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In vitro and in vivo approaches in the characterization of XTH gene products
KTH, School of Biotechnology (BIO), Glycoscience.
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

ABSTRACT

The xyloglucan endo-transglycosylase/hydrolase (XTH) genes are found in all vascular and some nonvascular plants. The XTH genes encode proteins which comprise a subfamily of glycoside hydrolase (GH) family 16 in the Carbohydrate-Active enZYmes (CAZY) classification. The XTH gene products are believed to play intrinsic role in cell wall modification during growth and development throughout the lifetime of the plant. In the present investigation, biochemical and reverse genetic approaches were used to better understand the functions of individual members of the XTH gene family of two important plants: the model organism Arabidopsis thaliana and the grain crop barley (Hordeum vulgare). A phylogenetic tree of the xyloglucan-active enzymes of GH16 has previously been constructed, where enzymes with similar activities have been shown to cluster together. Several members of phylogenetic Group I/II and III-B, predicted to exhibit xyloglucan endo-transglycosylase activity (EC 2.4.1.207) and members of Group III-A, predicted to exhibit xyloglucan endo-hydrolase activity (EC 3.2.1.151), were included to analyze the functional diversity of XTH gene products. A heterologous expression system using the yeast Pichia pastoris was found to be effective for recombinant protein production with a success rate of ca. 50%. XTH gene products were obtained in soluble and active forms for subsequent biochemical characterization.

In order to be able to screen larger numbers of protein producing clones, a fast and easy method is required to identify clones expressing active protein in high enough amounts. Thus, a miniaturized XET/XEH assay for high-throughput analysis was developed, which was able to identify activities with good precision and with a reduced time and materials consumption and a reduced work load.

Enzyme kinetic analysis indicated that the XET or XEH activity of all XTH gene products characterized in the present study corresponded to predictions based on the previously revised phylogenetic clustering. To gain insight into the biological function of the predominant XEHs AtXTH31 and AtXTH32, which are highly expressed in rapidly developing tissues, a reverse genetic approach was employed using T-DNA insertion lines of the A. thaliana Columbia ecotype. Genotypic and phenotypic characterization, together with in situ assays of XET and XEH activities, in single- and double-knock-out mutants indicated that these Group III-A enzymes are active in expanding tissues of the A. thaliana roots and hypocotyl.  Although suppression of in muro XEH activity was clearly observed in the double-knock-out, no significant growth phenotype was observed, with the exception that radicle emergence appeared to be faster than in the wild type plants.

Keywords: Arabidopis thaliana, Hordeum vulgare, plant cell wall, xyloglucan, glycoside hydrolase family 16, xyloglucan endo-transglycosylase/hydrolase gene family, xyloglucan endo-transglycosylase, xyloglucan endo-hydrolase, heterologous protein expression, Pichia pastoris, T-DNA insertion, in situ XET/XEH assay, high-throughput screening

Place, publisher, year, edition, pages
Stockholm: KTH , 2011. , x, 48 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2011: 1
Keyword [en]
Arabiodopsis thaliana, Hordeum vulgare, plant cell wall, xyloglucan, glycoside hydrolase family 16, xyloglucan endo-transglycosylase/hydrolase gene family, xyloglucan endo-hydrolase, heterologous protein expression, Pichia pastoris, T-DNA insertion, in situ XET/XEH assay, high-througput screening
National Category
Botany Biochemistry and Molecular Biology Biochemistry and Molecular Biology
Research subject
SRA - Molecular Bioscience
Identifiers
URN: urn:nbn:se:kth:diva-28222ISBN: 978-91-7415-848-9 (print)OAI: oai:DiVA.org:kth-28222DiVA: diva2:386091
Public defence
2011-02-02, FB54, Roslagstullsbacken 21, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
QC 20110114Available from: 2011-01-14 Created: 2011-01-12 Last updated: 2011-01-14Bibliographically approved
List of papers
1. Heterologous expression of diverse barley XTH genes in the yeast Pichia pastoris
Open this publication in new window or tab >>Heterologous expression of diverse barley XTH genes in the yeast Pichia pastoris
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2010 (English)In: PLANT BIOTECHNOLOGY, ISSN 1342-4580, Vol. 27, no 3, 251-258 p.Article in journal (Refereed) Published
Abstract [en]

Heterologous expression of plant genes, particularly those encoding carbohydrate-active enzymes such as glycoside hydrolases and glycosyl transferases, continues to be a major hurdle in the functional analysis of plant proteomes. Presently, there are few convenient systems for the production of recombinant plant enzymes in active form and at adequate levels for biochemical and structural characterization. The methylotrophic yeast Pichia pastoris is an attractive expression host due to its ease of manipulation and its capacity to perform post-translational protein modifications, such as N-glycosylation [Daly and Hearn (2005) J Mol Recognit 18: 119-138]. Here, we demonstrate the utility of the P. pastoris SMD1168H/pPICZ-alpha C system for the expression of a range of xyloglucan endo-transglycosylase/hydrolase (XTH) cDNAs from barley (Hordeum vulgare). Although stable transformants were readily obtained by positive selection for vector-induced antibiotic resistance for all of the nine constructs tested, only five isoforms were secreted as soluble proteins into the culture medium, four in active form. Furthermore, production levels of these five isoforms were found to be variable, depending on the transformant, which further underscores the necessity of screening multiple clones for expression of active enzyme. Failure to express certain XTH isoforms in P. pastoris could not be correlated with any conserved gene or protein sequence properties, and this precluded using rational sequence engineering to enhance heterologous expression of the cDNAs. Thus, while significant advances are reported here, systems for the heterologous production of plant proteins require further development.

Keyword
Pichia pastoris, plant protein expression, xyloglucan endo-transglycosylase/hydrolase (XTH) genes, XET, XEH
National Category
Plant Biotechnology
Identifiers
urn:nbn:se:kth:diva-28349 (URN)000280085800006 ()2-s2.0-77953215212 (Scopus ID)
Conference
Colloquium on Green Chemistry in Plants and Microorganisms Japanese Soc Promot Sci, Stockholm, SWEDEN, MAY 25, 2009
Note
QC 20110113Available from: 2011-01-13 Created: 2011-01-13 Last updated: 2011-03-01Bibliographically approved
2. Substrate specificity and catalytic mechanism of a xyloglucan xyloglucosyl transferase HvXET6 from barley (Hordeum vulgare L.)
Open this publication in new window or tab >>Substrate specificity and catalytic mechanism of a xyloglucan xyloglucosyl transferase HvXET6 from barley (Hordeum vulgare L.)
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2009 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 276, no 2, 437-456 p.Article in journal (Refereed) Published
Abstract [en]

A family 16 glycoside hydrolase, xyloglucan xyloglucosyl transferase (EC 2.4.1.207), also known as xyloglucan endotransglycosylase (XET), and designated isoenzyme HvXET6, was purified approximately 400-fold from extracts of young barley seedlings. The complete amino acid sequence of HvXET6 was deduced from the nucleotide sequence of a near full-length cDNA, in combination with tryptic peptide mapping. An additional five to six isoforms or post-translationally modified XET enzymes were detected in crude seedling extracts of barley. The HvXET6 isoenzyme was expressed in Pichia pastoris, characterized and compared with the previously purified native HvXET5 isoform. Barley HvXET6 has a similar apparent molecular mass of 33-35 kDa to the previously purified HvXET5 isoenzyme, but the two isoenzymes differ in their isoelectric points, pH optima, kinetic properties and substrate specificities. The HvXET6 isoenzyme catalyses transfer reactions between xyloglucans and soluble cellulosic substrates, using oligo-xyloglucosides as acceptors, but at rates that are significantly different from those observed for HvXET5. No hydrolytic activity could be detected with either isoenzyme. Comparisons of the reaction rates using xyloglucan or hydroxyethyl cellulose as donors and a series of cellodextrins as acceptors indicated that the acceptor site of HvXET can accommodate five glucosyl residues. Molecular modelling supported this conclusion and further confirmed the ability of the enzyme's active site to accommodate xyloglucan and cellulosic substrates. The two HvXETs followed a ping-pong (Bi, Bi) rather than a sequential reaction mechanism.

Keyword
glycoside hydrolase family 16, molecular modelling, phylogenetic, analyses, plant cell walls, reaction mechanisms, multiple sequence alignment, cell-wall, pichia-pastoris, saccharomyces-cerevisiae, endotransglycosylase, enzyme, oligosaccharides, family, proteins, linkage
Identifiers
urn:nbn:se:kth:diva-18069 (URN)10.1111/j.1742-4658.2008.06791.x (DOI)000261978000012 ()
Note
QC 20100525Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2017-12-12Bibliographically approved
3. Differences in enzymic properties of five recombinant xyloglucan endotransglucosylase/hydrolase (XTH) proteins of Arabidopsis thaliana
Open this publication in new window or tab >>Differences in enzymic properties of five recombinant xyloglucan endotransglucosylase/hydrolase (XTH) proteins of Arabidopsis thaliana
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2011 (English)In: Journal of Experimental Botany, ISSN 0022-0957, E-ISSN 1460-2431, Vol. 62, no 1, 261-271 p.Article in journal (Refereed) Published
Abstract [en]

Xyloglucan endotransglucosylase/hydrolases (XTHs) are cell wall enzymes that are able to graft xyloglucan chains to oligosaccharides or to other available xyloglucan chains and/or to hydrolyse xyloglucan chains. As they are involved in the modification of the load-bearing cell-wall components, they are believed to be very important in the regulation of growth and development. Given the large number (33) of XTH genes in Arabidopsis and the overlapping expression patterns, specific enzymic properties may be expected. Five predominantly root-expressed Arabidopsis thaliana XTHs belonging to subgroup I/II were analysed here. These represent two sets of closely related genes: AtXTH12 and 13 on the one hand (trichoblast-enriched) and AtXTH17, 18, and 19 on the other (expressed in nearly all cell types in the root). They were all recombinantly produced in the yeast Pichia pastoris and partially purified by ammonium sulphate precipitation before they were subsequently all subjected to a series of identical in vitro tests. The kinetic properties of purified AtXTH13 were investigated in greater detail to rule out interference with the assays by contaminating yeast proteins. All five proteins were found to exhibit only the endotransglucosylase (XET; EC 2.4.1.207) activity towards xyloglucan and non-detectable endohydrolytic (XEH; EC 3.2.1.151) activity. Their endotransglucosylase activity was preferentially directed towards xyloglucan and, in some cases, water-soluble cellulose acetate, rather than to mixed-linkage beta-glucan. Isoforms differed in optimum pH (5.0-7.5), in temperature dependence and in acceptor substrate preferences.

Keyword
Arabidopsis thaliana, Brassicaceae, cell elongation, cell wall, heterologous protein production, xyloglucan endotransglucosylase, hydrolase (XTH)
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-28187 (URN)10.1093/jxb/erq263 (DOI)000284951900022 ()
Funder
Swedish Research Council
Note
QC 20110111Available from: 2011-01-11 Created: 2011-01-10 Last updated: 2017-12-11Bibliographically approved
4. Group III-A XTH genes encode predominant xyloglucan endo hydrolase active in expanding tissues of Arabidopsis thaliana
Open this publication in new window or tab >>Group III-A XTH genes encode predominant xyloglucan endo hydrolase active in expanding tissues of Arabidopsis thaliana
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(English)Manuscript (preprint) (Other academic)
National Category
Genetics
Identifiers
urn:nbn:se:kth:diva-28352 (URN)
Note
QC 20110114Available from: 2011-01-14 Created: 2011-01-14 Last updated: 2011-04-01Bibliographically approved
5. Development of a high-throughput assay for screening xyloglucan endo-transglycosylase and endo-xyloglucanase expression in crude microbial supernatants
Open this publication in new window or tab >>Development of a high-throughput assay for screening xyloglucan endo-transglycosylase and endo-xyloglucanase expression in crude microbial supernatants
(English)Manuscript (preprint) (Other academic)
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-28353 (URN)
Note
QC 20110114Available from: 2011-01-14 Created: 2011-01-14 Last updated: 2011-01-14Bibliographically approved

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