular matrix proteins and arenaviruses, and is catalyzed by β-1,4-N-acetylglucosaminyltransferase 2 (POMGNT2). While another glycosyl transferase, β-1,4-N-acetylglucosaminyltransferase 1 (POMGNT1), has been shown to be promiscuous in extending O-mannosylated sites, POMGNT2 has been shown to display significant primary amino-acid selectivity near the site of O-mannosylation. Moreover, several single point mutations in POMGNT2 have been identified in patients with assorted dystroglycanopathies such as Walker–Warburg syndrome and limb girdle muscular dystrophy. To gain insight into POMGNT2 func...

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

α-Dystroglycan (α-DG) is uniquely modified on O-mannose sites by a repeating disaccharide (-Xylα1... more α-Dystroglycan (α-DG) is uniquely modified on O-mannose sites by a repeating disaccharide (-Xylα1,3-GlcAβ1,3-)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 α-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 infecti...

Download

A flexible, pan-species, multi-antigen platform for the detection and monitoring of SARS-CoV-2-specific antibody responses

The SARS-CoV-2 pandemic and the vaccination effort that is ongoing has created an unmet need for ... more The SARS-CoV-2 pandemic and the vaccination effort that is ongoing has created an unmet need for accessible, affordable, flexible and precise platforms for monitoring the induction, specificity and maintenance of virus-specific immune responses. Herein we validate a multiplex (Luminex-based) assay capable of detecting SARS-CoV-2-specific antibodies irrespective of host species, antibody isotype, and specimen type (e.g. plasma, serum, saliva or blood spots). The well-established precision of Luminex-based assays provides the ability to follow changes in antibody levels over time to many antigens, including multiple permutations of the most common SARS-CoV-2 antigens. This platform can easily measure antibodies known to correlate with neutralization activity as well as multiple non-SARS-CoV-2 antigens such as vaccines (e.g. Tetanus toxoid) or those from frequently encountered agents (influenza), which serve as stable reference points for quantifying the changing SARS-specific response...

Download

Kelley Moremen

Uploads

Papers by Kelley Moremen

Log In