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Characterization of specific domains of the cellulose and chitin synthases from pathogenic oomycetes
KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
2015 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Some oomycetes species are severe pathogens of fish or crops. As such, they are responsible for important losses in the aquaculture industry as well as in agriculture. Saprolegnia parasitica is a major concern in aquaculture as there is currently no method available for controlling the diseases caused by this microorganism. The cell wall is an extracellular matrix composed essentially of polysaccharides, whose integrity is required for oomycete viability. Thus, the enzymes involved in the biosynthesis of cell wall components, such as cellulose and chitin synthases, represent ideal targets for disease control. However, the biochemical properties of these enzymes are poorly understood, which limits our capacity to develop specific inhibitors that can be used for blocking the growth of pathogenic oomycetes.

In our work, we have used Saprolegnia monoica as a model species for oomycetes to characterize two types of domains that occur specifically in oomycete carbohydrate synthases: the Pleckstrin Homology (PH) domain of a cellulose synthase and the so-called ‘Microtubule Interacting and Trafficking’ (MIT) domain of chitin synthases. In addition, the chitin synthase activity of the oomycete phytopathogen Aphanomyces euteiches was characterized in vitro using biochemical approaches.

The results from our in vitro investigations revealed that the PH domain of the oomycete cellulose synthase binds to phosphoinositides, microtubules and F-actin. In addition, cell biology approaches were used to demonstrate that the PH domain co-localize with F-actin in vivo. The structure of the MIT domain of chitin synthase (CHS) 1 was solved by NMR. In vitro binding assays performed on recombinant MIT domains from CHS 1 and CHS 2 demonstrated that both proteins strongly interact with phosphatidic acid in vitro. These results were further supported by in silico data where biomimetic membranes composed of different phospholipids were designed for interaction studies. The use of a yeast-two-hybrid approach suggested that the MIT domain of CHS 2 interacts with the delta subunit of Adaptor Protein 3, which is involved in protein trafficking. These data support a role of the MIT domains in the cellular targeting of CHS proteins. Our biochemical data on the characterization of the chitin synthase activity of A. euteiches suggest the existence of two distinct enzymes responsible for the formation of water soluble and insoluble chitosaccharides, which is consistent with the existence of two putative CHS genes in the genome of this species.

Altogether our data support a role of the PH domain of cellulose synthase and MIT domains of CHS in membrane trafficking and cellular location.

Ort, förlag, år, upplaga, sidor
Stockholm: KTH Royal Institute of Technology, 2015. , s. viii, 90
Serie
TRITA-BIO-Report, ISSN 1654-2312 ; 2015:15
Nyckelord [en]
Cellulose biosynthesis; chitin biosynthesis; cellulose synthase genes; chitin synthase genes; oomycetes; Saprolegnia monoica; Microtubule Interacting and Trafficking (MIT) domain; Pleckstrin Homology (PH) domain
Nationell ämneskategori
Biokemi och molekylärbiologi
Forskningsämne
Bioteknologi
Identifikatorer
URN: urn:nbn:se:kth:diva-175375ISBN: 978-91-7595-690-9 (tryckt)OAI: oai:DiVA.org:kth-175375DiVA, id: diva2:860720
Disputation
2015-10-23, FB53, AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 13:00 (Engelska)
Opponent
Handledare
Anmärkning

QC 20151014

Tillgänglig från: 2015-10-14 Skapad: 2015-10-13 Senast uppdaterad: 2015-10-14Bibliografiskt granskad
Delarbeten
1. Radiometric and spectrophotometric in vitro assays of glycosyltransferases involved in plant cell wall carbohydrate biosynthesis
Öppna denna publikation i ny flik eller fönster >>Radiometric and spectrophotometric in vitro assays of glycosyltransferases involved in plant cell wall carbohydrate biosynthesis
2012 (Engelska)Ingår i: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 7, nr 9, s. 1634-1650Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Most of the glycosyltransferases (GTs) that catalyze the formation of plant cell wall carbohydrates remain to be biochemically characterized. This can be achieved only if specific assays are available for these enzymes. Here we present a protocol for in vitro assays of processive and nonprocessive membrane-bound GTs. The assays are either based on the use of radioactive nucleotide sugars (NDP sugars; e.g., UDP-[U-C-14] glucose) and the quantification of the radiolabeled monosaccharides incorporated into soluble or insoluble carbohydrates, or on the coupling of the GT reaction with that of pyruvate kinase (PK) and the oxidation of NADH by lactate dehydrogenase (LDH). The radiometric assays are more suitable for exploratory work on poorly characterized enzymes, whereas the spectrophotometric assays require the availability of highly enriched GTs. Both assays can be performed within 1 d, depending on the number of fractions to be assayed or reaction mixtures to be tested.

Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
urn:nbn:se:kth:diva-103376 (URN)10.1038/nprot.2012.089 (DOI)000308526300006 ()
Anmärkning

QC 20121016

Tillgänglig från: 2012-10-16 Skapad: 2012-10-11 Senast uppdaterad: 2019-05-08Bibliografiskt granskad
2. Functional characterization of the pleckstrin homology domain of a cellulose synthase from the Oomycete Saprolegnia monoica
Öppna denna publikation i ny flik eller fönster >>Functional characterization of the pleckstrin homology domain of a cellulose synthase from the Oomycete Saprolegnia monoica
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2012 (Engelska)Ingår i: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 417, nr 4, s. 1248-1253Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Some oomycetes, for instance Saprolegnia parasitica, are severe fish pathogens that cause important economic losses worldwide. Cellulose biosynthesis is a vital process for this class of microorganisms, but the corresponding molecular mechanisms are poorly understood. Of all cellulose synthesizing enzymes known, only some oomycete cellulose synthases contain a pleckstrin homology (PH) domain. Some human PH domains bind specifically to phosphoinositides, but most PH domains bind phospholipids in a non-specific manner. In addition, some PH domains interact with various proteins. Here we have investigated the function of the PH domain of cellulose synthase 2 from the oomycete Saprolegnia monoica (SmCesA2), a species closely related to S. parasitica. The SmCesA2 PH domain is similar to the C-terminal PH domain of the human protein TAPP1. It binds in vitro to phosphoinositides, F-actin and microtubules, and co-localizes with F-actin in vivo. Our results suggest a role of the SmCesA2 PH domain in the regulation, trafficking and/or targeting of the cell wall synthesizing enzyme.

Ort, förlag, år, upplaga, sidor
Academic Press, 2012
Nyckelord
Pleckstrin homology domain; Cellulose synthase; Cell wall biosynthesis; Oomycetes; Phosphoinositides; F-actin
Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
urn:nbn:se:kth:diva-34284 (URN)10.1016/j.bbrc.2011.12.118 (DOI)000300196100024 ()2-s2.0-84856215112 (Scopus ID)
Anmärkning
Updated from manuscript to article in journal. QC 20120306Tillgänglig från: 2011-05-31 Skapad: 2011-05-31 Senast uppdaterad: 2017-12-11Bibliografiskt granskad
3. Structural and functional characterization of the “Microtubule Interacting and Trafficking": domains of two oomycetes chitin synthases
Öppna denna publikation i ny flik eller fönster >>Structural and functional characterization of the “Microtubule Interacting and Trafficking": domains of two oomycetes chitin synthases
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(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
urn:nbn:se:kth:diva-175397 (URN)
Anmärkning

QS 2015

Tillgänglig från: 2015-10-14 Skapad: 2015-10-14 Senast uppdaterad: 2015-10-14Bibliografiskt granskad
4. Insight into the adsorption profiles of the Saprolegnia practica chitin synthase MIT domain on POPA and POPC membranes by molecular dynamics simulation studies
Öppna denna publikation i ny flik eller fönster >>Insight into the adsorption profiles of the Saprolegnia practica chitin synthase MIT domain on POPA and POPC membranes by molecular dynamics simulation studies
Visa övriga...
(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
urn:nbn:se:kth:diva-175398 (URN)
Anmärkning

QS 2015

Tillgänglig från: 2015-10-14 Skapad: 2015-10-14 Senast uppdaterad: 2015-10-14Bibliografiskt granskad
5. The phytopathogenic oomycete Aphanomyces euteiches contains two distinct N-acetylglucosaminyltransferase activities that form chitin-like saccharides in vitro
Öppna denna publikation i ny flik eller fönster >>The phytopathogenic oomycete Aphanomyces euteiches contains two distinct N-acetylglucosaminyltransferase activities that form chitin-like saccharides in vitro
(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
urn:nbn:se:kth:diva-175410 (URN)
Anmärkning

QS 2015

Tillgänglig från: 2015-10-14 Skapad: 2015-10-14 Senast uppdaterad: 2015-10-14Bibliografiskt granskad

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