title: Enzymatic properties and subcellular localization of Arabidopsis beta-N-acetylhexosaminidases.
abstract: Plant glycoproteins contain substantial amounts of paucimannosidic N-glycans lacking terminal GlcNAc residues at their nonreducing ends. It has been proposed that this is due to the action of beta-hexosaminidases during late stages of N-glycan processing or in the course of N-glycan turnover. We have now cloned the three putative beta-hexosaminidase sequences present in the Arabidopsis (Arabidopsis thaliana) genome. When heterologously expressed as soluble forms in Spodoptera frugiperda cells, the enzymes (termed HEXO1-3) could all hydrolyze the synthetic substrates p-nitrophenyl-2-acetamido-2-deoxy-beta-d-glucopyranoside, p-nitrophenyl-2-acetamido-2-deoxy-beta-d-galactopyranoside, 4-methylumbelliferyl-2-acetamido-2-deoxy-beta-d-glucopyranoside, and 4-methylumbelliferyl-6-sulfo-2-acetamido-2-deoxy-beta-d-glucopyranoside, albeit to a varying extent. HEXO1 to HEXO3 were further able to degrade pyridylaminated chitotriose, whereas pyridylaminated chitobiose was only cleaved by HEXO1. With N-glycan substrates, HEXO1 displayed a much higher specific activity than HEXO2 and HEXO3. Nevertheless, all three enzymes were capable of removing terminal GlcNAc residues from the alpha1,3- and alpha1,6-mannosyl branches of biantennary N-glycans without any strict branch preference. Subcellular localization studies with HEXO-fluorescent protein fusions transiently expressed in Nicotiana benthamiana plants showed that HEXO1 is a vacuolar protein. In contrast, HEXO2 and HEXO3 are mainly located at the plasma membrane. These results indicate that HEXO1 participates in N-glycan trimming in the vacuole, whereas HEXO2 and/or HEXO3 could be responsible for the processing of N-glycans present on secretory glycoproteins.
authors.affiliation: Institute of Applied Genetics and Cell Biology, BOKU-University of Natural Resources and Applied Life Sciences, A-1190 Vienna, Austria. richard.strasser@boku.ac.at
authors.forename: Richard
authors.initials: R
authors.lastname: Strasser
authors.forename: Jayakumar Singh
authors.initials: JS
authors.lastname: Bondili
authors.forename: Jennifer
authors.initials: J
authors.lastname: Schoberer
authors.forename: Barbara
authors.initials: B
authors.lastname: Svoboda
authors.forename: Eva
authors.initials: E
authors.lastname: Liebminger
authors.forename: Josef
authors.initials: J
authors.lastname: Glössl
authors.forename: Friedrich
authors.initials: F
authors.lastname: Altmann
authors.forename: Herta
authors.initials: H
authors.lastname: Steinkellner
authors.forename: Lukas
authors.initials: L
authors.lastname: Mach
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D018076:DNA, Complementary; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D011509:Proteoglycans / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera / Q000235:genetics / Q000378:metabolism*; D001619:beta-N-Acetylhexosaminidases / Q000235:genetics / Q000378:metabolism*
chemical_list: D018076:DNA, Complementary; D011509:Proteoglycans; D011994:Recombinant Proteins; D001619:beta-N-Acetylhexosaminidases