BOKU - Universität für Bodenkultur Wien - Forschungsinformationssystem

Logo BOKU-Forschungsportal

Gewählte Publikation:

Liebminger, E; Grass, J; Altmann, F; Mach, L; Strasser, R.
(2013): Characterizing the link between glycosylation state and enzymatic activity of the endo-â1,4-glucanase KORRIGAN1 from Arabidopsis thaliana.
J Biol Chem. 2013; 288(31):22270-22280 FullText FullText_BOKU

Abstract:
Defects in N-glycosylation and N-glycan processing frequently cause alterations in plant cell wall architecture, including changes in the structure of cellulose, which is the most abundant plant polysaccharide. KORRIGAN1 (KOR1) is a glycoprotein enzyme with an essential function during cellulose biosynthesis in Arabidopsis thaliana. KOR1 is a membrane-anchored endo-beta 1,4-glucanase and contains eight potential N-glycosylation sites in its extracellular domain. Here, we expressed A. thaliana KOR1 as a soluble, enzymatically active protein in insect cells and analyzed its N-glycosylation state. Structural analysis revealed that all eight potential N-glycosylation sites are utilized. Individual elimination of evolutionarily conserved N-glycosylation sites did not abolish proper KOR1 folding, but mutations of Asn-216, Asn-324, Asn-345, and Asn-567 resulted in considerably lower enzymatic activity. In contrast, production of wild-type KOR1 in the presence of the class I alpha-mannosidase inhibitor kifunensine, which abolished the conversion of KOR1 N-glycans into complex structures, did not affect the activity of the enzyme. To address N-glycosylation site occupancy and N-glycan composition of KOR1 under more natural conditions, we expressed a chimeric KOR1-Fc-GFP fusion protein in leaves of Nicotiana benthamiana. Although Asn-108 and Asn-133 carried oligomannosidic N-linked oligosaccharides, the six other glycosylation sites were modified with complex N-glycans. Interestingly, the partially functional KOR1 G429R mutant encoded by the A. thaliana rsw2-1 allele displayed only oligomannosidic structures when expressed in N. benthamiana, indicating its retention in the endoplasmic reticulum. In summary, our data indicate that utilization of several N-glycosylation sites is important for KOR1 activity, whereas the structure of the attached N-glycans is not critical.
Autor*innen der BOKU Wien:
Altmann Friedrich
Grass Josephine
Mach Lukas
Strasser Richard
Find related publications in this database (using NML MeSH Indexing)
Amino Acid Sequence -
Arabidopsis - enzymology
Arabidopsis Proteins - chemistry
Base Sequence -
Cellulase - chemistry
DNA Primers -
Glycosylation -
Membrane Proteins - chemistry
Molecular Sequence Data -
Mutagenesis, Site-Directed -
Tobacco - genetics



Altmetric:
© BOKU Wien Impressum