140 results on '"Jeong-Yeh Yang"'
Search Results
2. Glycan remodeled erythrocytes facilitate antigenic characterization of recent A/H3N2 influenza viruses
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Frederik Broszeit, Rosanne J. van Beek, Luca Unione, Theo M. Bestebroer, Digantkumar Chapla, Jeong-Yeh Yang, Kelley W. Moremen, Sander Herfst, Ron A. M. Fouchier, Robert P. de Vries, and Geert-Jan Boons
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Science - Abstract
Here, Broszeit et al. show that circulating A/H3N2 viruses have evolved binding specificity to α2,6-sialosides on extended LacNAc moieties and therefore cannot agglutinate erythrocytes. Applying glycan remodeling allows to install functional receptors on erythrocytes and promotes identification of newly circulating variants to facilitate vaccine design.
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- 2021
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3. Network inference from glycoproteomics data reveals new reactions in the IgG glycosylation pathway
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Elisa Benedetti, Maja Pučić-Baković, Toma Keser, Annika Wahl, Antti Hassinen, Jeong-Yeh Yang, Lin Liu, Irena Trbojević-Akmačić, Genadij Razdorov, Jerko Štambuk, Lucija Klarić, Ivo Ugrina, Maurice H. J. Selman, Manfred Wuhrer, Igor Rudan, Ozren Polasek, Caroline Hayward, Harald Grallert, Konstantin Strauch, Annette Peters, Thomas Meitinger, Christian Gieger, Marija Vilaj, Geert-Jan Boons, Kelley W. Moremen, Tatiana Ovchinnikova, Nicolai Bovin, Sakari Kellokumpu, Fabian J. Theis, Gordan Lauc, and Jan Krumsiek
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Science - Abstract
IgG glycosylation is an important factor in immune function, yet the molecular details of protein glycosylation remain poorly understood. The data-driven approach presented here uses large-scale plasma IgG mass spectrometry measurements to infer new biochemical reactions in the glycosylation pathway.
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- 2017
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4. Publisher Correction: Network inference from glycoproteomics data reveals new reactions in the IgG glycosylation pathway
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Elisa Benedetti, Maja Pučić-Baković, Toma Keser, Annika Wahl, Antti Hassinen, Jeong-Yeh Yang, Lin Liu, Irena Trbojević-Akmačić, Genadij Razdorov, Jerko Štambuk, Lucija Klarić, Ivo Ugrina, Maurice H. J. Selman, Manfred Wuhrer, Igor Rudan, Ozren Polasek, Caroline Hayward, Harald Grallert, Konstantin Strauch, Annette Peters, Thomas Meitinger, Christian Gieger, Marija Vilaj, Geert-Jan Boons, Kelley W. Moremen, Tatiana Ovchinnikova, Nicolai Bovin, Sakari Kellokumpu, Fabian J. Theis, Gordan Lauc, and Jan Krumsiek
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Science - Abstract
Correction to: Nature Communications (2017) 8:1231. doi:10.1038/s41467-017-01525-0
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- 2018
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5. Isoproterenol Increases Uncoupling, Glycolysis, and Markers of Beiging in Mature 3T3-L1 Adipocytes.
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Colette N Miller, Jeong-Yeh Yang, Emily England, Amelia Yin, Clifton A Baile, and Srujana Rayalam
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Medicine ,Science - Abstract
Beta-adrenergic activation stimulates uncoupling protein 1 (UCP1), enhancing metabolic rate. In vitro, most work has studied brown adipocytes, however, few have investigated more established adipocyte lines such as the murine 3T3-L1 line. To assess the effect of beta-adrenergic activation, mature 3T3-L1s were treated for 6 or 48 hours with or without isoproterenol (10 and 100 μM) following standard differentiation supplemented with thyroid hormone (T3; 1 nM). The highest dose of isoproterenol increased lipid content following 48 hours of treatment. This concentration enhanced UCP1 mRNA and protein expression. The increase in UCP1 following 48 hours of isoproterenol increased oxygen consumption rate. Further, coupling efficiency of the electron transport chain was disturbed and an enhancement of glycolytic rate was measured alongside this, indicating an attempt to meet the energy demands of the cell. Lastly, markers of beige adipocytes (protein content of CD137 and gene transcript of CITED1) were also found to be upregulated at 48 hours of isoproterenol treatment. This data indicates that mature 3T3-L1 adipocytes are responsive to isoproterenol and induce UCP1 expression and activity. Further, this finding provides a model for further pharmaceutical and nutraceutical investigation of UCP1 in 3T3-L1s.
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- 2015
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6. SSEA4-Positive Pig Induced Pluripotent Stem Cells are Primed for Differentiation into Neural Cells
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Jeong-Yeh Yang, Jennifer L. Mumaw, Yubing Liu, Steve L. Stice, and Franklin D. West
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Medicine - Published
- 2013
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7. Structural and biochemical insight into a modular β-1,4-galactan synthase in plants
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Pradeep Kumar Prabhakar, Jose Henrique Pereira, Rahil Taujale, Wanchen Shao, Vivek S. Bharadwaj, Digantkumar Chapla, Jeong-Yeh Yang, Yannick J. Bomble, Kelley W. Moremen, Natarajan Kannan, Michal Hammel, Paul D. Adams, Henrik V. Scheller, and Breeanna R. Urbanowicz
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Crop and Pasture Production ,Glycosyltransferases ,Plant Biology ,Plant Science ,Generic health relevance ,Galactans ,Article - Abstract
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, CBM95, 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 binding sites using deep evolutionary analysis, molecular simulations and biochemical studies. Combining all the results, a mechanism for GalS1 catalysis and a new model for pectic galactan side-chain addition are proposed.
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- 2023
8. Enzymatic Synthesis of Xylan Microparticles with Tunable Morphologies
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Peter J. Smith, Thomas M. Curry, Jeong-Yeh Yang, William J. Barnes, Samantha J. Ziegler, Ashutosh Mittal, Kelley W. Moremen, William S. York, Yannick J. Bomble, Maria J. Peña, and Breeanna R. Urbanowicz
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Biomaterials ,Polymers and Plastics ,Materials Chemistry ,Electronic, Optical and Magnetic Materials - Abstract
Xylans are a diverse family of hemicellulosic polysaccharides found in abundance within the cell walls of nearly all flowering plants. Unfortunately, naturally occurring xylans are highly heterogeneous, limiting studies of their synthesis and structure-function relationships. Here, we demonstrate that xylan synthase 1 from the charophyte alga
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- 2022
9. Appropriate aglycone modification significantly expands the glycan substrate acceptability of α1,6-fucosyltransferase (FUT8)
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Kelley W. Moremen, Lai-Xi Wang, Guanghui Zong, Chao Li, Jeong-Yeh Yang, Roushu Zhang, Bhargavi M. Boruah, Qiang Yang, and Digantkumar Chapla
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Glycan ,Glycosylation ,Ovalbumin ,Stereochemistry ,Gene Expression ,Peptide ,Context (language use) ,HIV Envelope Protein gp120 ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Article ,Substrate Specificity ,03 medical and health sciences ,chemistry.chemical_compound ,Polysaccharides ,Glycosyltransferase ,Animals ,Humans ,Moiety ,Erythropoietin ,Molecular Biology ,Fucosylation ,Fucose ,Glycoproteins ,030304 developmental biology ,chemistry.chemical_classification ,Fluorenes ,0303 health sciences ,biology ,Granulocyte-Macrophage Colony-Stimulating Factor ,Substrate (chemistry) ,Cell Biology ,Fucosyltransferases ,Peptide Fragments ,0104 chemical sciences ,carbohydrates (lipids) ,Kinetics ,HEK293 Cells ,Aglycone ,Carbohydrate Sequence ,chemistry ,HIV-1 ,biology.protein ,Chickens ,Mannose - Abstract
The α1,6-fucosyltransferase, FUT8, is the sole enzyme catalyzing the core-fucosylation of N-glycoproteins in mammalian systems. Previous studies using free N-glycans as acceptor substrates indicated that a terminal β1,2-GlcNAc moiety on the Man-α1,3-Man arm of N-glycan substrates is required for efficient FUT8-catalyzed core-fucosylation. In contrast, we recently demonstrated that, in a proper protein context, FUT8 could also fucosylate Man5GlcNAc2 without a GlcNAc at the non-reducing end. We describe here a further study of the substrate specificity of FUT8 using a range of N-glycans containing different aglycones. We found that FUT8 could fucosylate most of high-mannose and complex-type N-glycans, including highly branched N-glycans from chicken ovalbumin, when the aglycone moiety is modified with a 9-fluorenylmethyloxycarbonyl (Fmoc) moiety or in a suitable peptide/protein context, even if they lack the terminal GlcNAc moiety on the Man-α1,3-Man arm. FUT8 could also fucosylate paucimannose structures when they are on glycoprotein substrates. Such core-fucosylated paucimannosylation is a prominent feature of lysosomal proteins of human neutrophils and several types of cancers. We also found that sialylation of N-glycans significantly reduced their activity as a substrate of FUT8. Kinetic analysis demonstrated that Fmoc aglycone modification could either improve the turnover rate or decrease the KM value depending on the nature of the substrates, thus significantly enhancing the overall efficiency of FUT8 catalyzed fucosylation. Our results indicate that an appropriate aglycone context of N-glycans could significantly broaden the acceptor substrate specificity of FUT8 beyond what has previously been thought.
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- 2021
10. Crystal structures of β-1,4-N-acetylglucosaminyltransferase 2: structural basis for inherited muscular dystrophies
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Jeong Yeh Yang, Jeremy L. Praissman, William N. Lanzilotta, Digantkumar Chapla, Stephanie M. Halmo, Lance Wells, Danish Singh, and Kelley W. Moremen
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Glycosylation ,Stereochemistry ,Crystal structure ,Muscular Dystrophies ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Structural Biology ,Glycosyltransferase ,medicine ,Humans ,Muscular dystrophy ,Dystroglycans ,Walker–Warburg syndrome ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Binding Sites ,biology ,Chemistry ,Glycosyltransferases ,medicine.disease ,Research Papers ,Fusion protein ,HEK293 Cells ,Enzyme ,biology.protein ,030217 neurology & neurosurgery ,Protein Binding ,Limb-girdle muscular dystrophy - Abstract
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 function in humans, the enzyme was expressed as a soluble, secreted fusion protein by transient infection of HEK293 suspension cultures. Here, crystal structures of POMGNT2 (amino-acid residues 25–580) with and without UDP bound are reported. Consistent with a novel fold and a unique domain organization, no molecular-replacement model was available and phases were obtained through crystallization of a selenomethionine variant of the enzyme in the same space group. Tetragonal (space group P4212; unit-cell parameters a = b = 129.8, c = 81.6 Å, α = γ = β = 90°) crystals with UDP bound diffracted to 1.98 Å resolution and contained a single monomer in the asymmetric unit. Orthorhombic (space group P212121; unit-cell parameters a = 142.3, b = 153.9, c = 187.4 Å, α = γ = β = 90°) crystals were also obtained; they diffracted to 2.57 Å resolution and contained four monomers with differential glycosylation patterns and conformations. These structures provide the first rational basis for an explanation of the loss-of-function mutations and offer significant insights into the mechanics of this important human enzyme.
- Published
- 2021
11. Elucidating Human Milk Oligosaccharide biosynthetic genes through network-based multi-omics integration
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Lars Bode, Austin W. T. Chiang, Morey W. Haymond, Natalia Koga, Benjamin P. Kellman, Jeong-Yeh Yang, Nathan E. Lewis, Anne Richelle, Kelley W. Moremen, Julia A Najera, Digantkumar Chapla, Mahmoud A. Mohammad, Anders Bech Bruntse, and Bokan Bao
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Glycan ,Candidate gene ,Pediatric Research Initiative ,Systems biology ,Oligosaccharides ,General Physics and Astronomy ,Computational biology ,General Biochemistry, Genetics and Molecular Biology ,Genetics ,Humans ,Transcription factor ,health care economics and organizations ,Fucosylation ,Nutrition ,chemistry.chemical_classification ,Pediatric ,Multidisciplinary ,Milk, Human ,biology ,Glycosyltransferases ,Infant ,General Chemistry ,Oligosaccharide ,Milk ,chemistry ,biology.protein ,Multi omics ,Biosynthetic genes ,Human - Abstract
Human Milk Oligosaccharides (HMOs) are abundant carbohydrates fundamental to infant health and development. Although these oligosaccharides were discovered more than half a century ago, their biosynthesis in the mammary gland remains largely uncharacterized. Here, we used a systems biology framework that integrated glycan and RNA expression data to construct an HMO biosynthetic network and predict glycosyltransferases involved. To accomplish this, we constructed models describing the most likely pathways for the synthesis of the oligosaccharides accounting for >95% of the HMO content in human milk. Through our models, we propose candidate genes for elongation, branching, fucosylation, and sialylation of HMOs. We further explored selected enzyme activities through kinetic assay and their co-regulation through transcription factor analysis. These results provide the molecular basis of HMO biosynthesis necessary to guide progress in HMO research and application with the ultimate goal of understanding and improving infant health and development.Significance statementWith the HMO biosynthesis network resolved, we can begin to connect genotypes with milk types and thereby connect clinical infant, child and even adult outcomes to specific HMOs and HMO modifications. Knowledge of these pathways can simplify the work of synthetic reproduction of these HMOs providing a roadmap for improving infant, child, and overall human health with the specific application of a newly limitless source of nutraceuticals for infants and people of all ages.
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- 2022
12. Corrigendum to: Identification of key enzymes for pectin synthesis in seed mucilage
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Markus Günl, Kristen A. Engle, Sabine Dieluweit, Cătălin Voiniciuc, Björn Usadel, Debra Mohnen, Jeong-Yeh Yang, Maximilian Schmidt, and Kelley W. Moremen
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Regular Issue ,Hydrolases ,Physiology ,Arabidopsis ,macromolecular substances ,Plant Science ,Plant Mucilage ,Cell Wall ,Polysaccharides ,Genetics ,Glucuronosyltransferase ,Phylogeny ,chemistry.chemical_classification ,Arabidopsis Proteins ,Pectin synthesis ,Glycosyltransferases ,Articles ,Enzyme ,chemistry ,Mucilage ,Biochemistry ,Seeds ,Microscopy, Electron, Scanning ,Key (cryptography) ,Pectins ,Identification (biology) - Abstract
Pectin is a vital component of the plant cell wall and provides the molecular glue that maintains cell-cell adhesion, among other functions. As the most complex wall polysaccharide, pectin is composed of several covalently linked domains, such as homogalacturonan (HG) and rhamnogalacturonan I (RG I). Pectin has widespread uses in the food industry and has emerging biomedical applications, but its synthesis remains poorly understood. For instance, the enzymes that catalyze RG I elongation remain unknown. Recently, a coexpression- and sequence-based
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- 2021
13. Sparse isotope labeling for nuclear magnetic resonance (NMR) of glycoproteins using 13C-glucose
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Robert V Williams, Monique J. Rogals, Jeong-Yeh Yang, I. Jonathan Amster, James H. Prestegard, and Kelley W. Moremen
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Glycan ,Magnetic Resonance Spectroscopy ,Mammalian expression ,Biochemistry ,Endoglycosidase ,Regular Manuscripts ,03 medical and health sciences ,Nuclear magnetic resonance ,Animals ,Humans ,Nuclear Magnetic Resonance, Biomolecular ,Peptide sequence ,Glycoproteins ,030304 developmental biology ,Mammals ,chemistry.chemical_classification ,Carbon Isotopes ,0303 health sciences ,biology ,Isotope ,030302 biochemistry & molecular biology ,13c glucose ,Amino acid ,Glucose ,chemistry ,Isotope Labeling ,biology.protein ,Glycoprotein - Abstract
Preparation of samples for nuclear magnetic resonance (NMR) characterization of larger proteins requires enrichment with less abundant, NMR-active, isotopes such as 13C and 15N. This is routine for proteins that can be expressed in bacterial culture where low-cost isotopically enriched metabolic substrates can be used. However, it can be expensive for glycosylated proteins expressed in mammalian culture where more costly isotopically enriched amino acids are usually used. We describe a simple, relatively inexpensive procedure in which standard commercial media is supplemented with 13C-enriched glucose to achieve labeling of all glycans plus all alanines of the N-terminal domain of the highly glycosylated protein, CEACAM1. We demonstrate an ability to detect partially occupied N-glycan sites, sites less susceptible to processing by an endoglycosidase, and some unexpected truncation of the amino acid sequence. The labeling of both the protein (through alanines) and the glycans in a single culture requiring no additional technical expertise past standard mammalian expression requirements is anticipated to have several applications, including structural and functional screening of the many glycosylated proteins important to human health.
- Published
- 2020
14. A Glycan Array‐Based Assay for the Identification and Characterization of Plant Glycosyltransferases
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Max Peter Bartetzko, Michael G. Hahn, Digantkumar Chapla, Colin Ruprecht, Peter J. Smith, Hyunil Oh, Anna Lakhina, Maria J. Soto, Daniel Varón Silva, Mads Hartvig Clausen, Jeong-Yeh Yang, Deborah Senf, Fabian Pfrengle, Kelley W. Moremen, and Breeanna R. Urbanowicz
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glycan array ,Carbohydrates ,carbohydrates ,010402 general chemistry ,Nucleotide sugar ,01 natural sciences ,Catalysis ,Sugar nucleotide ,Cell wall ,chemistry.chemical_compound ,03 medical and health sciences ,Glycan array ,Biosynthesis ,Cell Wall ,Polysaccharides ,Glycosyltransferase ,plant cell wall ,Monosaccharide ,Sugar nucleotides ,Plant cell wall ,Research Articles ,030304 developmental biology ,2. Zero hunger ,chemistry.chemical_classification ,0303 health sciences ,biology ,Chemistry ,010405 organic chemistry ,Glycosyltransferases ,food and beverages ,General Chemistry ,General Medicine ,Plants ,Oligosaccharide ,0104 chemical sciences ,Biochemistry ,sugar nucleotides ,biology.protein ,Research Article - Abstract
Growing plants with modified cell wall compositions is a promising strategy to improve resistance to pathogens, increase biomass digestibility, and tune other important properties. In order to alter biomass architecture, a detailed knowledge of cell wall structure and biosynthesis is a prerequisite. We report here a glycan array‐based assay for the high‐throughput identification and characterization of plant cell wall biosynthetic glycosyltransferases (GTs). We demonstrate that different heterologously expressed galactosyl‐, fucosyl‐, and xylosyltransferases can transfer azido‐functionalized sugar nucleotide donors to selected synthetic plant cell wall oligosaccharides on the array and that the transferred monosaccharides can be visualized “on chip” by a 1,3‐dipolar cycloaddition reaction with an alkynyl‐modified dye. The opportunity to simultaneously screen thousands of combinations of putative GTs, nucleotide sugar donors, and oligosaccharide acceptors will dramatically accelerate plant cell wall biosynthesis research., Express and screen: A high‐throughput assay based on the application of azido‐functionalized sugar nucleotide donors on a synthetic glycan array for screening heterologously expressed plant glycosyltransferases was developed. The opportunity to express and screen large numbers of glycosyltransferases instead of rationally selected candidates will markedly accelerate the elucidation of plant cell wall biosynthetic pathways.
- Published
- 2020
15. Molecular Mechanism of Polysaccharide Acetylation by the Arabidopsis Xylan O-acetyltransferase XOAT1
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Stephanie A. Archer-Hartmann, Markus Alahuhta, Vivek S. Bharadwaj, Kelley W. Moremen, Yannick J. Bomble, Breeanna R. Urbanowicz, Jeong-Yeh Yang, Michael E. Himmel, Hsin-Tzu Wang, Maria J. Peña, William S. York, Vladimir V. Lunin, and Parastoo Azadi
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Models, Molecular ,0106 biological sciences ,0301 basic medicine ,Magnetic Resonance Spectroscopy ,Protein Conformation ,Stereochemistry ,Arabidopsis ,Plant Science ,Biology ,Arginine ,Crystallography, X-Ray ,01 natural sciences ,Catalysis ,In Brief ,03 medical and health sciences ,Protein structure ,Acetyltransferases ,Cell Wall ,Polysaccharides ,Catalytic Domain ,Glucuronoxylan ,Catalytic triad ,Humans ,Research Articles ,chemistry.chemical_classification ,Arabidopsis Proteins ,Membrane Proteins ,food and beverages ,Acetylation ,Cell Biology ,biology.organism_classification ,Xylan acetylation ,HEK293 Cells ,030104 developmental biology ,chemistry ,Acetyltransferase ,Mutation ,Xylans ,Oxyanion hole ,010606 plant biology & botany - Abstract
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-O-acetylation 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 α/β/α 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.
- Published
- 2020
16. Human CEACAM1 N-domain dimerization is independent from glycan modifications
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Meagan Belcher Dufrisne, Nicole Swope, Marissa Kieber, Jeong-Yeh Yang, Ji Han, Jason Li, Kelley W. Moremen, James H. Prestegard, and Linda Columbus
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Structural Biology ,Antigens, CD ,Polysaccharides ,Humans ,Molecular Biology ,Cell Adhesion Molecules ,Dimerization ,Article - Abstract
Carcinoembryonic cellular adhesion molecules (CEACAMs) serve diverse roles in cell signaling, proliferation, and survival and are made up of one or several immunoglobulin (Ig)-like ectodomains glycosylated in vivo. The physiological oligomeric state and how it contributes to protein function are central to understanding CEACAMs. Two putative dimer conformations involving different CEACAM1 N-terminal Ig-like domain (CCM1) protein faces (ABED and GFCC'C″) were identified from crystal structures. GFCC'C″ was identified as the dominant CCM1 solution dimer, but ambiguity regarding the effect of glycosylation on dimer formation calls its physiological relevance into question. We present the first crystal structure of minimally glycosylated CCM1 in the GFCC'C″ dimer conformation and characterization in solution by continuous-wave and double electron-electron resonance electron paramagnetic resonance spectroscopy. Our results suggest the GFCC'C″ dimer is dominant in solution with different levels of glycosylation, and structural conservation and co-evolved residues support that the GFCC'C″ dimer is conserved across CEACAMs.
- Published
- 2021
17. NMR Resonance Assignment Methodology: Characterizing Large Sparsely Labeled Glycoproteins
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Alexander Eletsky, James H. Prestegard, Laura C. Morris, Jeong Yeh Yang, Robert J. Woods, Kelley W. Moremen, Fang Tian, and Gordon R. Chalmers
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Models, Molecular ,Protein Conformation ,Stereochemistry ,Sialyltransferase ,Substrate analog ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Molecular dynamics ,0302 clinical medicine ,Protein structure ,Structural Biology ,Animals ,Humans ,Nuclear Magnetic Resonance, Biomolecular ,Molecular Biology ,Glycoproteins ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,biology ,Chemistry ,Chemical shift ,Relaxation (NMR) ,Active site ,Sialyltransferases ,Rats ,HEK293 Cells ,biology.protein ,Glycoprotein ,Algorithms ,Software ,030217 neurology & neurosurgery - Abstract
Characterization of proteins using NMR methods begins with assignment of resonances to specific residues. This is usually accomplished using sequential connectivities between nuclear pairs in proteins uniformly labeled with NMR active isotopes. This becomes impractical for larger proteins, and especially for proteins that are best expressed in mammalian cells, including glycoproteins. Here an alternate protocol for the assignment of NMR resonances of sparsely labeled proteins, namely ones labeled with a single amino acid type, or a limited subset of types, isotopically enriched with (15)N or (13)C, is described. The protocol is based on comparison of data collected using extensions of simple two-dimensional NMR experiments (correlated chemical shifts, nuclear Overhauser effects, residual dipolar couplings) to predictions from molecular dynamics trajectories that begin with known protein structures. Optimal pairing of predicted and experimental values is facilitated by a software package that employs a genetic algorithm, ASSIGN_SLP_MD. The approach is applied to the 36kDa luminal domain of the sialyltransferase, rST6Gal1, in which all phenylalanines are labeled with (15)N, and the results are validated by elimination of resonances via single-point mutations of selected phenylalanines to tyrosines. Assignment allows the use of previously published paramagnetic relaxation enhancements to evaluate placement of a substrate analog in the active site of this protein. The protocol will open the way to structural characterization of the many glycosylated and other proteins that are best expressed in mammalian cells.
- Published
- 2019
18. Measurement of residual dipolar couplings in methyl groups via carbon detection
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Kelley W. Moremen, Jeong-Yeh Yang, James H. Prestegard, Robert V Williams, and I. Jonathan Amster
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0301 basic medicine ,Glycosylation ,Magnetic Resonance Spectroscopy ,Protonation ,010402 general chemistry ,Residual ,Methylation ,01 natural sciences ,Biochemistry ,Article ,03 medical and health sciences ,Molecule ,Nuclear Magnetic Resonance, Biomolecular ,Spectroscopy ,Carbon Isotopes ,Spins ,Chemistry ,Proteins ,Pulse sequence ,Carbon ,0104 chemical sciences ,Characterization (materials science) ,Dipole ,030104 developmental biology ,Chemical physics ,Macromolecule - Abstract
Residual dipolar couplings (RDCs) provide both structural and dynamical information useful in the characterization of biological macromolecules. While most data come from the interaction of simple pairs of directly bonded spin-1/2 nuclei ((1)H-(15)N, (1)H-(13)C, (1)H-(1)H), it is possible to acquire data from interactions among the multiple spins of (13)C-labeled methyl groups ((1)H(3)-(13)C). This is especially important because of the advantages that observation of (13)C-labled methyl groups offers in working with very large molecules. Here we consider some of the options for measurement of methyl RDCs in large and often fully protonated proteins and arrive at a pulse sequence that exploits both J-modulation and direct detection of (13)C. Its utility is illustrated by application to a fully protonated two domain fragment from the mammalian glycoprotein, Robo1, (13)C-methyl-labeled in all valines.
- Published
- 2019
19. Synergistic Phytochemicals Fail to Protect Against Ovariectomy Induced Bone Loss in Rats
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Mary Anne Della-Fera, Srujana Rayalam, Natalie M. Hohos, Emily W Kelso, Diane L. Hartzell, Suresh Ambati, Jeong-Yeh Yang, Colette N. Miller, Clifton A. Baile, Emily R. Trunnell, and Erica F. Bass
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medicine.drug_class ,Ovariectomy ,Phytochemicals ,Osteoporosis ,Medicine (miscellaneous) ,Genistein ,030209 endocrinology & metabolism ,Pharmacology ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Bone Density ,Animals ,Humans ,Medicine ,030212 general & internal medicine ,Osteoporosis, Postmenopausal ,Adiposity ,Nutrition and Dietetics ,business.industry ,Drug Synergism ,medicine.disease ,Rats, Inbred F344 ,Rats ,Menopause ,Osteopenia ,Zoledronic acid ,medicine.anatomical_structure ,chemistry ,Phytochemical ,Resveratrol ,Estrogen ,Drug Therapy, Combination ,Female ,Quercetin ,Cortical bone ,business ,medicine.drug - Abstract
Menopause induces a loss of bone as a result of estrogen deficiency. Despite pharmaceutical options for the treatment of osteopenia and osteoporosis, many aging women use dietary supplements with estrogenic activity to prevent bone loss and other menopausal-related symptoms. Such supplements are yet to be tested for efficacy against a Food and Drug Administration (FDA) approved medication for menopausal bone loss such as zoledronic acid (ZA). The postmenopausal rat model was used to investigate the efficacy of various synergistic phytochemical blends mixed into the diet for 16 weeks. Retired-breeder, Fischer 344 rats were randomly assigned to sham or ovariectomy surgery and 4 treatment groups: ZA; genistein supplementation; and a low dose and high dose blend of genistein, resveratrol, and quercetin. Ovariectomy resulted in a loss of both trabecular and cortical bone which was prevented with ZA. The phytochemical blends tested were unable to reverse these losses. Despite the lack of effectiveness in preventing bone loss, a significant dose-response trend was observed in the phytochemical-rich diets in bone adipocyte number compared to ovariectomized control rats. Data from this study indicate that estrogenic phytochemicals are not as efficacious as ZA in preventing menopausal-related bone loss but may have beneficial effects on bone marrow adiposity in rats.
- Published
- 2018
20. Identification of Key Enzymes for Pectin Synthesis in Seed Mucilage
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Sabine Dieluweit, Kristen A. Engle, Maximilian Schmidt, Kelley W. Moremen, Cătălin Voiniciuc, Debra Mohnen, Bjoern Usadel, Jeong-Yeh Yang, and Markus Günl
- Subjects
0106 biological sciences ,0301 basic medicine ,food.ingredient ,Pectin ,biology ,Physiology ,Chemistry ,Mutant ,food and beverages ,Plant Science ,biology.organism_classification ,01 natural sciences ,Xylan ,Cell wall ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,food ,Mucilage ,Biochemistry ,Arabidopsis ,Genetics ,Arabidopsis thaliana ,Hemicellulose ,010606 plant biology & botany - Abstract
Pectin is a vital component of the plant cell wall and provides the molecular glue that maintains cell-cell adhesion, among other functions. As the most complex wall polysaccharide, pectin is composed of several covalently linked domains, such as homogalacturonan (HG) and rhamnogalacturonan I (RG I). Pectin has widespread uses in the food industry and has emerging biomedical applications, but its synthesis remains poorly understood. For instance, the enzymes that catalyze RG I elongation remain unknown. Recently, a coexpression- and sequence-based MUCILAGE-RELATED (MUCI) reverse genetic screen uncovered hemicellulose biosynthetic enzymes in the Arabidopsis (Arabidopsis thaliana) seed coat. Here, we use an extension of this strategy to identify MUCI70 as the founding member of a glycosyltransferase family essential for the accumulation of seed mucilage, a gelatinous wall rich in unbranched RG I. Detailed biochemical and histological characterization of two muci70 mutants and two galacturonosyltransferase11 (gaut11) mutants identified MUCI70 and GAUT11 as required for two distinct RG I domains in seed mucilage. We demonstrate that, unlike MUCI70, GAUT11 catalyzes HG elongation in vitro and, thus, likely is required for the synthesis of an HG region important for RG I elongation. Analysis of a muci70 gaut11 double mutant confirmed that MUCI70 and GAUT11 are indispensable for the production and release of the bulk of mucilage RG I and for shaping the surface morphology of seeds. In addition, we uncover relationships between pectin and hemicelluloses and show that xylan is essential for the elongation of at least one RG I domain.
- Published
- 2018
21. Comparison of human poly-N-acetyl-lactosamine synthase structure with GT-A fold glycosyltransferases supports a modular assembly of catalytic subsites
- Author
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Hyun W. Kim, Zachary A. Wood, Jeong-Yeh Yang, Kelley W. Moremen, Justin H. Sanders, and Renuka Kadirvelraj
- Subjects
0301 basic medicine ,Glycan ,Glycosylation ,Stereochemistry ,Disaccharide ,N-Acetylglucosaminyltransferases ,Nucleotide sugar ,Biochemistry ,glycosyltransferase ,Catalysis ,Substrate Specificity ,03 medical and health sciences ,chemistry.chemical_compound ,Glycolipid ,Glycosyltransferase ,Humans ,enzyme mechanism ,Binding site ,Molecular Biology ,Binding Sites ,030102 biochemistry & molecular biology ,biology ,substrate recognition ,SEC-MALS, size-exclusion chromatography coupled with multiangle light scattering ,Glycosyltransferases ,Amino Sugars ,Cell Biology ,Editors' Pick ,Acceptor ,HEK293 Cells ,030104 developmental biology ,chemistry ,Chromatography, Gel ,biology.protein ,Research Article - Abstract
Poly-N-acetyl-lactosamine (poly-LacNAc) structures are composed of repeating [-Galβ(1,4)-GlcNAcβ(1,3)-]n glycan extensions. They are found on both N- and O-glycoproteins and glycolipids and play an important role in development, immune function, and human disease. The majority of mammalian poly-LacNAc is synthesized by the alternating iterative action of β1,3-N-acetylglucosaminyltransferase 2 (B3GNT2) and β1,4-galactosyltransferases. B3GNT2 is in the largest mammalian glycosyltransferase family, GT31, but little is known about the structure, substrate recognition, or catalysis by family members. Here we report the structures of human B3GNT2 in complex with UDP:Mg2+ and in complex with both UDP:Mg2+ and a glycan acceptor, lacto-N-neotetraose. The B3GNT2 structure conserves the GT-A fold and the DxD motif that coordinates a Mg2+ ion for binding the UDP-GlcNAc sugar donor. The acceptor complex shows interactions with only the terminal Galβ(1,4)-GlcNAcβ(1,3)- disaccharide unit, which likely explains the specificity for both N- and O-glycan acceptors. Modeling of the UDP-GlcNAc donor supports a direct displacement inverting catalytic mechanism. Comparative structural analysis indicates that nucleotide sugar donors for GT-A fold glycosyltransferases bind in similar positions and conformations without conserving interacting residues, even for enzymes that use the same donor substrate. In contrast, the B3GNT2 acceptor binding site is consistent with prior models suggesting that the evolution of acceptor specificity involves loops inserted into the stable GT-A fold. These observations support the hypothesis that GT-A fold glycosyltransferases employ coevolving donor, acceptor, and catalytic subsite modules as templates to achieve the complex diversity of glycan linkages in biological systems.
- Published
- 2020
22. Heterologous expression of plant glycosyltransferases for biochemistry and structural biology
- Author
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Peter J. Smith, William J. Barnes, Maria J. Peña, Breeanna R. Urbanowicz, Hsin-Tzu Wang, Pradeep Kumar Prabhakar, Kelley W. Moremen, and Jeong-Yeh Yang
- Subjects
CAZy ,Glycosylation ,Arabidopsis ,Biology ,Biochemistry ,Article ,Cell wall ,03 medical and health sciences ,chemistry.chemical_compound ,Cell Wall ,Protein purification ,Endopeptidases ,Lignin ,Humans ,Hemicellulose ,Glucans ,030304 developmental biology ,0303 health sciences ,food and beverages ,Glycosyltransferases ,Xyloglucan ,HEK293 Cells ,chemistry ,Structural biology ,Xylans ,Heterologous expression - Abstract
Much of the carbon captured by photosynthesis is converted into the polysaccharides that constitute plant cell walls. These complex macrostructures are composed of cellulose, hemicellulose, and pectins, together with small amounts of structural proteins, minerals, and in many cases lignin. Wall components assemble and interact with one another to produce dynamic structures with many capabilities, including providing mechanical support to plant structures and determining plant cell shape and size. Despite their abundance, major gaps in our knowledge of the synthesis of the building blocks of these polymers remain, largely due to ineffective methods for expression and purification of active synthetic enzymes for in vitro biochemical analyses. The hemicellulosic polysaccharide, xyloglucan, comprises up to 25% of the dry weight of primary cell walls in plants. Most of the knowledge about the glycosyltransferases (GTs) involved in the xyloglucan biosynthetic pathway has been derived from the identification and carbohydrate analysis of knockout mutants, lending little information on how the catalytic biosynthesis of xyloglucan occurs in planta. In this chapter we describe methods for the heterologous expression of plant GTs using the HEK293 expression platform. As a demonstration of the utility of this platform, nine xyloglucan-relevant GTs from three different CAZy families were evaluated, and methods for expression, purification, and construct optimization are described for biochemical and structural characterization.
- Published
- 2020
23. Rational enzyme design for controlled functionalization of acetylated xylan for cell-free polymer biosynthesis
- Author
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Breeanna R. Urbanowicz, Kelley W. Moremen, Yannick J. Bomble, Hsin-Tzu Wang, Vivek S. Bharadwaj, Jeong-Yeh Yang, and Thomas M. Curry
- Subjects
animal structures ,Polymers and Plastics ,Stereochemistry ,Arabidopsis ,Xylan (coating) ,macromolecular substances ,Molecular Dynamics Simulation ,Article ,chemistry.chemical_compound ,Acetyltransferases ,Catalytic Domain ,Catalytic triad ,Materials Chemistry ,Hemicellulose ,Arabidopsis Proteins ,Organic Chemistry ,technology, industry, and agriculture ,Membrane Proteins ,food and beverages ,Substrate (chemistry) ,Acetylation ,Xylan binding ,Protein engineering ,Molecular Docking Simulation ,carbohydrates (lipids) ,chemistry ,Docking (molecular) ,Mutation ,Mutagenesis, Site-Directed ,Xylans ,Protein Binding - Abstract
Xylan O-acetyltransferase 1 (XOAT1) is involved in O-acetylating the backbone of hemicellulose xylan. Recent structural analysis of XOAT1 showed two unequal lobes forming a cleft that is predicted to accommodate and position xylan acceptors into proximity with the catalytic triad. Here, we used docking and molecular dynamics simulations to investigate the optimal orientation of xylan in the binding cleft of XOAT1 and identify putative key residues (Gln445 and Arg444 on Minor lobe & Asn312, Met311 and Asp403 on Major lobe) involved in substrate interactions. Site-directed mutagenesis coupled with biochemical analyses revealed the major lobe of XOAT1 is important for xylan binding. Mutation of single key residues yielded XOAT1 variants with various enzymatic efficiencies that are applicable to one-pot synthesis of xylan polymers with different degrees of O-acetylation. Taken together, our results demonstrate the effectiveness of computational modeling in guiding enzyme engineering aimed at modulating xylan and redesigning plant cell walls.
- Published
- 2021
24. A photo-cross-linking GlcNAc analog enables covalent capture of N-linked glycoprotein-binding partners on the cell surface
- Author
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Connie M. Arthur, Kelley W. Moremen, Sean R. Stowell, Han Wu, Amberlyn M. Wands, Jennifer J. Kohler, Jeong-Yeh Yang, Parastoo Azadi, Emanuela Capota, and Asif Shajahan
- Subjects
Glycan ,Glycosylation ,Surface Properties ,Protein subunit ,Clinical Biochemistry ,Proteomics ,Biochemistry ,Article ,Acetylglucosamine ,chemistry.chemical_compound ,Polysaccharides ,Drug Discovery ,Humans ,Molecular Biology ,Cells, Cultured ,Galectin ,Glycoproteins ,Pharmacology ,chemistry.chemical_classification ,biology ,Glycoprotein binding ,Cell Membrane ,Photochemical Processes ,carbohydrates (lipids) ,Cross-Linking Reagents ,chemistry ,Diazirine ,biology.protein ,Molecular Medicine ,Glycoprotein - Abstract
N-glycans are displayed on cell-surface proteins and can engage in direct binding interactions with membrane-bound and secreted glycan-binding proteins (GBPs). Biochemical identification and characterization of glycan-mediated interactions is often made difficult by low binding affinities. Here we describe the metabolic introduction of a diazirine photo-cross-linker onto N-acetylglucosamine (GlcNAc) residues of N-linked glycoproteins on cell surfaces. We characterize sites at which diazirine-modified GlcNAc is incorporated, as well as modest perturbations to glycan structure. We show that diazirine-modified GlcNAc can be used to covalently cross-link two extracellular GBPs, galectin-1 and cholera toxin subunit B, to cell-surface N-linked glycoproteins. The extent of cross-linking correlates with display of the preferred glycan ligands for the GBPs. In addition, covalently cross-linked complexes could be isolated, and protein components of cross-linked N-linked glycoproteins were identified by proteomics analysis. This method may be useful in the discovery and characterization of binding interactions that depend on N-glycans.
- Published
- 2020
25. hFUT1-Based Live-Cell Assay To Profile α1–2-Fucoside-Enhanced Influenza Virus A Infection
- Author
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Peng Wu, Yi Yang, Chenhua Yu, Geramie Grande, Digantkumar Chapla, Ken Izumori, Senlian Hong, Natalie Reigh, Jeong-Yeh Yang, Jia Xie, and Kelley W. Moremen
- Subjects
0301 basic medicine ,Microbiological Techniques ,Glycan ,Cell Survival ,Host tropism ,Hemagglutinin (influenza) ,Hemagglutinin Glycoproteins, Influenza Virus ,CHO Cells ,Biology ,01 natural sciences ,Biochemistry ,Virus ,Epitope ,Article ,Madin Darby Canine Kidney Cells ,03 medical and health sciences ,Epitopes ,Cricetulus ,Dogs ,Influenza, Human ,Animals ,Humans ,Avidity ,Glycosides ,Cell Engineering ,Tropism ,Fucose ,Host cell surface ,010405 organic chemistry ,General Medicine ,Fucosyltransferases ,Virology ,0104 chemical sciences ,Viral Tropism ,030104 developmental biology ,Influenza A virus ,Host-Pathogen Interactions ,biology.protein ,Molecular Medicine ,sense organs - Abstract
Host cell surface glycans play critical roles in influenza virus A (IVA) infection ranging from modulation of IVA attachment to membrane fusion and host tropism. Approaches for quick and sensitive profile of viral avidity toward a specific type of host cell glycan can contribute to the understanding of tropism switching among different IVA strains. Here, we developed a method based on chemoenzymatic glycan engineering to investigate the possible involvement of α1-2-fucosides in IVA infections. Using a truncated human fucosyltransferase 1 (hFUT1), we created α1-2-fucosides in situ on host cells to assess their influence on the host cell binding to IVA hemagglutinin and the susceptibility of host cells toward IVA-induced killing. We discovered that the newly created α1-2-fucosides on host cells enhanced the infection of several human pandemic IVA subtypes either directly or indirectly. These findings suggest that glycan epitopes other than sialic acid should also be considered for assessing the human pandemic risk of this viral pathogen.
- Published
- 2020
26. Structure of the Xylan O-Acetyltransferase AtXOAT1 Reveals Molecular Insight into Polysaccharide Acetylation in Plants
- Author
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Stephanie A. Archer-Hartmann, Yannick J. Bomble, Parastoo Azadi, Vivek S. Bharadwaj, Markus Alahuhta, Kelley W. Moremen, Vladimir V. Lunin, Jeong-Yeh Yang, Hsin-Tzu Wang, Maria J. Peña, William S. York, Michael E. Himmel, and Breeanna R. Urbanowicz
- Subjects
chemistry.chemical_classification ,0303 health sciences ,010304 chemical physics ,Chemistry ,Stereochemistry ,food and beverages ,01 natural sciences ,Xylan acetylation ,Cell wall ,03 medical and health sciences ,Residue (chemistry) ,Acetylation ,Glucuronoxylan ,Acetyltransferase ,0103 physical sciences ,Catalytic triad ,Oxyanion hole ,030304 developmental biology - Abstract
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.
- Published
- 2020
27. Structural Characterization of a Heparan Sulfate Pentamer Interacting with LAR-Ig1-2
- Author
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Kelley W. Moremen, James H. Prestegard, John G. Flanagan, Jeong-Yeh Yang, and Qi Gao
- Subjects
0301 basic medicine ,Protein Folding ,Pentamer ,Protein domain ,Immunoglobulin domain ,Crystallography, X-Ray ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Protein Domains ,Polysaccharides ,Humans ,Chondroitin sulfate ,Tyrosine ,Nuclear Magnetic Resonance, Biomolecular ,Chemistry ,Receptor-Like Protein Tyrosine Phosphatases, Class 2 ,Heparan sulfate ,0104 chemical sciences ,Molecular Docking Simulation ,HEK293 Cells ,030104 developmental biology ,Fondaparinux ,Receptor-Like Protein Tyrosine Phosphatases ,Biophysics ,Protein folding ,Heparitin Sulfate - Abstract
Leukocyte common antigen-related (LAR) protein is one of the type IIa receptor protein tyrosine phosphatases (RPTPs), which are important for signal transduction in biological processes including axon growth and regeneration. Glycosaminoglycan chains, including heparan sulfate (HS) and chondroitin sulfate (CS), act as ligands that regulate LAR signaling. Here, we report the structural characterization of the first two immunoglobulin domains (Ig1-2) of LAR interacting with an HS pentasaccharide (GlcNS6S-GlcA-GlcNS3,6S-IdoA2S-GlcNS6S-OME, fondaparinux) using multiple solution-based NMR methods. In the course of the study, we extended an assignment strategy useful for sparsely labeled proteins expressed in mammalian cell culture supplemented with a single type of isotopically enriched amino acid ((15)N-lysine in this case) by including paramagnetic perturbations to NMR resonances. The folded two domain structure for LAR-Ig1-2 seen in previous crystal structures has been validated in solution using residual dipolar coupling data, and a combination of chemical shift perturbation on titration of LAR-Ig1-2 with fondaparinux, saturation transfer difference (STD) spectra and transferred nuclear Overhauser effect (trNOEs) have been employed in the docking program, HADDOCK, to generate models for the LAR-fondaparinux complex. These models are further analyzed by post-processing energetic analysis to identify key binding interactions. In addition to providing insight into the ligand interaction mechanisms of type IIa RPTPs and the origin of opposing effects of CS and HS ligands, these results may assist in future design of therapeutic compounds for nervous system repair.
- Published
- 2018
28. Structural, mutagenic and in silico studies of xyloglucan fucosylation in Arabidopsis thaliana suggest a water‐mediated mechanism
- Author
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Maria J. Peña, Sami T. Tuomivaara, William S. York, Michael E. Himmel, Yannick J. Bomble, Kelley W. Moremen, Breeanna R. Urbanowicz, Markus Alahuhta, Jeong Yeh Yang, Shuo Wang, Vladimir V. Lunin, Michael F. Crowley, and Vivek S. Bharadwaj
- Subjects
0301 basic medicine ,Glycosylation ,Fucosyltransferase ,In silico ,Arabidopsis ,Plant Science ,Molecular Dynamics Simulation ,Crystallography, X-Ray ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Glycosyltransferase ,Genetics ,Arabidopsis thaliana ,Glucans ,Fucosylation ,biology ,Arabidopsis Proteins ,Mutagenesis ,Water ,Active site ,Cell Biology ,Fucosyltransferases ,biology.organism_classification ,Models, Structural ,Xyloglucan ,030104 developmental biology ,chemistry ,Biochemistry ,biology.protein ,Xylans - Abstract
The mechanistic underpinnings of the complex process of plant polysaccharide biosynthesis are poorly understood, largely because of the resistance of glycosyltransferase (GT) enzymes to structural characterization. In Arabidopsis thaliana, a glycosyl transferase family 37 (GT37) fucosyltransferase 1 (AtFUT1) catalyzes the regiospecific transfer of terminal 1,2-fucosyl residues to xyloglucan side chains - a key step in the biosynthesis of fucosylated sidechains of galactoxyloglucan. We unravel the mechanistic basis for fucosylation by AtFUT1 with a multipronged approach involving protein expression, X-ray crystallography, mutagenesis experiments and molecular simulations. Mammalian cell culture expressions enable the sufficient production of the enzyme for X-ray crystallography, which reveals the structural architecture of AtFUT1 in complex with bound donor and acceptor substrate analogs. The lack of an appropriately positioned active site residue as a catalytic base leads us to propose an atypical water-mediated fucosylation mechanism facilitated by an H-bonded network, which is corroborated by mutagenesis experiments as well as detailed atomistic simulations.
- Published
- 2017
29. Structural Aspects of Glycosylated Protein–Glycan Interactions by NMR
- Author
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Robert V Williams, Digantkumar Chapla, I Jon Amster, Jeong-Yeh Yang, Gordon R. Chalmers, Alexandre Eletsky, James H. Prestegard, Laura C. Morris, and Kelley W. Moremen
- Subjects
Glycan ,Glycosylated protein ,biology ,Biochemistry ,Chemistry ,Genetics ,biology.protein ,Molecular Biology ,Biotechnology - Published
- 2019
30. Association mapping, transcriptomics, and transient expression identify candidate genes mediating plant-pathogen interactions in a tree
- Author
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Gerald A. Tuskan, Kerrie Barry, Jeff H. Chang, Erika Lindquist, Jin Zhang, Juan Franco-Coronado, Yongil Yang, Robert Brueggeman, Priya Ranjan, Kelley W. Moremen, Jeong-Yeh Yang, Jeremy Schmutz, Jin-Gui Chen, Breeanna R. Urbanowicz, Jared M. LeBoldus, Sara S. Jawdy, Kelsey L. Søndreli, Wellington Muchero, Vasanth R. Singan, Nivi Abraham, and Alexandra J. Weisberg
- Subjects
0106 biological sciences ,0301 basic medicine ,Populus trichocarpa ,Candidate gene ,disease resistance ,Locus (genetics) ,Receptors, Cell Surface ,Plant disease resistance ,01 natural sciences ,Mannose-Binding Lectin ,Chromosomes, Plant ,Chromosomes ,Transcriptome ,03 medical and health sciences ,Gene Expression Regulation, Plant ,Receptors ,Genetics ,septoria canker ,Plant Immunity ,Allele ,Association mapping ,association mapping ,Alleles ,Plant Proteins ,Plant Diseases ,Multidisciplinary ,biology ,Gene Expression Profiling ,fungi ,food and beverages ,Chromosome Mapping ,Plant ,15. Life on land ,Biological Sciences ,biology.organism_classification ,Gene expression profiling ,030104 developmental biology ,Populus ,Gene Expression Regulation ,Genetic Loci ,Saccharomycetales ,Cell Surface ,Host-Pathogen Interactions ,invasive disease ,Protein Kinases ,010606 plant biology & botany - Abstract
Significance International trade has resulted in the introduction of plant diseases into natural ecosystems around the world. These introductions have potentially catastrophic impacts on ecosystem structure and function. Leveraging genomic tools, natural variation within a tree species, and a high-throughput phenotyping platform, we present a framework that can be broadly applied to rapidly identify candidate genes associated with resistance and susceptibility to introduced plant diseases. The unprecedented speed and accuracy with which the candidate genes can be identified in woody trees demonstrates the potential of genomics to mitigate the impacts of invasive diseases on forest health., Invasive microbes causing diseases such as sudden oak death negatively affect ecosystems and economies around the world. The deployment of resistant genotypes for combating introduced diseases typically relies on breeding programs that can take decades to complete. To demonstrate how this process can be accelerated, we employed a genome-wide association mapping of ca. 1,000 resequenced Populus trichocarpa trees individually challenged with Sphaerulina musiva, an invasive fungal pathogen. Among significant associations, three loci associated with resistance were identified and predicted to encode one putative membrane-bound L-type receptor-like kinase and two receptor-like proteins. A susceptibility-associated locus was predicted to encode a putative G-type D-mannose–binding receptor-like kinase. Multiple lines of evidence, including allele analysis, transcriptomics, binding assays, and overexpression, support the hypothesized function of these candidate genes in the P. trichocarpa response to S. musiva.
- Published
- 2018
31. Appropriate aglycone modification significantly expands the glycan substrate acceptability of a1,6-fucosyltransferase (FUT8).
- Author
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Roushu Zhang, Qiang Yang, Boruah, Bhargavi M., Guanghui Zong, Chao Li, Chapla, Digantkumar, Jeong-Yeh Yang, Moremen, Kelley W., and Lai-Xi Wang
- Subjects
MOIETIES (Chemistry) ,FUCOSYLATION ,AGLYCONES ,NEUTROPHILS ,OVALBUMINS ,PROTEINS ,GLYCANS - Abstract
The a1,6-fucosyltransferase, FUT8, is the sole enzyme catalyzing the core-fucosylation of N-glycoproteins in mammalian systems. Previous studies using free N-glycans as acceptor substrates indicated that a terminal ß1,2-GlcNAc moiety on the Man-a1,3-Man arm of N-glycan substrates is required for efficient FUT8-catalyzed core-fucosylation. In contrast, we recently demonstrated that, in a proper protein context, FUT8 could also fucosylate Man
5 GlcNAc2 without a GlcNAc at the non-reducing end. We describe here a further study of the substrate specificity of FUT8 using a range of N-glycans containing different aglycones. We found that FUT8 could fucosylate most of high-mannose and complex-type N-glycans, including highly branched N-glycans from chicken ovalbumin, when the aglycone moiety is modified with a 9-fluorenylmethyloxycarbonyl (Fmoc) moiety or in a suitable peptide/protein context, even if they lack the terminal GlcNAc moiety on the Man-a1,3-Man arm. FUT8 could also fucosylate paucimannose structures when they are on glycoprotein substrates. Such core-fucosylated paucimannosylation is a prominent feature of lysosomal proteins of human neutrophils and several types of cancers. We also found that sialylation of N-glycans significantly reduced their activity as a substrate of FUT8. Kinetic analysis demonstrated that Fmoc aglycone modification could either improve the turnover rate or decrease the KM value depending on the nature of the substrates, thus significantly enhancing the overall efficiency of FUT8 catalyzed fucosylation. Our results indicate that an appropriate aglycone context of N-glycans could significantly broaden the acceptor substrate specificity of FUT8 beyond what has previously been thought. [ABSTRACT FROM AUTHOR]- Published
- 2021
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32. Correction: Glycosylation alters dimerization properties of a cell-surface signaling protein, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1)
- Author
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You Zhuo, Jeong-Yeh Yang, Kelley W. Moremen, and James H. Prestegard
- Subjects
Cell Biology ,Molecular Biology ,Biochemistry - Published
- 2020
33. Paramagnetic Tag for Glycosylation Sites in Glycoproteins: Structural Constraints on Heparan Sulfate Binding to Robo1
- Author
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Kelley W. Moremen, Geert-Jan Boons, Jeong-Yeh Yang, Qi Gao, Yuejie Zhao, Digantkumar Chapla, Maria J Moure, I. Jonathan Amster, Laura C. Morris, James H. Prestegard, Chengli Zong, and Alexander Eletsky
- Subjects
0301 basic medicine ,Glycosylation ,Stereochemistry ,Nerve Tissue Proteins ,Ligands ,Biochemistry ,Article ,Turn (biochemistry) ,Cyclic N-Oxides ,03 medical and health sciences ,chemistry.chemical_compound ,Residue (chemistry) ,Moiety ,Humans ,Receptors, Immunologic ,Nuclear Magnetic Resonance, Biomolecular ,Glycoproteins ,chemistry.chemical_classification ,Electron Spin Resonance Spectroscopy ,General Medicine ,Heparan sulfate ,Molecular Docking Simulation ,030104 developmental biology ,HEK293 Cells ,chemistry ,Click chemistry ,Molecular Medicine ,Heparan sulfate binding ,Click Chemistry ,Heparitin Sulfate ,Glycoprotein ,Protein Binding - Abstract
An enzyme- and click chemistry-mediated methodology for the site-specific nitroxide spin labeling of glycoproteins has been developed and applied. The procedure relies on the presence of single N-glycosylation sites that are present natively in proteins or that can be engineered into glycoproteins by mutational elimination of all but one glycosylation site. Recombinantly expressing glycoproteins in HEK293S (GnT1-) cells results in N-glycans with high-mannose structures that can be processed to leave a single GlcNAc residue. This can in turn be modified by enzymatic addition of a GalNAz residue that is subject to reaction with an alkyne-carrying TEMPO moiety using copper (I)-catalyzed Click Chemistry. To illustrate the procedure, we have made an application to a two-domain construct of Robo1, a protein that carries a single N-glycosylation site in its N-terminal domains. The construct has also been labeled with (15)N at amide nitrogens of lysine residues to provide a set of sites that are used to derive an effective location of the paramagnetic nitroxide moiety of the TEMPO group. This, in turn, allowed measurements of paramagnetic perturbations to the spectra of a new high affinity heparan sulfate ligand. Calculation of distance constraints from these data facilitated determination of an atomic level model for the docked complex.
- Published
- 2018
34. A two-phase model for the non-processive biosynthesis of homogalacturonan polysaccharides by the GAUT1:GAUT7 complex
- Author
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Kelley W. Moremen, Debra Mohnen, Jeong-Yeh Yang, Melani A. Atmodjo, Breeanna R. Urbanowicz, Robert A. Amos, and Sivakumar Pattathil
- Subjects
0301 basic medicine ,Enzyme complex ,Glycan ,Static Electricity ,Arabidopsis ,Biochemistry ,Models, Biological ,Substrate Specificity ,Cell wall ,03 medical and health sciences ,Glycosyltransferase ,Humans ,Glucuronosyltransferase ,Molecular Biology ,chemistry.chemical_classification ,biology ,Molecular Structure ,Arabidopsis Proteins ,Substrate (chemistry) ,Cell Biology ,Processivity ,Oligosaccharide ,Uridine Diphosphate Sugars ,030104 developmental biology ,HEK293 Cells ,chemistry ,Biophysics ,biology.protein ,Enzymology ,Pectins ,Elongation - Abstract
Homogalacturonan (HG) is a pectic glycan in the plant cell wall that contributes to plant growth and development and cell wall structure and function, and interacts with other glycans and proteoglycans in the wall. HG is synthesized by the galacturonosyltransferase (GAUT) gene family. Two members of this family, GAUT1 and GAUT7, form a heteromeric enzyme complex in Arabidopsis thaliana. Here, we established a heterologous GAUT expression system in HEK293 cells and show that co-expression of recombinant GAUT1 with GAUT7 results in the production of a soluble GAUT1:GAUT7 complex that catalyzes elongation of HG products in vitro. The reaction rates, progress curves, and product distributions exhibited major differences dependent upon small changes in the degree of polymerization (DP) of the oligosaccharide acceptor. GAUT1:GAUT7 displayed >45-fold increased catalytic efficiency with DP11 acceptors relative to DP7 acceptors. Although GAUT1:GAUT7 synthesized high-molecular-weight polymeric HG (>100 kDa) in a substrate concentration–dependent manner typical of distributive (nonprocessive) glycosyltransferases with DP11 acceptors, reactions primed with short-chain acceptors resulted in a bimodal product distribution of glycan products that has previously been reported as evidence for a processive model of GT elongation. As an alternative to the processive glycosyltransfer model, a two-phase distributive elongation model is proposed in which a slow phase, which includes the de novo initiation of HG and elongation of short-chain acceptors, is distinguished from a phase of rapid elongation of intermediate- and long-chain acceptors. Upon reaching a critical chain length of DP11, GAUT1:GAUT7 elongates HG to high-molecular-weight products.
- Published
- 2018
35. Identification of an algal xylan synthase indicates that there is functional orthology between algal and plant cell wall biosynthesis
- Author
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Christian Peter Poulsen, Peter Ulvskov, Curtis G. Wilkerson, Malene Hessellund Dinesen, Michael Melkonian, Helle Juel Martens, Jonatan U. Fangel, Peter J. Smith, Breeanna R. Urbanowicz, Paul Dupree, Jesper Harholt, Kelley W. Moremen, Henrik Vibe Scheller, Jeong Yeh Yang, Maria J. Peña, Jacob Krüger Jensen, Gane Ka-Shu Wong, Marta Busse-Wicher, Dupree, Paul [0000-0001-9270-6286], and Apollo - University of Cambridge Repository
- Subjects
0301 basic medicine ,animal structures ,Physiology ,Evolution ,Charophyceae ,Amino Acid Motifs ,Plant Biology & Botany ,Plant Science ,macromolecular substances ,Biology ,xylan ,Cell wall ,Transcriptome ,Evolution, Molecular ,03 medical and health sciences ,Algae ,Phylogenetics ,Cell Wall ,Plant Cells ,Botany ,evolution ,Humans ,Pentosyltransferases ,IRX10 ,Gene ,Phylogeny ,Phylogenetic tree ,Full Paper ,Agricultural and Veterinary Sciences ,Research ,food and beverages ,Molecular ,Full Papers ,Biological Sciences ,biology.organism_classification ,Xylan ,Biosynthetic Pathways ,Klebsormidium nitens ,030104 developmental biology ,HEK293 Cells ,XYS1 ,Klebsormidium flaccidum ,Heterologous expression ,biosynthesis - Abstract
© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust Insights into the evolution of plant cell walls have important implications for comprehending these diverse and abundant biological structures. In order to understand the evolving structure–function relationships of the plant cell wall, it is imperative to trace the origin of its different components. The present study is focused on plant 1,4-β-xylan, tracing its evolutionary origin by genome and transcriptome mining followed by phylogenetic analysis, utilizing a large selection of plants and algae. It substantiates the findings by heterologous expression and biochemical characterization of a charophyte alga xylan synthase. Of the 12 known gene classes involved in 1,4-β-xylan formation, XYS1/IRX10 in plants, IRX7, IRX8, IRX9, IRX14 and GUX occurred for the first time in charophyte algae. An XYS1/IRX10 ortholog from Klebsormidium flaccidum, designated K. flaccidumXYLAN SYNTHASE-1 (KfXYS1), possesses 1,4-β-xylan synthase activity, and 1,4-β-xylan occurs in the K. flaccidum cell wall. These data suggest that plant 1,4-β-xylan originated in charophytes and shed light on the origin of one of the key cell wall innovations to occur in charophyte algae, facilitating terrestrialization and emergence of polysaccharide-based plant cell walls.
- Published
- 2018
36. Pig Induced Pluripotent Stem Cell-Derived Neural Rosettes Developmentally Mimic Human Pluripotent Stem Cell Neural Differentiation
- Author
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Robin L. Webb, Rachel West, Kai Wang, Erin T. Jordan, Franklin D. West, Amalia Gallegos-Cardenas, Jeong-Yeh Yang, and Steven L. Stice
- Subjects
Stage-Specific Embryonic Antigens ,Rosette Formation ,Swine ,Neurogenesis ,Induced Pluripotent Stem Cells ,ved/biology.organism_classification_rank.species ,Retinoic acid ,Biology ,Cell therapy ,chemistry.chemical_compound ,Neural Stem Cells ,Species Specificity ,Animals ,Humans ,Induced pluripotent stem cell ,Model organism ,Neural cell ,Cells, Cultured ,Homeodomain Proteins ,Genetics ,Otx Transcription Factors ,ved/biology ,Cell Biology ,Hematology ,Cell biology ,Neuroepithelial cell ,chemistry ,Octamer Transcription Factor-3 ,Neural development ,Developmental Biology - Abstract
For diseases of the brain, the pig (Sus scrofa) is increasingly being used as a model organism that shares many anatomical and biological similarities with humans. We report that pig induced pluripotent stem cells (iPSC) can recapitulate events in early mammalian neural development. Pig iPSC line (POU5F1(high)/SSEA4(low)) had a higher potential to form neural rosettes (NR) containing neuroepithelial cells than either POU5F1(low)/SSEA4(low) or POU5F1(low)/SSEA4(high) lines. Thus, POU5F1 and SSEA4 pluripotency marker profiles in starting porcine iPSC populations can predict their propensity to form more robust NR populations in culture. The NR were isolated and expanded in vitro, retaining their NR morphology and neuroepithelial molecular properties. These cells expressed anterior central nervous system fate markers OTX2 and GBX2 through at least seven passages, and responded to retinoic acid, promoting a more posterior fate (HOXB4+, OTX2-, and GBX2-). These findings offer insight into pig iPSC development, which parallels the human iPSC in both anterior and posterior neural cell fates. These in vitro similarities in early neural differentiation processes support the use of pig iPSC and differentiated neural cells as a cell therapy in allogeneic porcine neural injury and degeneration models, providing relevant translational data for eventual human neural cell therapies.
- Published
- 2015
37. Comparison of human poly-N-acetyl-lactosamine synthase structure with GT-A fold glycosyltransferases supports a modular assembly of catalytic subsites.
- Author
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Kadirvelraj, Renuka, Jeong-Yeh Yang, Kim, Hyun W., Sanders, Justin H., Moremen, Kelley W., and Wood, Zachary A.
- Subjects
- *
GLYCOSYLTRANSFERASES , *GLYCOLIPIDS , *GLYCANS , *BIOLOGICAL systems , *BINDING sites , *HUMAN beings , *SUGAR - Abstract
Poly-N-acetyl-lactosamine (poly-LacNAc) structures are composed of repeating [-Galβ(1,4)-GlcNAcβ(1,3)-]n glycan extensions. They are found on both N- and O-glycoproteins and glycolipids and play an important role in development, immune function, and human disease. The majority of mammalian poly-LacNAc is synthesized by the alternating iterative action of β1,3-N-acetylglucosaminyltransferase 2 (B3GNT2) and β1,4-galactosyltransferases. B3GNT2 is in the largest mammalian glycosyltransferase family, GT31, but little is known about the structure, substrate recognition, or catalysis by family members. Here we report the structures of human B3GNT2 in complex with UDP:Mg2+ and in complex with both UDP:Mg2+ and a glycan acceptor, lacto-N-neotetraose. The B3GNT2 structure conserves the GT-A fold and the DxD motif that coordinates aMg2+ ion for binding the UDP-GlcNAc sugar donor. The acceptor complex shows interactions with only the terminal Galβ(1,4)-GlcNAcβ(1,3)-disaccharide unit, which likely explains the specificity for both N- and O-glycan acceptors. Modeling of the UDP-GlcNAc donor supports a direct displacement inverting catalytic mechanism. Comparative structural analysis indicates that nucleotide sugar donors for GT-A fold glycosyltransferases bind in similar positions and conformations without conserving interacting residues, even for enzymes that use the same donor substrate. In contrast, the B3GNT2 acceptor binding site is consistent with prior models suggesting that the evolution of acceptor specificity involves loops inserted into the stable GT-A fold. These observations support the hypothesis that GT-A fold glycosyltransferases employ coevolving donor, acceptor, and catalytic subsite modules as templates to achieve the complex diversity of glycan linkages in biological systems. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
38. A dietary phytochemical blend prevents liver damage associated with adipose tissue mobilization in ovariectomized rats
- Author
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Suresh Ambati, Srujana Rayalam, Jeong-Yeh Yang, Colette N. Miller, Clifton A. Baile, Mary Anne Della-Fera, and Tucker Avra
- Subjects
medicine.medical_specialty ,Nutrition and Dietetics ,business.industry ,Endocrinology, Diabetes and Metabolism ,Fatty liver ,Medicine (miscellaneous) ,Adipose tissue ,Genistein ,medicine.disease ,chemistry.chemical_compound ,Endocrinology ,Phytochemical ,chemistry ,Lipotoxicity ,Internal medicine ,medicine ,Ovariectomized rat ,Vitamin D and neurology ,business ,Quercetin - Abstract
Objective Menopausal reduction in estrogen causes increased adipose accumulation, leading many to turn to dietary supplements to prevent and treat such changes. Enhanced adipose mobilization stimulated by some supplements can increase the risk of non-alcoholic fatty liver disease (NAFLD). Cytoprotective and anti-obesity compounds may prevent the lipotoxicity associated with mobilization. Methods A phytochemical blend was tested in aged, ovariectomized rats. Rats were given the AIN-93M basal diet or a diet containing varying doses of phytochemicals with 2.4 IU/g vitamin D [diet 1: 1000 mg/kg genistein (G); diet 2: 500 mg/kg (G), 200 mg/kg resveratrol (R), and 1000 mg/kg quercetin (Q); diet 3: 1000 mg/kg (G), 400 mg/kg (R), and 2000 mg/kg (Q)]. Results Serum free fatty acids and hepatic triglycerides were elevated with diets 2 and 3. Despite this increase, the phytochemical blends did not increase apoptotic, cell repair, or remodeling gene expression. The highest phytochemical dose prevented increases in serum alanine aminotransferase. Conclusions Adverse hepatic effects secondary to ovariectomy were mitigated through the inclusion of a dietary phytochemical blend in aged ovariectomized rats. The use of such compounds may not only help with weight management and disease risk in menopausal women, but may also prevent the lipotoxicity in NAFLD.
- Published
- 2014
39. Expression system for structural and functional studies of human glycosylation enzymes
- Author
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Digantkumar Chapla, Harry J. Gilbert, Shuo Wang, Melissa Stuart, Zhongwei Gao, Rosa, Alison V. Nairn, Farhad Forouhar, Jason Steel, Heather A. Moniz, Joshua LaBaer, Gagandeep Kaur Gahlay, S.C. Wu, Donald L. Jarvis, Liang Tong, Lu Meng, Roy W. Johnson, Christoph Geisler, Jeong-Yeh Yang, Annapoorani Ramiah, Pradeep Kumar Prabhakar, Jayaraman Seetharaman, and Kelley W. Moremen
- Subjects
0301 basic medicine ,Glycosylation ,Insecta ,Glycoside Hydrolases ,Sialyltransferase ,Genetic Vectors ,010402 general chemistry ,Crystallography, X-Ray ,01 natural sciences ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Glycosyltransferase ,Cytidine Monophosphate ,Animals ,Humans ,Glycoside hydrolase ,Molecular Biology ,chemistry.chemical_classification ,Expression vector ,biology ,Glycobiology ,Gene Expression Profiling ,Cell Biology ,Fusion protein ,Recombinant Proteins ,Sialyltransferases ,0104 chemical sciences ,Kinetics ,030104 developmental biology ,Enzyme ,HEK293 Cells ,Biochemistry ,chemistry ,biology.protein ,Sulfotransferases ,Baculoviridae - Abstract
Vertebrate glycoproteins and glycolipids are synthesized in complex biosynthetic pathways localized predominantly within membrane compartments of the secretory pathway. The enzymes that catalyze these reactions are exquisitely specific, yet few have been extensively characterized because of challenges associated with their recombinant expression as functional products. We used a modular approach to create an expression vector library encoding all known human glycosyltransferases, glycoside hydrolases, and sulfotransferases, as well as other glycan-modifying enzymes. We then expressed the enzymes as secreted catalytic domain fusion proteins in mammalian and insect cell hosts, purified and characterized a subset of the enzymes, and determined the structure of one enzyme, the sialyltransferase ST6GalNAcII. Many enzymes were produced at high yields and at similar levels in both hosts, but individual protein expression levels varied widely. This expression vector library will be a transformative resource for recombinant enzyme production, broadly enabling structure-function studies and expanding applications of these enzymes in glycochemistry and glycobiology.
- Published
- 2017
40. AGlycan Array-Based Assay for the Identification and Characterization of Plant Glycosyltransferases.
- Author
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Ruprecht, Colin, Bartetzko, Max P., Senf, Deborah, Lakhina, Anna, Smith, Peter J., Soto, Maria J., Oh, Hyunil, Jeong-Yeh Yang, Chapla, Digantkumar, Varon Silva, Daniel, Clausen, Mads H., Hahn, Michael G., Moremen, Kelley W., Urbanowicz, Breeanna R., and Pfrengle, Fabian
- Subjects
PLANT identification ,PLANT cell walls ,GLYCOSYLTRANSFERASES ,DYE-sensitized solar cells ,RING formation (Chemistry) ,PHOTOVOLTAIC cells ,MONOSACCHARIDES ,SUGAR - Abstract
Growing plants with modified cell wall compositions is a promising strategy to improve resistance to pathogens, increase biomass digestibility, and tune other important properties. In order to alter biomass architecture, a detailed knowledge of cell wall structure and biosynthesis is a prerequisite. We report here a glycan array‐based assay for the high‐throughput identification and characterization of plant cell wall biosynthetic glycosyltransferases (GTs). We demonstrate that different heterologously expressed galactosyl‐, fucosyl‐, and xylosyltransferases can transfer azido‐functionalized sugar nucleotide donors to selected synthetic plant cell wall oligosaccharides on the array and that the transferred monosaccharides can be visualized “on chip” by a 1,3‐dipolar cycloaddition reaction with an alkynyl‐modified dye. The opportunity to simultaneously screen thousands of combinations of putative GTs, nucleotide sugar donors, and oligosaccharide acceptors will dramatically accelerate plant cell wall biosynthesis research. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
41. α-1,3-Galactosyltransferase Knockout Pig Induced Pluripotent Stem Cells: A Cell Source for the Production of Xenotransplant Pigs
- Author
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Jessica M. Hutcheson, Steven L. Stice, Yubing Liu, C Robert Dove, Franklin D. West, Jennifer Mumaw, Yangqing Lu, Jeong Yeh Yang, and Ping Yu
- Subjects
Homeobox protein NANOG ,Swine ,Xenotransplantation ,medicine.medical_treatment ,Induced Pluripotent Stem Cells ,Transplantation, Heterologous ,Gene Expression ,Biology ,SOX2 ,medicine ,Animals ,Humans ,Induced pluripotent stem cell ,Cells, Cultured ,Cell Biology ,Galactosyltransferases ,Antigens, Differentiation ,Embryonic stem cell ,Cell biology ,Gene Knockdown Techniques ,Immunology ,Somatic cell nuclear transfer ,Stem cell ,Reprogramming ,Stem Cell Transplantation ,Transcription Factors ,Developmental Biology ,Biotechnology - Abstract
The shortage of human organs and tissues for transplant has led to significant interest in xenotransplantation of pig tissues for human patients. However, transplantation of pig organs results in an acute immune rejection, leading to death of the organ within minutes. The α-1,3-galactosyltransferase (GALT) gene has been knocked out in pigs to reduce rejection, yet additional genes need to be modified to ultimately make pig tissue immunocompatible with humans. The development of pig induced pluripotent stem cells (piPSCs) from GALT knockout (GALT-KO) tissue would provide an excellent cell source for complex genetic manipulations (e.g., gene targeting) that often require highly robust and proliferative cells. In this report, we generated GALT-KO piPSCs by the overexpression of POU5F1, SOX2, NANOG, LIN28, KLF-4, and C-MYC reprogramming genes. piPSCs showed classical stem cell morphology and characteristics, expressing integrated reprogramming genes in addition to the pluripotent markers AP, SSEA1, and SSEA4. GALT-KO piPSCs were highly proliferative and possessed doubling times and telomerase activity similar to human embryonic stem cells. These results demonstrated successful reprogramming of GALT-KO fibroblasts into GALT-KO piPSCs. GALT-KO piPSCs are potentially an excellent immortal cell source for the generation of pigs with complex genetic modifications for xenotransplantation, somatic cell nuclear transfer, or chimera formation.
- Published
- 2013
42. Glycosylation Alters Dimerization Properties of a Cell-surface Signaling Protein, Carcinoembryonic Antigen-related Cell Adhesion Molecule 1 (CEACAM1)
- Author
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James H. Prestegard, You Zhuo, Jeong-Yeh Yang, and Kelley W. Moremen
- Subjects
0301 basic medicine ,Glycosylation ,Dimer ,Cell ,Glycobiology and Extracellular Matrices ,Biochemistry ,Protein Structure, Secondary ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Antigens, CD ,medicine ,Molecule ,Humans ,Cell adhesion ,Protein Structure, Quaternary ,Molecular Biology ,Nuclear Magnetic Resonance, Biomolecular ,Cell adhesion molecule ,Chemistry ,Cell Biology ,Adhesion ,carbohydrates (lipids) ,030104 developmental biology ,medicine.anatomical_structure ,HEK293 Cells ,Residual dipolar coupling ,030220 oncology & carcinogenesis ,Biophysics ,Additions and Corrections ,Protein Multimerization ,Cell Adhesion Molecules - Abstract
Human carcinoembryonic antigen-related cell adhesion molecule 1 (C?/Au: EACAM1) is a cell-surface signaling molecule involved in cell adhesion, proliferation, and immune response. It is also implicated in cancer angiogenesis, progression, and metastasis. This diverse set of effects likely arises as a result of the numerous homophilic and heterophilic interactions that CEACAM1 can have with itself and other molecules. Its N-terminal Ig variable (IgV) domain has been suggested to be a principal player in these interactions. Previous crystal structures of the β-sandwich-like IgV domain have been produced using Escherichia coli-expressed material, which lacks native glycosylation. These have led to distinctly different proposals for dimer interfaces, one involving interactions of ABED β-strands and the other involving GFCC′C″ β-strands, with the former burying one prominent glycosylation site. These structures raise questions as to which form may exist in solution and what the effect of glycosylation may have on this form. Here, we use NMR cross-correlation measurements to examine the effect of glycosylation on CEACAM1-IgV dimerization and use residual dipolar coupling (RDC) measurements to characterize the solution structure of the non-glycosylated form. Our findings demonstrate that even addition of a single N-linked GlcNAc at potential glycosylation sites inhibits dimer formation. Surprisingly, RDC data collected on E. coli expressed material in solution indicate that a dimer using the non-glycosylated GFCC′C″ interface is preferred even in the absence of glycosylation. The results open new questions about what other factors may facilitate dimerization of CEACAM1 in vivo, and what roles glycosylation may play in heterophylic interactions.
- Published
- 2016
43. Structural basis for regulation of human calcium-sensing receptor by magnesium ions and an unexpected tryptophan derivative co-agonist
- Author
-
Anthony L. Schilmiller, Tuo Zhang, Kenneth Huang, Jenny J. Yang, Rakshya Gorkhali, Jian Hu, Cassandra Lynn Miller, Edward M. Brown, Kelley W. Moremen, Shuo Wang, Jeong Yeh Yang, Juan Zou, and Chen Zhang
- Subjects
0301 basic medicine ,inorganic chemicals ,Models, Molecular ,G protein ,Calcium Sensing Receptor ,Molecular Conformation ,Mg2+ ,Ligands ,Biochemistry ,Cell Line ,03 medical and health sciences ,chemistry.chemical_compound ,L-Phe ,Structure-Activity Relationship ,0302 clinical medicine ,Heterotrimeric G protein ,Aromatic amino acids ,TNCA ,Humans ,Magnesium ,Magnesium ion ,Research Articles ,G protein-coupled receptor ,chemistry.chemical_classification ,Ions ,Multidisciplinary ,Chemistry ,Tryptophan ,SciAdv r-articles ,Ligand (biochemistry) ,3. Good health ,Amino acid ,EC50 ,030104 developmental biology ,Binding Affinity ,Mutation ,Crystal Structure ,Calcium-sensing receptor ,Receptors, Calcium-Sensing ,030217 neurology & neurosurgery ,Research Article ,Protein Binding - Abstract
Structural and functional characterization of the extracellular domain of the human CaSR with bound Mg2+ and a tryptophan derivative., Ca2+-sensing receptors (CaSRs) modulate calcium and magnesium homeostasis and many (patho)physiological processes by responding to extracellular stimuli, including divalent cations and amino acids. We report the first crystal structure of the extracellular domain (ECD) of human CaSR bound with Mg2+ and a tryptophan derivative ligand at 2.1 Å. The structure reveals key determinants for cooperative activation by metal ions and aromatic amino acids. The unexpected tryptophan derivative was bound in the hinge region between two globular ECD subdomains, and represents a novel high-affinity co-agonist of CaSR. The dissection of structure-function relations by mutagenesis, biochemical, and functional studies provides insights into the molecular basis of human diseases arising from CaSR mutations. The data also provide a novel paradigm for understanding the mechanism of CaSR-mediated signaling that is likely shared by the other family C GPCR [G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptor] members and can facilitate the development of novel CaSR-based therapeutics.
- Published
- 2016
44. Metabolomic Response of Human Embryonic Stem Cell-Derived Germ-like Cells After Exposure to Steroid Hormones
- Author
-
W. Matthew Henderson, Ping Yu, Steven L. Stice, Jeong-Yeh Yang, Mary Alice Smith, and Franklin D. West
- Subjects
medicine.medical_specialty ,Cell type ,medicine.medical_treatment ,Endogeny ,Biology ,Toxicology ,Steroid ,Metabolomics ,Internal medicine ,medicine ,Humans ,Testosterone ,Embryonic Stem Cells ,Progesterone ,Cell Proliferation ,Estradiol ,Flow Cytometry ,Embryonic stem cell ,Cell biology ,Steroid hormone ,Germ Cells ,Endocrinology ,medicine.anatomical_structure ,Germ cell ,Hormone - Abstract
To assess the potential risks of human exposure to endocrine active compounds (EACs), the mechanisms of toxicity must first be identified and characterized. Currently, there are no robust in vitro models for identifying the mechanisms of toxicity in germ cells resulting from EAC exposure. Human embryonic stem cells can differentiate into numerous functional cell types including germ-like cells (GLCs). These cells possess characteristics indicative of a germ cell state, suggesting they offer a novel system to investigate the consequences of chemical exposure on normal germ cell processes. To characterize these processes, a metabolomic-based approach was employed to determine the response of GLCs following exposure to 0.001, 0.01, 0.1, 1, 10, or 100µM estradiol, testosterone, or progesterone for 48h. Following exposure, cellular extracts underwent gas chromatography coupled with mass spectrometry analysis. Models were then constructed using principal component analysis on acquired spectra to discriminate among steroid hormones as well as doses for each hormone. t-test comparisons generated a preliminary list of metabolites that were statistically significant in GLC's biochemical response to these steroid hormones. Steroid hormone exposures caused fluxes in intracellular pathways such as amino acid synthesis and metabolism, fatty acid synthesis, as well as cholesterol and lipoprotein metabolism. Further pathway analysis, based on these identified metabolites, will aid in modeling the response of GLCs to endogenous steroid hormones and allow for identification of biomarkers delineating germ cell-based developmental and reproductive pathways.
- Published
- 2012
45. Effect of resveratrol on fat mobilization
- Author
-
Jeong-Yeh Yang, Diane L. Hartzell, Ching-Yi Lai, Clifton A. Baile, Mary Anne Della-Fera, Srujana Rayalam, and Charlotte Andersen
- Subjects
medicine.medical_specialty ,General Neuroscience ,Genistein ,Adipose tissue ,Biology ,Resveratrol ,General Biochemistry, Genetics and Molecular Biology ,chemistry.chemical_compound ,Endocrinology ,History and Philosophy of Science ,chemistry ,Adipogenesis ,Internal medicine ,Adipocyte ,Lipogenesis ,medicine ,Ovariectomized rat ,Lipolysis - Abstract
Higher levels of body fat are associated with increased risk for development of numerous adverse health conditions. Phytochemicals are potential agents to inhibit differentiation of preadipocytes, stimulate lipolysis, and induce apoptosis of existing adipocytes, thereby reducing adipose tissue mass. Resveratrol decreased adipogenesis and viability in maturing preadipocytes; these effects were mediated not only through down-regulating adipocyte specific transcription factors and enzymes but also by genes that modulate mitochondrial function. Additionally, resveratrol increased lipolysis and reduced lipogenesis in mature adipocytes. In addition, combining resveratrol with other natural products produced synergistic activities from actions on multiple molecular targets in the adipocyte life cycle. Treatment of mice with resveratrol alone was shown to improve resistance to weight gain caused by a high-fat diet. Moreover, dietary supplementation of aged ovariectomized rats with a combination of resveratrol and vitamin D, quercetin, and genistein not only decreased weight gain but also inhibited bone loss. Combining several phytochemicals, including resveratrol, or using them as templates for synthesizing new drugs, provides a large potential for using phytochemicals to target adipocyte adipogenesis, apoptosis, and lipolysis.
- Published
- 2011
46. Publisher Correction: Network inference from glycoproteomics data reveals new reactions in the IgG glycosylation pathway
- Author
-
Manfred Wuhrer, Ozren Polasek, Igor Rudan, Ivo Ugrina, Antti Hassinen, Harald Grallert, Kelley W. Moremen, Thomas Meitinger, Elisa Benedetti, Lucija Klaric, Tatiana V. Ovchinnikova, Sakari Kellokumpu, Fabian J. Theis, Nicolai V. Bovin, Konstantin Strauch, Jerko Štambuk, Toma Keser, Maurice H. J. Selman, Christian Gieger, Jan Krumsiek, Irena Trbojević-Akmačić, Jeong-Yeh Yang, Annette Peters, Maja Pučić-Baković, Gordan Lauc, Annika Wahl, Lin Liu, Caroline Hayward, Genadij Razdorov, Geert-Jan Boons, and Marija Vilaj
- Subjects
Multidisciplinary ,Published Erratum ,business.industry ,Science ,General Physics and Astronomy ,Inference ,General Chemistry ,Computational biology ,Biology ,Igg glycosylation ,Article ,General Biochemistry, Genetics and Molecular Biology ,Glycoproteomics ,Text mining ,lcsh:Q ,business ,lcsh:Science - Abstract
Immunoglobulin G (IgG) is a major effector molecule of the human immune response, and aberrations in IgG glycosylation are linked to various diseases. However, the molecular mechanisms underlying protein glycosylation are still poorly understood. We present a data-driven approach to infer reactions in the IgG glycosylation pathway using large-scale mass-spectrometry measurements. Gaussian graphical models are used to construct association networks from four cohorts. We find that glycan pairs with high partial correlations represent enzymatic reactions in the known glycosylation pathway, and then predict new biochemical reactions using a rule-based approach. Validation is performed using data from a GWAS and results from three in vitro experiments. We show that one predicted reaction is enzymatically feasible and that one rejected reaction does not occur in vitro. Moreover, in contrast to previous knowledge, enzymes involved in our predictions colocalize in the Golgi of two cell lines, further confirming the in silico predictions., IgG glycosylation is an important factor in immune function, yet the molecular details of protein glycosylation remain poorly understood. The data-driven approach presented here uses large-scale plasma IgG mass spectrometry measurements to infer new biochemical reactions in the glycosylation pathway.
- Published
- 2018
47. Octanoate and Decanoate Induce Apoptosis in 3T3-L1 Adipocytes
- Author
-
Suresh Ambati, Jeong-Yeh Yang, Clifton A. Baile, Mary Anne Della-Fera, and Srujana Rayalam
- Subjects
medicine.medical_specialty ,Cell Survival ,Medicine (miscellaneous) ,Apoptosis ,Biology ,Mice ,chemistry.chemical_compound ,3T3-L1 Cells ,Adipocyte ,Internal medicine ,Adipocytes ,medicine ,Animals ,Viability assay ,bcl-2-Associated X Protein ,chemistry.chemical_classification ,Reactive oxygen species ,Nutrition and Dietetics ,Caspase 3 ,Cytochrome c ,Decanoates ,Cytochromes c ,3T3-L1 ,Molecular biology ,Mitochondria ,Enzyme ,Endocrinology ,Proto-Oncogene Proteins c-bcl-2 ,chemistry ,Cell culture ,biology.protein ,Caprylates ,Poly(ADP-ribose) Polymerases ,Reactive Oxygen Species ,Signal Transduction - Abstract
The effect of octanoate and decanoate, respectively, eight- and 10-carbon medium-chain fatty acids (MCFAs), on apoptotic signaling in 3T3-L1 adipocytes was investigated. 3T3-L1 adipocytes were treated with various concentrations of octanoate or decanoate. Cell viability, apoptosis, and expression of apoptosis-related proteins were investigated. Results indicated that both octanoate and decanoate decreased viability, increased apoptosis, and increased reactive oxygen species production. Immunoblotting analysis showed an increase in the levels of cytoplasmic cytochrome c and cleaved poly(ADP-ribose) polymerase by octanoate and decanoate. Concomitantly, we observed that pro-caspase-3 was decreased, resulting in the induced accumulation of the cleaved form of caspase-3 by both octanoate and decanoate. In addition, both octanoate and decanoate increased the expression of pro-apoptotic Bax with an accompanied decrease of anti-apoptotic Bcl-2. These results show that octanoate and decanoate mediate adipocyte apoptosis via a caspase-dependent mitochondrial pathway in 3T3-L1 adipocytes. MCFAs thus decrease adipocyte number by initiating the apoptotic process in 3T3-L1 adipocytes.
- Published
- 2009
48. Anti-Obesity Effects of Xanthohumol Plus Guggulsterone in 3T3-L1 Adipocytes
- Author
-
Jeong-Yeh Yang, Hea Jin Park, Srujana Rayalam, Mary Anne Della-Fera, Suresh Ambati, and Clifton A. Baile
- Subjects
medicine.medical_specialty ,Cell Survival ,Lipolysis ,Down-Regulation ,Gene Expression ,Medicine (miscellaneous) ,Apoptosis ,Biology ,Mice ,chemistry.chemical_compound ,Western blot ,Pregnenediones ,3T3-L1 Cells ,Internal medicine ,Adipocytes ,medicine ,Animals ,Obesity ,Viability assay ,Commiphora ,bcl-2-Associated X Protein ,Flavonoids ,Propiophenones ,Adipogenesis ,Nutrition and Dietetics ,medicine.diagnostic_test ,Plant Extracts ,3T3-L1 ,Lipid Metabolism ,Endocrinology ,Proto-Oncogene Proteins c-bcl-2 ,chemistry ,Cell culture ,Caspases ,Xanthohumol ,Drug Therapy, Combination ,Guggulsterone ,Anti-Obesity Agents ,Phytotherapy - Abstract
Xanthohumol (XN) and guggulsterone (GS) have each been shown to inhibit adipogenesis and induce apoptosis in adipocytes. In the present study effects of the combination of XN + GS on 3T3-L1 adipocyte apoptosis and adipogenesis were investigated. Mature adipocytes were treated with XN and GS individually and in combination. XN and GS individually decreased cell viability, but XN + GS caused an enhanced decrease in viability and potentiated induction of apoptosis. Likewise, XN + GS caused a potentiated increase in caspase-3/7 activation, whereas neither of the compounds showed any effect individually. In addition, western blot analysis revealed that XN + GS increased Bax expression and decreased Bcl-2 expression, whereas individual compounds did not show any significant effect. XN and GS both decreased lipid accumulation. Individually, XN at 1.5 microM and GS at 3.12 microM decreased lipid accumulation by 26 +/- 4.5% (P.001) each, whereas XN1.5 + GS3.12 decreased lipid accumulation by 78.2 +/- 1.8% (P.001). Moreover, expression of the adipocyte-specific proteins was down-regulated with XN1.5 + GS3.12, but no effect was observed with the individual compounds. Finally, XN + GS caused an enhanced stimulation of lipolysis. Thus, combination of XN and GS is more potent in exerting anti-obesity effects than additive effects of the individual compounds.
- Published
- 2009
49. Combined Effects of Genistein, Quercetin, and Resveratrol in Human and 3T3-L1 Adipocytes
- Author
-
Jeong-Yeh Yang, Hea Jin Park, Clifton A. Baile, Dorothy B. Hausman, Srujana Rayalam, Mary Anne Della-Fera, and Suresh Ambati
- Subjects
medicine.medical_specialty ,Medicine (miscellaneous) ,Genistein ,Apoptosis ,Glycerolphosphate Dehydrogenase ,Phytoestrogens ,Resveratrol ,Antioxidants ,Mice ,chemistry.chemical_compound ,3T3-L1 Cells ,Adipocyte ,Internal medicine ,Stilbenes ,Adipocytes ,medicine ,Animals ,Humans ,Obesity ,Viability assay ,Cells, Cultured ,Adipogenesis ,Nutrition and Dietetics ,Drug Synergism ,3T3-L1 ,Lipids ,Endocrinology ,chemistry ,Drug Therapy, Combination ,Female ,Quercetin ,Anti-Obesity Agents - Abstract
The natural compounds genistein (G), quercetin (Q), and resveratrol (R) have been reported to each exhibit anti-adipogenic activities in adipocytes and antiproliferative and pro-apoptotic activities in several cell types. We studied the combined effects of G, Q, and R on adipogenesis and apoptosis in primary human adipocytes (HAs) and 3T3-L1 murine adipocyte (MAs). Combined treatment with 6.25 microM G, 12.5 microM Q, and 12.5 microM R during the 14-day differentiation period caused an enhanced inhibition of lipid accumulation in maturing HAs that was greater than the responses to individual compounds and to the calculated additive response. Glycerol 3-phosphate dehydrogenase activity, a marker of late adipocyte differentiation, was decreased markedly in HAs treated with the combination of G+Q+R. In addition, combined treatment with 50 microM G, 100 microM Q, and 100 microM R for 3 days decreased cell viability and induced apoptosis in early- and mid- phase maturing and lipid-filled mature HAs. In contrast, no compound alone induced apoptosis. Oil Red O stain and Hoechst 33342 stain were performed to confirm the effects on lipid accumulation and apoptosis, respectively. We also determined whether MAs responded to the combination treatment similarly to HAs. As in HAs, G+Q+R treatment decreased lipid accumulation in maturing MAs and increased apoptosis in pre- and lipid-filled mature MAs more than the responses to G, Q, and R when used separately. These results show that lower concentrations of combined treatments with several natural compounds may be useful for treatments for obesity through the suppression of adipogenesis and enhanced adipocyte apoptosis.
- Published
- 2008
50. Resveratrol induces apoptosis and inhibits adipogenesis in 3T3-L1 adipocytes
- Author
-
Mary Anne Della-Fera, Srujana Rayalam, Clifton A. Baile, Suresh Ambati, and Jeong-Yeh Yang
- Subjects
Pharmacology ,medicine.medical_specialty ,biology ,SIRT3 ,food and beverages ,3T3-L1 ,Resveratrol ,chemistry.chemical_compound ,Endocrinology ,chemistry ,Apoptosis ,Adipogenesis ,Adipocyte ,Internal medicine ,Sirtuin ,biology.protein ,medicine ,Viability assay - Abstract
Resveratrol, a phytoallexin, has recently been reported to slow aging by acting as a sirtuin activator. Resveratrol also has a wide range of pharmacological effects on adipocytes. In this study, we investigated the effects of resveratrol on adipogenesis and apoptosis using 3T3-L1 cells. In mature adipocytes, 100 and 200 µM resveratrol decreased cell viability dose-dependently by 23 ± 2.7%, and 75.3 ± 2.8% (p < 0.0001), respectively, after 48 h treatment, and 100 µM resveratrol increased apoptosis by 76 ± 8.7% (p < 0.0001). Resveratrol at 25 and 50 µM decreased lipid accumulation in maturing preadipocytes significantly by 43 ± 1.27% and 94.3 ± 0.3% (p < 0.0001) and decreased cell viability by 25 ± 1.3% and 70.4 ± 1.6% (p < 0.0001), respectively. In order to understand the anti-adipogenic effects of resveratrol, maturing 3T3-L1 preadipocytes were treated with 25 µM resveratrol and the change in the expression of several adipogenic transcription factors and enzymes was investigated using real-time RT-PCR. Resveratrol down-regulated the expression of PPARγ, C/EBPα, SREBP-1c, FAS, HSL, LPL and up-regulated the expression of genes regulating mitochondrial activity (SIRT3, UCP1 and Mfn2). These results indicate that resveratrol may alter fat mass by directly affecting cell viability and adipogenesis in maturing preadipocytes and inducing apoptosis in adipocytes and thus may have applications for the treatment of obesity. Copyright © 2008 John Wiley & Sons, Ltd.
- Published
- 2008
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