Change search
ReferencesLink to record
Permanent link

Direct link
Identification and Preliminary Characterization of a New Chemical Affecting Glucosyltransferase Activities Involved in Plant Cell Wall Biosynthesis
KTH, School of Biotechnology (BIO), Glycoscience.
KTH, School of Biotechnology (BIO), Glycoscience.
Show others and affiliations
2008 (English)In: MOLECULAR PLANT, ISSN 1674-2052, Vol. 1, no 6, 977-989 p.Article in journal (Refereed) Published
Abstract [en]

Chemical genetics as a part of chemical genomics is a powerful and fast developing approach to dissect biological processes that may be difficult to characterize using conventional genetics because of gene redundancy or lethality and, in the case of polysaccharide biosynthesis, plant flexibility. Polysaccharide synthetic enzymes are located in two main compartments-the Golgi apparatus and plasma membrane-and can be studied in vitro using membrane fractions. Here, we first developed a high-throughput assay that allowed the screening of a library of chemicals with a potential effect on glycosyltransferase activities. Out of the 4800 chemicals screened for their effect on Golgi glucosyltransferases, 66 compounds from the primary screen had an effect on carbohydrate biosynthesis. Ten of these compounds were confirmed to inhibit glucose incorporation after a second screen. One compound exhibiting a strong inhibition effect (ID 6240780 named chemical A) was selected and further studied. it reversibly inhibits the transfer of glucose from UDPglucose by Golgi membranes, but activates the plasma membrane-bound callose synthase. The inhibition effect is dependent on the chemical structure of the compound, which does not affect endomembrane morphology of the plant cells, but causes changes in cell wall composition. Chemical A represents a novel drug with a great potential for the study of the mechanisms of Golgi and plasma membrane-bound glucosyltransferases.

Place, publisher, year, edition, pages
2008. Vol. 1, no 6, 977-989 p.
Keyword [en]
cell walls, callose synthase, chemical screening and identification, glycosyltransferase, Golgi, plasma membrane, suspension-cultured-cells, arabidopsis-thaliana, golgi-apparatus, xyloglucan biosynthesis, fucosyl-transferase, molecular-cloning, cellulose, mutant, isoxaben, pea
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
URN: urn:nbn:se:kth:diva-18137DOI: 10.1093/mp/ssn055ISI: 000262858000009ScopusID: 2-s2.0-70349593386OAI: diva2:336183

QC 20100525

Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2014-07-10Bibliographically approved
In thesis
1. Analyzing the properties and biosynthesis of β-glucans from Gluconacetobacter and poplar
Open this publication in new window or tab >>Analyzing the properties and biosynthesis of β-glucans from Gluconacetobacter and poplar
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Glucans are polysaccharides integral to many materials and biological functions. Under the umbrella of Biomime, the Swedish Center for Biomimetic Fiber Engineering, this work has aimed to improve basic understanding of the biosynthesis of such glucans. This has been achieved through direct investigation of cellulose structure, and by developing the tools to analyze glucan biosynthesis. Notably we have identified a novel chemical effector of glucan synthesis processes and developed a proteomic toolkit useful for analyzing membrane-bound glycosyltransferases, the enzyme group responsible for glucan biosynthesis. During this work, glucan synthesis has been studied using both Gluconacetobacter and Populus cell suspension cultures.

Publication I. Gluconacetobacter cellulose (BC) was used as a base to create a novel and well characterized nano-material with improved mechanical properties. This novel composite of BC and hydroxyethylcellulose (HEC) had improved tensile strength compared to pure BC. Through thorough study utilizing dispersion measurements, electron microscopy, nuclear magnetic resonance and X-ray diffraction it was shown that the improved properties derived from a layer of HEC coating each fibril.

Publication II. Bacterial cellulose was labeled in specific positions with 13C (C4 and C6). These samples were analyzed by CP/MAS NMR along with cellulose samples from cotton and Halocynthia sp. For each sample spectral fitting was performed and general properties of crystal allomorph composition and fibril widths were determined. Calculations were also made for water accessible surfaces of the fibrils. The results showed that water accessible C4 surface signals are reflective of the allomorph composition of the sample, along with a distorted signal that derives due to fibril imperfections. Water accessible surface signals from the C6 region are instead derived from rotamer conformations of the C6 hydroxymethyl groupsfrom glucose residues.

In Publication III, a high-throughput screen was used to identify an inhibitor of Golgi-derived glycosyltransferase activity, termed chemical A. The structural basis for inhibition was determined and in vitro assays of callose synthesis were performed. The in vitro assays revealed chemical A to also be an activator of callose synthesis. To understand this activation kinetic studies were performed, showing that chemical A is a mixed type of activator, which can bind either the free enzyme or the enzyme-substrate complex. Chemical A has uses in chemical genetics for dissecting processes involving callose synthesis, such as stress response and cell-plate formation.

In publication IV, we present an in-house developed platform for proteomics with a distributed processing model. This in-house system has been central to many proteomics tasks, including for those presented in publication V, and is being distributed as the Automated Proteomics Pipeline (APP).

In publication V, conditions for enrichment of Detergent-Resistant Microdomains (DRM) have been optimized for Populus trichocarpa cell cultures. The proteins enriched in DRM were identified using mass spectrometry based proteomics, and a functional model for DRM was proposed. This model involves proteins specialized in stress response, including callose synthase, and cell signaling. This further strengthens the arguments for DRMs as sites of specific cellular functions and confirms they play a role in glucan synthesis.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2014. 66 p.
TRITA-BIO-Report, ISSN 1654-2312 ; 2014:8
Cellulose synthesis, Bioinformatics, Proteomics, Cell wall
National Category
Biological Sciences Bioinformatics and Systems Biology
urn:nbn:se:kth:diva-147932 (URN)978-91-7595-184-3 (ISBN)
Public defence
2014-09-05, FR4, AlbaNova, Roslagstullsbacken 21, Stockholm, 13:00 (English)

QC 20140710

Available from: 2014-07-10 Created: 2014-07-09 Last updated: 2014-07-10Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full textScopus

Search in DiVA

By author/editor
Malm, ErikBulone, Vincent
By organisation
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)

Search outside of DiVA

GoogleGoogle Scholar
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Altmetric score

Total: 29 hits
ReferencesLink to record
Permanent link

Direct link