23 results on '"Ramiah, Annapoorani"'
Search Results
2. Structural basis for heparan sulfate co-polymerase action by the EXT1–2 complex
- Author
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Li, Hua, Chapla, Digantkumar, Amos, Robert A., Ramiah, Annapoorani, Moremen, Kelley W., and Li, Huilin
- Published
- 2023
- Full Text
- View/download PDF
3. Human N -acetylglucosaminyltransferase II substrate recognition uses a modular architecture that includes a convergent exosite
- Author
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Kadirvelraj, Renuka, Yang, Jeong-Yeh, Sanders, Justin H., Liu, Lin, Ramiah, Annapoorani, Prabhakar, Pradeep Kumar, Boons, Geert-Jan, Wood, Zachary A., and Moremen, Kelley W.
- Published
- 2018
4. Abstract 1524: Modular Economical One-Pot Multienzyme Synthesis of Complex Glycans
- Author
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Huang, Chin, primary, Gao, Zhongwei, additional, Ramiah, Annapoorani, additional, Zheng, Zhifeng, additional, and Moremen, Kelley, additional
- Published
- 2023
- Full Text
- View/download PDF
5. Abstract 1388: Structural basis for heparan sulfate co-polymerase action by the EXT1-2 complex
- Author
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Moremen, Kelley, primary, Li, Hua, additional, Chapla, Digantkumar, additional, Amos, Robert, additional, Ramiah, Annapoorani, additional, and Li, Huilin, additional
- Published
- 2023
- Full Text
- View/download PDF
6. Characterizing human α-1,6-fucosyltransferase (FUT8) substrate specificity and structural similarities with related fucosyltransferases
- Author
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Boruah, Bhargavi M, Kadirvelraj, Renuka, Liu, Lin, Ramiah, Annapoorani, Li, Chao, Zong, Guanghui, Bosman, Gerlof P, Yang, Jeong-Yeh, Wang, Lai-Xi, Boons, Geert-Jan, Wood, Zachary A, Moremen, Kelley W, Afd Chemical Biology and Drug Discovery, Sub Chemical Biology and Drug Discovery, Chemical Biology and Drug Discovery, Afd Chemical Biology and Drug Discovery, Sub Chemical Biology and Drug Discovery, and Chemical Biology and Drug Discovery
- Subjects
0301 basic medicine ,Glycan ,Protein Folding ,Fucosyltransferase ,Glycobiology and Extracellular Matrices ,Crystallography, X-Ray ,Biochemistry ,glycosyltransferase ,Substrate Specificity ,Fucosyltransferases ,03 medical and health sciences ,N-linked glycosylation ,Protein Domains ,Humans ,enzyme mechanism ,Binding site ,Molecular Biology ,Fucosylation ,030102 biochemistry & molecular biology ,biology ,Glycobiology ,Chemistry ,substrate recognition ,Active site ,Cell Biology ,030104 developmental biology ,HEK293 Cells ,Structural Homology, Protein ,biology.protein - Abstract
Mammalian Asn-linked glycans are extensively processed as they transit the secretory pathway to generate diverse glycans on cell surface and secreted glycoproteins. Additional modification of the glycan core by α-1,6-fucose addition to the innermost GlcNAc residue (core fucosylation) is catalyzed by an α-1,6-fucosyltransferase (FUT8). The importance of core fucosylation can be seen in the complex pathological phenotypes of FUT8 null mice, which display defects in cellular signaling, development, and subsequent neonatal lethality. Elevated core fucosylation has also been identified in several human cancers. However, the structural basis for FUT8 substrate specificity remains unknown. Here, using various crystal structures of FUT8 in complex with a donor substrate analog, and with four distinct glycan acceptors, we identify the molecular basis for FUT8 specificity and activity. The ordering of three active site loops corresponds to an increased occupancy for bound GDP, suggesting an induced-fit folding of the donor-binding subsite. Structures of the various acceptor complexes were compared with kinetic data on FUT8 active site mutants and with specificity data from a library of glycan acceptors to reveal how binding site complementarity and steric hindrance can tune substrate affinity. The FUT8 structure was also compared with other known fucosyltransferases to identify conserved and divergent structural features for donor and acceptor recognition and catalysis. These data provide insights into the evolution of modular templates for donor and acceptor recognition among GT-B fold glycosyltransferases in the synthesis of diverse glycan structures in biological systems.
- Published
- 2020
7. Integrated Approach to Identify Heparan Sulfate Ligand Requirements of Robo1
- Author
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Zong, Chengli, Huang, Rongrong, Condac, Eduard, Chiu, Yulun, Xiao, Wenyuan, Li, Xiuru, Lu, Weigang, Ishihara, Mayumi, Wang, Shuo, Ramiah, Annapoorani, Stickney, Morgan, Azadi, Parastoo, Amster, I Jonathan, Moremen, Kelley W, Wang, Lianchun, Sharp, Joshua S, Boons, Geert-Jan, Sub Chemical pharmacology, Sub Algemeen Scheikunde, Medicinal Chemistry and Chemical Biology, Sub Chemical pharmacology, Sub Algemeen Scheikunde, and Medicinal Chemistry and Chemical Biology
- Subjects
0301 basic medicine ,Size-exclusion chromatography ,Nerve Tissue Proteins ,Ligands ,010402 general chemistry ,Mass spectrometry ,01 natural sciences ,Biochemistry ,Chemical synthesis ,DNA-binding protein ,Article ,Catalysis ,03 medical and health sciences ,chemistry.chemical_compound ,Colloid and Surface Chemistry ,Cell Movement ,ROBO1 ,Humans ,Receptors, Immunologic ,Receptor ,Chromatography ,Chemistry ,General Chemistry ,Heparan sulfate ,Ligand (biochemistry) ,0104 chemical sciences ,030104 developmental biology ,Heparitin Sulfate ,Protein Binding - Abstract
An integrated methodology is described to establish ligand requirements for heparan sulfate (HS) binding proteins based on a workflow in which HS octasaccharides are produced by partial enzymatic degradation of natural HS followed by size exclusion purification, affinity enrichment using an immobilized HS-binding protein of interest, putative structure determination of isolated compounds by a hydrophilic interaction chromatography-high-resolution mass spectrometry platform, and chemical synthesis of well-defined HS oligosaccharides for structure-activity relationship studies. The methodology was used to establish the ligand requirements of human Roundabout receptor 1 (Robo1), which is involved in a number of developmental processes. Mass spectrometric analysis of the starting octasaccharide mixture and the Robo1-bound fraction indicated that Robo1 has a preference for a specific set of structures. Further analysis was performed by sequential permethylation, desulfation, and pertrideuteroacetylation followed by online separation and structural analysis by MS/MS. Sequences of tetrasaccharides could be deduced from the data, and by combining the compositional and sequence data, a putative octasaccharide ligand could be proposed (GlA-GlcNS6S-IdoA-GlcNS-IdoA2S-GlcNS6S-IdoA-GlcNAc6S). A modular synthetic approach was employed to prepare the target compound, and binding studies by surface plasmon resonance (SPR) confirmed it to be a high affinity ligand for Robo1. Further studies with a number of tetrasaccharides confirmed that sulfate esters at C-6 are critical for binding, whereas such functionalities at C-2 substantially reduce binding. High affinity ligands were able to reverse a reduction in endothelial cell migration induced by Slit2-Robo1 signaling.
- Published
- 2016
8. Structural Aspects of Heparan Sulfate Binding to Robo1-Ig1-2
- Author
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Gao, Qi, Chen, Cheng-Yu, Zong, Chengli, Wang, Shuo, Ramiah, Annapoorani, Prabhakar, Pradeep, Morris, Laura C, Boons, Geert-Jan, Moremen, Kelley W, Prestegard, James H, Medicinal Chemistry and Chemical Biology, and Medicinal Chemistry and Chemical Biology
- Subjects
0301 basic medicine ,Glycosylation ,Magnetic Resonance Spectroscopy ,Protein family ,Static Electricity ,Nerve Tissue Proteins ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Tetramer ,ROBO1 ,Binding site ,Receptors, Immunologic ,chemistry.chemical_classification ,Molecular Structure ,Chemistry ,General Medicine ,Nuclear magnetic resonance spectroscopy ,Heparan sulfate ,0104 chemical sciences ,Amino acid ,Molecular Docking Simulation ,030104 developmental biology ,Molecular Medicine ,Heparan sulfate binding ,Heparitin Sulfate - Abstract
Roundabout 1, or Robo1, is a cell surface signaling molecule important in axon guidance. Its interaction with heparan sulfate (HS) and members of the Slit protein family is essential to its activity, making characterization of these interactions by structural methods, such as NMR, highly desirable. However, the fact that Robo1 is a glycosylated protein prevents employment of commonly used bacterial hosts for expression of properly glycosylated forms with the uniform (15)N, (13)C, and (2)H labeling needed for NMR studies. Here, we apply an alternative methodology, based on labeling with a single amino acid type and high structural content NMR data, to characterize a two-domain construct of glycosylated Robo1 (Robo1-Ig1-2) interacting with a synthetic HS tetramer (IdoA-GlcNS6S-IdoA2S-GlcNS6S-(CH2)5NH2). Significant chemical shift perturbations of the crosspeak from K81 on titration with the tetramer provide initial evidence for the location of a binding site and allow determination of a 255 μM disassociation constant. The binding epitopes, bound conformation, and binding site placement of the HS tetramer have been further characterized by saturation transfer difference (STD), transferred nuclear Overhauser effect (trNOE), and paramagnetic perturbation experiments. A model of the complex has been generated using constraints derived from the various NMR experiments. Postprocessing energetic analysis of this model provides a rationale for the role each glycan residue plays in the binding event, and examination of the binding site in the context of a previous Robo-Slit structure provides a rationale for modulation of Robo-Slit interactions by HS.
- Published
- 2016
9. Expression system for structural and functional studies of human glycosylation enzymes
- Author
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Moremen, Kelley W, primary, Ramiah, Annapoorani, additional, Stuart, Melissa, additional, Steel, Jason, additional, Meng, Lu, additional, Forouhar, Farhad, additional, Moniz, Heather A, additional, Gahlay, Gagandeep, additional, Gao, Zhongwei, additional, Chapla, Digantkumar, additional, Wang, Shuo, additional, Yang, Jeong-Yeh, additional, Prabhakar, Pradeep Kumar, additional, Johnson, Roy, additional, Rosa, Mitche dela, additional, Geisler, Christoph, additional, Nairn, Alison V, additional, Seetharaman, Jayaraman, additional, Wu, Sheng-Cheng, additional, Tong, Liang, additional, Gilbert, Harry J, additional, LaBaer, Joshua, additional, and Jarvis, Donald L, additional
- Published
- 2017
- Full Text
- View/download PDF
10. Enzymatic Basis for N-Glycan Sialylation: STRUCTURE OF RAT α2,6-SIALYLTRANSFERASE (ST6GAL1) REVEALS CONSERVED AND UNIQUE FEATURES FOR GLYCAN SIALYLATION*
- Author
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Meng, Lu, Forouhar, Farhad, Thieker, David, Gao, Zhongwei, Ramiah, Annapoorani, Moniz, Heather, Xiang, Yong, Seetharaman, Jayaraman, Milaninia, Sahand, Su, Min, Bridger, Robert, Veillon, Lucas, Azadi, Parastoo, Kornhaber, Gregory, Wells, Lance, Montelione, Gaetano T., Woods, Robert J., Tong, Liang, and Moremen, Kelley W.
- Subjects
Binding Sites ,Bacteria ,Protein Conformation ,Swine ,Glycobiology and Extracellular Matrices ,Molecular Dynamics Simulation ,Crystallography, X-Ray ,Sialyltransferases ,Rats ,carbohydrates (lipids) ,Molecular Docking Simulation ,Structure-Activity Relationship ,Polysaccharides ,embryonic structures ,cardiovascular system ,Sialic Acids ,Animals ,beta-D-Galactoside alpha 2-6-Sialyltransferase - Abstract
Glycan structures on glycoproteins and glycolipids play critical roles in biological recognition, targeting, and modulation of functions in animal systems. Many classes of glycan structures are capped with terminal sialic acid residues, which contribute to biological functions by either forming or masking glycan recognition sites on the cell surface or secreted glycoconjugates. Sialylated glycans are synthesized in mammals by a single conserved family of sialyltransferases that have diverse linkage and acceptor specificities. We examined the enzymatic basis for glycan sialylation in animal systems by determining the crystal structures of rat ST6GAL1, an enzyme that creates terminal α2,6-sialic acid linkages on complex-type N-glycans, at 2.4 Å resolution. Crystals were obtained from enzyme preparations generated in mammalian cells. The resulting structure revealed an overall protein fold broadly resembling the previously determined structure of pig ST3GAL1, including a CMP-sialic acid-binding site assembled from conserved sialylmotif sequence elements. Significant differences in structure and disulfide bonding patterns were found outside the sialylmotif sequences, including differences in residues predicted to interact with the glycan acceptor. Computational substrate docking and molecular dynamics simulations were performed to predict and evaluate the CMP-sialic acid donor and glycan acceptor interactions, and the results were compared with kinetic analysis of active site mutants. Comparisons of the structure with pig ST3GAL1 and a bacterial sialyltransferase revealed a similar positioning of donor, acceptor, and catalytic residues that provide a common structural framework for catalysis by the mammalian and bacterial sialyltransferases.
- Published
- 2013
11. Crystal structure and mutagenesis studies of mammalian fucosyltransferase‐9 (FUT9) reveals the catalytic core and substrate specificity
- Author
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Boruah, Bhargavi M, primary, Kadirvalraj, Renu, additional, Wang, Shuo, additional, Ramiah, Annapoorani, additional, Wood, Zachary A, additional, and Moremen, Kelley W, additional
- Published
- 2016
- Full Text
- View/download PDF
12. An investigation on ZnO photocatalysed oxidation of uracil
- Author
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M.R. Dhananjeyan, Rajalingam Renganathan, and Ramiah Annapoorani
- Subjects
Reaction mechanism ,Langmuir ,Aqueous solution ,General Chemical Engineering ,General Physics and Astronomy ,Uracil ,General Chemistry ,Photochemistry ,Catalysis ,chemistry.chemical_compound ,Reaction rate constant ,chemistry ,Photocatalysis ,Uracil glycol - Abstract
The semiconductor ZnO mediated photo-oxidation (λirr = 365 nm) of a biologically important compound, uracil, carried out in aqueous suspension reveals certain features: (i) intial rate dependence on [uracil]o, (ii) Langmuir adsorption behavior, (iii) initial linear dependence of rate on the amount of ZnO followed by saturation behavior at higher values, and (iv) influence of pH and [Cu2+] on rate. The involvement of some radical intermediates in the reaction was hinted at by the rate retarding influence of [acrylamide]. A mechanism involving the oxidation of uracil induced by OH and hvb+ leading to the formation of uracil glycol is proposed.
- Published
- 1997
13. High Structural Resolution Hydroxyl Radical Protein Footprinting Reveals an Extended Robo1-Heparin Binding Interface
- Author
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Li, Zixuan, primary, Moniz, Heather, additional, Wang, Shuo, additional, Ramiah, Annapoorani, additional, Zhang, Fuming, additional, Moremen, Kelley W., additional, Linhardt, Robert J., additional, and Sharp, Joshua S., additional
- Published
- 2015
- Full Text
- View/download PDF
14. B4GAT1 is the priming enzyme for the LARGE-dependent functional glycosylation of α-dystroglycan
- Author
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Praissman, Jeremy L, primary, Live, David H, additional, Wang, Shuo, additional, Ramiah, Annapoorani, additional, Chinoy, Zoeisha S, additional, Boons, Geert-Jan, additional, Moremen, Kelley W, additional, and Wells, Lance, additional
- Published
- 2014
- Full Text
- View/download PDF
15. Author response: B4GAT1 is the priming enzyme for the LARGE-dependent functional glycosylation of α-dystroglycan
- Author
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Praissman, Jeremy L, primary, Live, David H, additional, Wang, Shuo, additional, Ramiah, Annapoorani, additional, Chinoy, Zoeisha S, additional, Boons, Geert-Jan, additional, Moremen, Kelley W, additional, and Wells, Lance, additional
- Published
- 2014
- Full Text
- View/download PDF
16. Integrated Approach to Identify Heparan Sulfate Ligand Requirements of Robo1.
- Author
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Chengli Zong, Rongrong Huang, Condac, Eduard, Yulun Chiu, Wenyuan Xiao, Xiuru Li, Weigang Lu, Mayumi Ishihara, Shuo Wang, Ramiah, Annapoorani, Stickney, Morgan, Azadi, Parastoo, Amster, I. Jonathan, Moremen, Kelley W., Lianchun Wang, Sharp, Joshua S., and Boons, Geert-Jan
- Published
- 2016
- Full Text
- View/download PDF
17. Expression system for structural and functional studies of human glycosylation enzymes
- Author
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Moremen, Kelley W, Ramiah, Annapoorani, Stuart, Melissa, Steel, Jason, Meng, Lu, Forouhar, Farhad, Moniz, Heather A, Gahlay, Gagandeep, Gao, Zhongwei, Chapla, Digantkumar, Wang, Shuo, Yang, Jeong-Yeh, Prabhakar, Pradeep Kumar, Johnson, Roy, Rosa, Mitche dela, Geisler, Christoph, Nairn, Alison V, Seetharaman, Jayaraman, Wu, Sheng-Cheng, Tong, Liang, Gilbert, Harry J, LaBaer, Joshua, and Jarvis, Donald L
- 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
- 2018
- Full Text
- View/download PDF
18. Enzymatic Basis for N-Glycan Sialylation
- Author
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Meng, Lu, primary, Forouhar, Farhad, additional, Thieker, David, additional, Gao, Zhongwei, additional, Ramiah, Annapoorani, additional, Moniz, Heather, additional, Xiang, Yong, additional, Seetharaman, Jayaraman, additional, Milaninia, Sahand, additional, Su, Min, additional, Bridger, Robert, additional, Veillon, Lucas, additional, Azadi, Parastoo, additional, Kornhaber, Gregory, additional, Wells, Lance, additional, Montelione, Gaetano T., additional, Woods, Robert J., additional, Tong, Liang, additional, and Moremen, Kelley W., additional
- Published
- 2013
- Full Text
- View/download PDF
19. Mutations in the Alpha 1,2-Mannosidase Gene, MAN1B1, Cause Autosomal-Recessive Intellectual Disability
- Author
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Rafiq, Muhammad Arshad, primary, Kuss, Andreas W., additional, Puettmann, Lucia, additional, Noor, Abdul, additional, Ramiah, Annapoorani, additional, Ali, Ghazanfar, additional, Hu, Hao, additional, Kerio, Nadir Ali, additional, Xiang, Yong, additional, Garshasbi, Masoud, additional, Khan, Muzammil Ahmad, additional, Ishak, Gisele E., additional, Weksberg, Rosanna, additional, Ullmann, Reinhard, additional, Tzschach, Andreas, additional, Kahrizi, Kimia, additional, Mahmood, Khalid, additional, Naeem, Farooq, additional, Ayub, Muhammad, additional, Moremen, Kelley W., additional, Vincent, John B., additional, Ropers, Hans Hilger, additional, Ansar, Muhammad, additional, and Najmabadi, Hossein, additional
- Published
- 2011
- Full Text
- View/download PDF
20. High Structural Resolution Hydroxyl Radical Protein Footprinting Reveals an Extended Robo1-Heparin Binding Interface.
- Author
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Zixuan Li, Moniz, Heather, Shuo Wang, Ramiah, Annapoorani, Zhang, Fuming, Moremen, Kelley W., Linhardt, Robert J., and Sharp, Joshua S.
- Subjects
- *
HYDROXYL group , *PROTEIN research , *CENTRAL nervous system , *POLYSACCHARIDES , *HEPARIN , *CHARGE exchange - Abstract
Interaction of transmembrane receptors of the Robo family and the secreted protein Slit provides important signals in the development of the central nervous system and regulation of axonal midline crossing. Heparan sulfate, a sulfated linear polysaccharide modified in a complex variety of ways, serves as an essential co-receptor in Slit-Robo signaling. Previous studies have shown that closely related heparin octasaccharides bind to Drosophila Robo directly, and surface plasmon resonance analysis revealed that Robo1 binds more tightly to full-length unfractionated heparin. For the first time, we utilized electron transfer dissociation-based high spatial resolution hydroxyl radical protein footprinting to identify two separate binding sites for heparin interaction with Robo1: one binding site at the previously identified site for heparin dp8 and a second binding site at the N terminus of Robo1 that is disordered in the x-ray crystal structure. Mutagenesis of the identified N-terminal binding site exhibited a decrease in binding affinity as measured by surface plasmon resonance and heparin affinity chromatography. Footprinting also indicated that heparin binding induces a minor change in the conformation and/or dynamics of the Ig2 domain, but no major conformational changes were detected. These results indicate a second low affinity binding site in the Robo-Slit complex as well as suggesting the role of the Ig2 domain of Robo1 in heparin-mediated signal transduction. This study also marks the first use of electron transfer dissociationbased high spatial resolution hydroxyl radical protein footprinting, which shows great utility for the characterization of protein- carbohydrate complexes. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
21. Enzymatic Basis for N-Glycan Sialylation.
- Author
-
Lu Meng, Forouhar, Farhad, Thieker, David, Zhongwei Gao, Ramiah, Annapoorani, Moniz, Heather, Yong Xiang, Seetharaman, Jayaraman, Milaninia, Sahand, Min Su, Bridger, Robert, Veillon, Lucas, Azadi, Parastoo, Kornhaber, Gregory, Wells, Lance, Montelione, Gaetano T., Woods, Robert J., Liang Tong, and Moremen, Kelley W.
- Subjects
- *
ENZYMES , *CATALYSTS , *GLYCANS , *CARBOHYDRATES , *GLYCOPROTEINS - Abstract
Glycan structures on glycoproteins and glycolipids play critical roles in biological recognition, targeting, and modulation of functions in animal systems. Many classes of glycan structures are capped with terminal sialic acid residues, which contribute to biological functions by either forming or masking glycan recognition sites on the cell surface or secreted glycoconjugates. Sialylated glycans are synthesized in mammals by a single conserved family of sialyltransferases that have diverse linkage and acceptor specificities. We examined the enzymatic basis for glycan sialylation in animal systems by determining the crystal structures of rat ST6GAL1, an enzyme that creates terminal a2,6-sialic acid linkages on complex-type N-glycans, at 2.4 A resolution. Crystals were obtained from enzyme preparations generated in mammalian cells. The resulting structure revealed an overall protein fold broadly resembling the previously determined structure of pig ST3GAL1, including a CMP-sialic acid-binding site assembled from conserved sialylmotif sequence elements. Significant differences in structure and disulfide bonding patterns were found outside the sialylmotif sequences, including differences in residues predicted to interact with the glycan acceptor. Computational substrate docking and molecular dynamics simulations were performed to predict and evaluate the CMP-sialic acid donor and glycan acceptor interactions, and the results were compared with kinetic analysis of active site mutants. Comparisons of the structure with pig ST3GAL1 and a bacterial sialyltransferase revealed a similar positioning of donor, acceptor, and catalytic residues that provide a common structural framework for catalysis by the mammalian and bacterial sialyltransferases. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
22. Characterizing human α-1,6-fucosyltransferase (FUT8) substrate specificity and structural similarities with related fucosyltransferases.
- Author
-
Boruah BM, Kadirvelraj R, Liu L, Ramiah A, Li C, Zong G, Bosman GP, Yang JY, Wang LX, Boons GJ, Wood ZA, and Moremen KW
- Subjects
- Crystallography, X-Ray, HEK293 Cells, Humans, Protein Domains, Structural Homology, Protein, Substrate Specificity, Fucosyltransferases chemistry, Protein Folding
- Abstract
Mammalian Asn-linked glycans are extensively processed as they transit the secretory pathway to generate diverse glycans on cell surface and secreted glycoproteins. Additional modification of the glycan core by α-1,6-fucose addition to the innermost GlcNAc residue (core fucosylation) is catalyzed by an α-1,6-fucosyltransferase (FUT8). The importance of core fucosylation can be seen in the complex pathological phenotypes of FUT8 null mice, which display defects in cellular signaling, development, and subsequent neonatal lethality. Elevated core fucosylation has also been identified in several human cancers. However, the structural basis for FUT8 substrate specificity remains unknown.Here, using various crystal structures of FUT8 in complex with a donor substrate analog, and with four distinct glycan acceptors, we identify the molecular basis for FUT8 specificity and activity. The ordering of three active site loops corresponds to an increased occupancy for bound GDP, suggesting an induced-fit folding of the donor-binding subsite. Structures of the various acceptor complexes were compared with kinetic data on FUT8 active site mutants and with specificity data from a library of glycan acceptors to reveal how binding site complementarity and steric hindrance can tune substrate affinity. The FUT8 structure was also compared with other known fucosyltransferases to identify conserved and divergent structural features for donor and acceptor recognition and catalysis. These data provide insights into the evolution of modular templates for donor and acceptor recognition among GT-B fold glycosyltransferases in the synthesis of diverse glycan structures in biological systems., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Boruah et al.)
- Published
- 2020
- Full Text
- View/download PDF
23. Structural Aspects of Heparan Sulfate Binding to Robo1-Ig1-2.
- Author
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Gao Q, Chen CY, Zong C, Wang S, Ramiah A, Prabhakar P, Morris LC, Boons GJ, Moremen KW, and Prestegard JH
- Subjects
- Glycosylation, Heparitin Sulfate chemistry, Magnetic Resonance Spectroscopy, Molecular Docking Simulation, Molecular Structure, Nerve Tissue Proteins chemistry, Receptors, Immunologic chemistry, Static Electricity, Roundabout Proteins, Heparitin Sulfate metabolism, Nerve Tissue Proteins metabolism, Receptors, Immunologic metabolism
- Abstract
Roundabout 1, or Robo1, is a cell surface signaling molecule important in axon guidance. Its interaction with heparan sulfate (HS) and members of the Slit protein family is essential to its activity, making characterization of these interactions by structural methods, such as NMR, highly desirable. However, the fact that Robo1 is a glycosylated protein prevents employment of commonly used bacterial hosts for expression of properly glycosylated forms with the uniform
15 N,13 C, and2 H labeling needed for NMR studies. Here, we apply an alternative methodology, based on labeling with a single amino acid type and high structural content NMR data, to characterize a two-domain construct of glycosylated Robo1 (Robo1-Ig1-2) interacting with a synthetic HS tetramer (IdoA-GlcNS6S-IdoA2S-GlcNS6S-(CH2 )5 NH2 ). Significant chemical shift perturbations of the crosspeak from K81 on titration with the tetramer provide initial evidence for the location of a binding site and allow determination of a 255 μM disassociation constant. The binding epitopes, bound conformation, and binding site placement of the HS tetramer have been further characterized by saturation transfer difference (STD), transferred nuclear Overhauser effect (trNOE), and paramagnetic perturbation experiments. A model of the complex has been generated using constraints derived from the various NMR experiments. Postprocessing energetic analysis of this model provides a rationale for the role each glycan residue plays in the binding event, and examination of the binding site in the context of a previous Robo-Slit structure provides a rationale for modulation of Robo-Slit interactions by HS.- Published
- 2016
- Full Text
- View/download PDF
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