An Artificial Golgi: Controlled GAG Synthesis and Screening

Summary

Principal Investigator: ROBERT J contact LINHARDT
Abstract: DESCRIPTION (provided by applicant): The Golgi is the organelle responsible for the posttranslational modification of proteins. Glycosylation, the most prominent posttranslational modification, results in the biosynthesis of glycoproteins and proteoglycans (PGs). Glycosylation is not template driven and it is hypothesized that highly ordered cellular glycosylation is regulated by the unique substrate specificities of the biosynthetic enzymes involved and the Golgi's compartmental structures housing the biosynthetic enzymes. The glycosaminoglycan (GAG) chains of PGs are among the most structurally complex and biologically important glycans. GAG structure, particularly heparan sulfate (HS), carries immense amounts of biological information. HS has been found to play an important role in fibroblast growth factor (FGF)-fibroblast growth factor receptor (FGFR) signaling complexes. The FGF signaling system is important in developmental biology and in cancer. Previous studies performed have shown that HS activates different FGF7FGFR complexes in a sequence dependent manner. These studies suggest that there is a discrete structure-activity relationship (SAR) for HS. The determination of HS-SAR, however, is extremely challenging, as there are no straightforward methods for the synthesis of HS having sufficient domain size and a wide variety of defined sequences. We have begun to address this challenge by designing an artificial Golgi organelle capable of the controlled synthesis of structurally defined, bioactive GAGs. This artificial organelle is based on a newly developed digital microfluidic platform designed to simulate the fluid membrane-based structure of the natural Golgi. This artificial Golgi has begun to provide the precise temporal and spatial control needed for the synthesis of GAGs of defined sequences. The artificial Golgi we have designed is amenable to computer automation and can be interfaced with on-line electrospray ionization-liquid chromatography/mass spectrometry analysis, facilitating high-throughput synthesis and analysis of GAGs. The five specific aims of this project are: 1. Investigate the biosynthesis of HS in a controlled and automated fashion using an artificial Golgi;2. Synthesize a limited combinatorial library of 1080 HS decasaccharide structural subtypes using the artificial Golgi;3. Screen this limited HS library for its ability to signal through FGF;4. Synthesize an HS decasaccharide library expanded around these hits to optimize bioactivity;and 5. Determine the specificity and potency of the HS decasaccharides to signal through the 126 possible FGFWFGFR signaling complexes and establish their SAR The outcome of these studies will be the development of a platform for the controlled synthesis of defined glycan libraries. In vitro high-throughput screening techniques will be established for investigating the biological activity of these glycans. Using these methodologies, we expect to identify potential therapeutic glycans, their sequences, and the specific enzymatic synthesis parameters required for their synthesis. PUBLIC HEALTH RELEVANCE: The proposed effort impacts human health by developing an artificial Golgi to synthesize a library of heparan sulfates and heparan sulfate oligosaccharides. This artificial Golgi uses a digital microfluidic platform for synthesis and screen activity on a cell microarray to develop a structure activity relationship for fibroblast growth factor signaling.
Funding Period: ----------------2009 - ---------------2011-
more information: NIH RePORT

Top Publications

  1. pmc Immobilization of heparin: approaches and applications
    Saravanababu Murugesan
    Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, USA
    Curr Top Med Chem 8:80-100. 2008
  2. pmc Synthesis of gold and silver nanoparticles stabilized with glycosaminoglycans having distinctive biological activities
    Melissa M Kemp
    Department of Biology, Rensselaer Polytechnic Institute, Troy, New York 12180, USA
    Biomacromolecules 10:589-95. 2009
  3. pmc Ultraperformance liquid chromatography with electrospray ionization ion trap mass spectrometry for chondroitin disaccharide analysis
    Kemal Solakyildirim
    Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA
    Anal Biochem 397:24-8. 2010
  4. ncbi Polysaccharides and phytochemicals: a natural reservoir for the green synthesis of gold and silver nanoparticles
    Y Park
    Inje University, College of Pharmacy, Gimhae, Republic of Korea
    IET Nanobiotechnol 5:69-78. 2011
  5. pmc Ozonolysis of the double bond of the unsaturated uronate residue in low-molecular-weight heparin and K5 heparosan
    Sayaka Masuko
    Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, NY 12180, USA
    Carbohydr Res 346:1962-6. 2011
  6. pmc Hyphenated techniques for the analysis of heparin and heparan sulfate
    Bo Yang
    Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA
    Anal Bioanal Chem 399:541-57. 2011
  7. pmc Conductive cable fibers with insulating surface prepared by coaxial electrospinning of multiwalled nanotubes and cellulose
    Minoru Miyauchi
    Rensselaer Nanotechnology Center, Rensselaer Polytechnic Institute, 110 Eighth Street, Troy, New York 12180, USA
    Biomacromolecules 11:2440-5. 2010
  8. pmc Proteoglycomics: recent progress and future challenges
    Mellisa Ly
    Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, New York, USA
    OMICS 14:389-99. 2010
  9. pmc High-resolution preparative separation of glycosaminoglycan oligosaccharides by polyacrylamide gel electrophoresis
    Tatiana N Laremore
    Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, NY 12180, USA
    Anal Biochem 401:236-41. 2010
  10. pmc Chemoenzymatic synthesis of the next generation of ultralow MW heparin therapeutics
    Sayaka Masuko
    Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA
    Future Med Chem 4:289-96. 2012

Detail Information

Publications11

  1. pmc Immobilization of heparin: approaches and applications
    Saravanababu Murugesan
    Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, USA
    Curr Top Med Chem 8:80-100. 2008
    ..In the applications reviewed, immobilized heparin is compared with soluble heparin for efficient and versatile use in each of the various applications...
  2. pmc Synthesis of gold and silver nanoparticles stabilized with glycosaminoglycans having distinctive biological activities
    Melissa M Kemp
    Department of Biology, Rensselaer Polytechnic Institute, Troy, New York 12180, USA
    Biomacromolecules 10:589-95. 2009
    ..In conclusion, gold and silver nanoparticles complexed with heparin demonstrated effective anticoagulant and anti-inflammatory efficacy, having potential in various local applications...
  3. pmc Ultraperformance liquid chromatography with electrospray ionization ion trap mass spectrometry for chondroitin disaccharide analysis
    Kemal Solakyildirim
    Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA
    Anal Biochem 397:24-8. 2010
    ..UPLC separation technology uses small particle diameter, short column length, and elevated column temperature to obtain high resolution and sensitivity. Hexylamine (15 mM) was selected as the optimal ion-pairing reagent...
  4. ncbi Polysaccharides and phytochemicals: a natural reservoir for the green synthesis of gold and silver nanoparticles
    Y Park
    Inje University, College of Pharmacy, Gimhae, Republic of Korea
    IET Nanobiotechnol 5:69-78. 2011
    ..The focus of this review is the application of polysaccharides and phytochemicals in the green synthesis of gold and silver nanoparticles to afford biocomposites with novel uses in nanomedicine and as nanocomposites...
  5. pmc Ozonolysis of the double bond of the unsaturated uronate residue in low-molecular-weight heparin and K5 heparosan
    Sayaka Masuko
    Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, NY 12180, USA
    Carbohydr Res 346:1962-6. 2011
    ..This reaction also has potential applications in the chemoenzymatic synthesis of bioengineered heparin from Escherichia coli-derived K5 heparosan...
  6. pmc Hyphenated techniques for the analysis of heparin and heparan sulfate
    Bo Yang
    Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA
    Anal Bioanal Chem 399:541-57. 2011
    ..This review focuses on recent efforts to develop hyphenated techniques for the analysis of heparin and heparan sulfate...
  7. pmc Conductive cable fibers with insulating surface prepared by coaxial electrospinning of multiwalled nanotubes and cellulose
    Minoru Miyauchi
    Rensselaer Nanotechnology Center, Rensselaer Polytechnic Institute, 110 Eighth Street, Troy, New York 12180, USA
    Biomacromolecules 11:2440-5. 2010
    ..Fiber mat conductivity increased with increasing ratio of MWNT in the fibers with a maximum conductivity of 10.7 S/m obtained at 45 wt % MWNT loading...
  8. pmc Proteoglycomics: recent progress and future challenges
    Mellisa Ly
    Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, New York, USA
    OMICS 14:389-99. 2010
    ..Unique challenges in the characterization of glycosaminoglycan component of proteoglycans are discussed, with emphasis on the many analytical tools used and the types of information they provide...
  9. pmc High-resolution preparative separation of glycosaminoglycan oligosaccharides by polyacrylamide gel electrophoresis
    Tatiana N Laremore
    Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, NY 12180, USA
    Anal Biochem 401:236-41. 2010
    ..The described method is straightforward and time-efficient, affording size-homogeneous oligosaccharides that can be used in sequencing, protein binding, and other structure-function relationship studies...
  10. pmc Chemoenzymatic synthesis of the next generation of ultralow MW heparin therapeutics
    Sayaka Masuko
    Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA
    Future Med Chem 4:289-96. 2012
    ..In this article, we discuss the current methods and recent advances in heparin and low MW heparin syntheses and the recent successful chemoenzymatic preparation of ultralow MW heparins...