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Structural and functional characterization of the microtubule interacting and trafficking domains of two oomycete chitin synthases
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.ORCID iD: 0000-0003-2965-2873
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2016 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 283, no 16, 3072-3088 p.Article in journal (Refereed) Published
Abstract [en]

Chitin synthases (Chs) are responsible for the synthesis of chitin, a key structural cell wall polysaccharide in many organisms. They are essential for growth in certain oomycete species, some of which are pathogenic to diverse higher organisms. Recently, a Microtubule Interacting and Trafficking (MIT) domain, which is not found in any fungal Chs, has been identified in some oomycete Chs proteins. Based on experimental data relating to the binding specificity of other eukaryotic MIT domains, there was speculation that this domain may be involved in the intracellular trafficking of Chs proteins. However, there is currently no evidence for this or any other function for the MIT domain in these enzymes. To attempt to elucidate their function, MIT domains from two Chs enzymes from the oomycete Saprolegnia monoica were cloned, expressed and characterized. Both were shown to interact strongly with the plasma membrane component phosphatidic acid, and to have additional putative interactions with proteins thought to be involved in protein transport and localization. Aiding our understanding of these data, the structure of the first MIT domain from a carbohydrate-active enzyme (MIT1) was solved by NMR, and a model structure of a second MIT domain (MIT2) was built by homology modelling. Our results suggest a potential function for these MIT domains in the intracellular transport and/or regulation of Chs enzymes in the oomycetes. 

Place, publisher, year, edition, pages
2016. Vol. 283, no 16, 3072-3088 p.
Keyword [en]
chitin synthase, microtubule interacting and trafficking domain, NMR structure, oomycete, phospholipids
National Category
Biophysics
Research subject
Biophysics
Identifiers
URN: urn:nbn:se:su:diva-131279DOI: 10.1111/febs.13794OAI: oai:DiVA.org:su-131279DiVA: diva2:937198
Available from: 2016-06-15 Created: 2016-06-15 Last updated: 2016-09-01Bibliographically approved
In thesis
1. Structure and lipid interactions of membrane-associated glycosyltransferases: Cationic patches and anionic lipids regulate biomembrane binding of both GT-A and GT-B enzymes
Open this publication in new window or tab >>Structure and lipid interactions of membrane-associated glycosyltransferases: Cationic patches and anionic lipids regulate biomembrane binding of both GT-A and GT-B enzymes
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis concerns work on structure and membrane interactions of enzymes involved in lipid synthesis, biomembrane and cell wall regulation and cell defense processes. These proteins, known as glycosyltransferases (GTs), are involved in the transfer of sugar moieties from nucleotide sugars to lipids or chitin polymers. Glycosyltransferases from three types of organisms have been investigated; one is responsible for vital lipid synthesis in Arabidopsis thaliana (atDGD2) and adjusts the lipid content in biomembranes if the plant experiences stressful growth conditions. This enzyme shares many structural features with another GT found in gram-negative bacteria (WaaG). WaaG is however continuously active and involved in synthesis of the protective lipopolysaccharide layer in the cell walls of Escherichia coli. The third type of enzymes investigated here are chitin synthases (ChS) coupled to filamentous growth in the oomycete Saprolegnia monoica. I have investigated two ChS-derived MIT domains that may be involved in membrane interactions within the endosomal pathway.

From analysis of the three-dimensional structure and the amino-acid sequence, some important regions of these very large proteins were selected for in vitro studies. By the use of an array of biophysical methods (e.g. Nuclear Magnetic Resonance, Fluorescence and Circular Dichroism spectroscopy) and directed sequence analyses it was possible to shed light on some important details regarding the structure and membrane-interacting properties of the GTs. The importance of basic amino-acid residues and hydrophobic anchoring segments, both generally and for the abovementioned proteins specifically, is discussed. Also, the topology and amino-acid sequence of GT-B enzymes of the GT4 family are analyzed with emphasis on their biomembrane association modes. The results presented herein regarding the structural and lipid-interacting properties of GTs aid in the general understanding of glycosyltransferase activity. Since GTs are involved in a high number of biochemical processes in vivo it is of outmost importance to understand the underlying processes responsible for their activity, structure and interaction events. The results are likely to be useful for many applications and future experimental design within life sciences and biomedicine.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2016. 81 p.
Keyword
glycosyltransferase, monotopic membrane proteins, galactolipids, NMR, chitin synthase, DGD2, LPS, WaaG, MIT domain
National Category
Biophysics
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-131084 (URN)978-91-7649-435-6 (ISBN)
External cooperation:
Public defence
2016-09-16, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
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Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.

Available from: 2016-08-24 Created: 2016-06-13 Last updated: 2016-09-01Bibliographically approved

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