Enzymatic properties and subcellular localization of Arabidopsis beta-N-acetylhexosaminidases.
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.
Strasser
Richard
R
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
Bondili
Jayakumar Singh
JS
Schoberer
Jennifer
J
Svoboda
Barbara
B
Liebminger
Eva
E
Glössl
Josef
J
Altmann
Friedrich
F
Steinkellner
Herta
H
Mach
Lukas
L
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*
D018076:DNA, Complementary; D011509:Proteoglycans; D011994:Recombinant Proteins; D001619:beta-N-Acetylhexosaminidases