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International audience; N-Glycans are widely distributed in living organisms but represent only a small fraction of the carbohydrates found in plants. This probably explains why they have not previously been considered as substrates exploited by phytopathogenic bacteria during plant infection. Xanthomonas campestris pv. campestris, the causal agent of black rot disease of Brassica plants, possesses a specific system for GlcNAc utilization expressed during host plant infection. This system encompasses a cluster of eight genes (nixE to nixL) encoding glycoside hydrolases (GHs). In this paper, we have characterized the enzymatic activities of these GHs and demonstrated their involvement in sequential degradation of a plant N-glycan using a N-glycopeptide containing two GlcNAcs, three mannoses, one fucose, and one xylose (N2M3FX) as a substrate. The removal of the α-1,3-mannose by the α-mannosidase NixK (GH92) is a prerequisite for the subsequent action of the β-xylosidase NixI (GH3), which is involved in the cleavage of the β-1,2-xylose, followed by the α-mannosidase NixJ (GH125), which removes the α-1,6-mannose. These data, combined to the subcellular localization of the enzymes, allowed us to propose a model of N-glycopeptide processing by X. campestris pv. campestris. This study constitutes the first evidence suggesting N-glycan degradation by a plant pathogen, a feature shared with human pathogenic bacteria. Plant N-glycans should therefore be included in the repertoire of molecules putatively metabolized by phytopathogenic bacteria during their life cycle.

In the accompanying paper [Mellor, Neville, Harvey, Platt, Dwek and Butters (2004) Biochem. J. 381, 861–866] we treated HL60 cells with N-alk(en)yl-deoxynojirimycin (DNJ) compounds to inhibit glucosphingolipid (GSL) biosynthesis and identified a number of non-GSL-derived, small, free oligosaccharides (FOS) most likely produced due to inhibition of the oligosaccharide-processing enzymes α-glucosidases I and II. When HL60 cells were treated with concentrations of N-alk(en)ylated DNJ analogues that inhibited GSL biosynthesis completely, N-butyl- and N-nonyl-DNJ inhibited endoplasmic reticulum (ER) glucosidases I and II, but octadecyl-DNJ did not, probably due to the lack of ER lumen access for this novel, long-chain derivative. Glucosidase inhibition resulted in the appearance of free Glc1–3Man structures, which is evidence of Golgi glycoprotein endomannosidase processing of oligosaccharides with retained glucose residues. Additional large FOS was also detected in cells following a 16 h treatment with N-butyl- and N-nonyl-DNJ. When these FOS structures (>30, including >20 species not present in control cells) were characterized by enzyme digests and MALDI-TOF (matrix-assisted laser-desorption ionization–time-of-flight) MS, all were found to be polymannose-type oligosaccharides, of which the majority were glucosylated and had only one reducing terminal GlcNAc (N-acetylglucosamine) residue (FOS-GlcNAc1), demonstrating a cytosolic location. These results support the proposal that the increase in glucosylated FOS results from enzyme-mediated cytosolic cleavage of oligosaccharides from glycoproteins exported from the ER because of misfolding or excessive retention. Importantly, the present study characterizes the cellular properties of DNJs further and demonstrates that side-chain modifications allow selective inhibition of protein and lipid glycosylation pathways. This represents the most detailed characterization of the FOS structures arising from ER α-glucosidase inhibition to date.

The lysosomal system is up-regulated in the brain of patients with Alzheimer's Disease (AD), as demonstrated by previous experiments carried out in postmortem samples of brain patients. In this paper we provide evidence that an up-regulation of lysosomal glycohydrolases (alpha-D-mannosidase, beta-D-hexosaminidase, and beta-D-galactosidase) takes place in skin fibroblasts from AD patients affected either by sporadic or familial forms and is detectable also in presymptomatic subjects carrying the above mutations but healthy at the time of skin biopsy. This increase of enzyme activity is consequent to a transcriptional up-regulation. The oncogene Ras appears to be involved in the regulation of enzymatic activity. A parallel increase of Ras transcript and Ras protein, without an increase of p44/p42 MAPK activation was revealed in the same AD fibroblasts. An activation of p38 MAPK already described to occur in neurodegenerative diseases such as Alzheimer's, was also found in fibroblasts derived from AD patients. High levels of expression of the constitutively active form of Ras in normal or AD fibroblasts induced glycohydrolases up-regulation. Overall results demonstrated that glycohydrolases up-regulation, as well as Ras up-regulation, are early markers of AD, detectable at peripheral level, and good candidates to be exploited for diagnostic purposes. These data also provide the first proof for a role of Ras in regulating lysosomal glycohydrolases expression.