The two endo-β-N-acetylglucosaminidase genes from Arabidopsis thaliana encode cytoplasmic enzymes controlling free N-glycan levels
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Endo-β-N-acetylglucosaminidases (ENGases) cleave N-glycans from proteins and/or peptides by hydrolyzing the O-glycosidic linkage between the two core-N-acetylglucosamine (GlcNAc) residues. Although, two homologous genes potentially encoding ENGases have been identified in Arabidopsis thaliana, their respective substrate specificity, their subcellular and their organ specific localization was hitherto unknown. In order to investigate the role of ENGases in this model plant species, we transiently expressed the two A. thaliana genes in Nicotiana benthamiana and determined the substrate specificities, as well as the Km values, of the purified recombinant enzymes. The assumed predominantly cytosolic localisation of both enzymes, here referred to as AtENGase85A and AtENGase85B, was determined by confocal microscopy of plant leaves expressing the respective GFP-fusion constructs. For the individual characterization of the two enzymes expression patterns in planta, single knock-out plants were selected for both genes. Although both enzymes are present in most organs, only AtENGase85A (At5g05460) was expressed in stems and no ENGase activity was detected in siliques. A double knock-out was generated by crossing but—like single knock-out plants—no apparent phenotype was observed. In contrast, in this double knock-out, free N-glycans carrying a single GlcNAc at the reducing end are completely absent and their counterparts with two GlcNAc—visible only at a trace level in wild type—accumulated dramatically.
KeywordsEndo-N-acetylglucosaminidase Arabidopsis thaliana Free N-glycan ERAD
Peptide-N 4-(N-acetyl-β-glucosaminyl)asparagine amidase
This project was funded by the Fonds zur Förderung der wissenschaftlichen Forschung (P20132-B16 to Renaud Léonard). We thank Clemens Gruber for the MS analysis of N-glycosylation sites, Richard Strasser (BOKU, Vienna, Austria) for supplying p21GT and Sabine Lhernould (Université de Limoges, France) for her guidance in free N-glycan preparation.
- Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B (2009) The Carbohydrate-Active EnZymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Res Database issue: D233–238Google Scholar
- Kimura Y (2007) Structural and functional features of plant glycoprotein glycans. In: Kamerling JP (ed) Comprehensive Glycoscience, Elsevier, Amsterdam, vol 3, pp 61–78Google Scholar
- Nakamura K, Inoue M, Maeda M, Nakano R, Hosoi K, Fujiyama K, Kimura Y (2009) Molecular cloning and gene expression analysis of tomato endo-beta-N-acetylglucosaminidase, an endoglycosidase involved in the production of high-mannose type free N-glycans during tomato fruit ripening. Biosci Biotechnol Biochem 73:461–464PubMedCrossRefGoogle Scholar
- Rademacher T, Sack M, Arcalis E, Stadlmann J, Balzer S, Altmann F, Quendler H, Stiegler G, Kunert R, Fischer R, Stoger E (2008) Recombinant antibody 2G12 produced in maize endosperm efficiently neutralizes HIV-1 and contains predominantly single-GlcNAc N-glycans. Plant Biotechnol J 6:189–201PubMedCrossRefGoogle Scholar
- Takegawa K, Yamabe K, Fujita K, Tabuchi M, Mita M, Izu H, Watanabe A, Asada Y, Sano M, Kondo A, Kato I, Iwahara S (1997) Cloning, sequencing, and expression of Arthrobacter protophormiae endo-beta-N-acetylglucosaminidase in Escherichia coli. Arch Biochem Biophys 338:22–28PubMedCrossRefGoogle Scholar