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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Enzymatic Properties and Subcellular Localization of Arabidopsis -N-Acetylhexosaminidases^1,[W],[OA]

Institute of Applied Genetics and Cell Biology (R.S., J.S., B.S., E.L., J.G., H.S., L.M.) and Department of Chemistry (J.S.B., F.A.), BOKU-University of Natural Resources and Applied Life Sciences, A–1190 Vienna, Austria

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 -hexosaminidases during late stages of N-glycan processing or in the course of N-glycan turnover. We have now cloned the three putative -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--D-glucopyranoside, p-nitrophenyl-2-acetamido-2-deoxy--D-galactopyranoside, 4-methylumbelliferyl-2-acetamido-2-deoxy--D-glucopyranoside, and 4-methylumbelliferyl-6-sulfo-2-acetamido-2-deoxy--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 1,3- and 1,6-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.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Richard Strasser (richard.strasser{at}boku.ac.at).

^[W] The online version of this article contains Web-only data.

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www.plantphysiol.org/cgi/doi/10.1104/pp.107.101162

^* Corresponding author; e-mail richard.strasser{at}boku.ac.at.

Received April 17, 2007; accepted July 10, 2007; published July 20, 2007.

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