Dataset Information

Non-lysosomal Degradation of Singly Phosphorylated Oligosaccharides Initiated by the Action of a Cytosolic Endo-?-N-acetylglucosaminidase.

ABSTRACT: Phosphorylated oligosaccharides (POSs) are produced by the degradation of dolichol-linked oligosaccharides (DLOs) by an unclarified mechanism in mammalian cells. Although POSs are exclusively found in the cytosol, their intracellular fates remain unclear. Our findings indicate that POSs are catabolized via a non-lysosomal glycan degradation pathway that involves a cytosolic endo-?-N-acetylglucosaminidase (ENGase). Quantitative and structural analyses of POSs revealed that ablation of the ENGase results in the significant accumulation of POSs with a hexasaccharide structure composed of Man?1,2Man?1,3(Man?1,6)Man?1,4GlcNAc?1,4GlcNAc.In vitroENGase assays revealed that the presence of an ?1,2-linked mannose residue facilitates the hydrolysis of POSs by the ENGase. Liquid chromatography-mass spectrometric analyses and fluorescent labeling experiments show that such POSs contain one phosphate group at the reducing end. These results indicate that ENGase efficiently hydrolyzes POSs that are larger than Man4GlcNAc2-P, generating GlcNAc-1-P and neutral Gn1-type free oligosaccharides. These results provide insight into important aspects of the generation and degradation of POSs.

SUBMITTER: Harada Y

PROVIDER: S-EPMC4825009 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Non-lysosomal Degradation of Singly Phosphorylated Oligosaccharides Initiated by the Action of a Cytosolic Endo-β-N-acetylglucosaminidase.

Harada Yoichiro Y Huang Chengcheng C Yamaki Satoshi S Dohmae Naoshi N Suzuki Tadashi T

The Journal of biological chemistry 20160208 15

Phosphorylated oligosaccharides (POSs) are produced by the degradation of dolichol-linked oligosaccharides (DLOs) by an unclarified mechanism in mammalian cells. Although POSs are exclusively found in the cytosol, their intracellular fates remain unclear. Our findings indicate that POSs are catabolized via a non-lysosomal glycan degradation pathway that involves a cytosolic endo-β-N-acetylglucosaminidase (ENGase). Quantitative and structural analyses of POSs revealed that ablation of the ENGase ...[more]

PMID: 26858256

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Project description:In the endoplasmic reticulum-associated degradation system of plant and animal cells, high-mannose type free N-glycans (HMT-FNGs) are produced from misfolded glycoproteins prior to proteasomal degradation, and two enzymes, cytosolic peptide:N-glycanase (cPNGase) and endo-β-N-acetylglucosaminidase (endo-β-GlcNAc-ase), are involved in the deglycosylation. Although the physiological functions of these FNGs in plant growth and development remain to be elucidated, detailed characterization of cPNGase and endo-β-GlcNAc-ase is required. In our previous work, we described the purification, characterization, and subcellular distribution of some plant endo-β-GlcNAc-ases and preliminarily reported the gene information of rice endo-β-GlcNAc-ase (Endo-Os). Furthermore, we analyzed the changes in gene expression of endo-β-GlcNAc-ase during tomato fruit maturation and constructed a mutant line of Arabidopsis thaliana, in which the two endo-β-GlcNAc-ase genes were knocked-out based on the Endo-Os gene. In this report, we describe the purification, characterization, amino acid sequence, and gene cloning of Endo-Os in detail. Purified Endo-Os, with an optimal pH of 6.5, showed high activity for high-mannose type N-glycans bearing the Manα1-2Manα1-3Manβ1 unit; this substrate specificity was almost the same as that of other plant endo-β-GlcNAc-ases, suggesting that Endo-Os plays a critical role in the production of HTM-FNGs in the cytosol. Electrospray ionization-mass spectrometry analysis of the tryptic peptides revealed 17 internal amino acid sequences, including the C terminus; the N-terminal sequence could not be identified due to chemical modification. These internal amino acid sequences were consistent with the amino acid sequence (UniProt ID: Q5W6R1) deduced from the Oryza sativa cDNA clone AK112067 (gene ID: Os05g0346500). Recombinant Endo-Os expressed in Escherichia coli using cDNA showed the same enzymatic properties as those of native Endo-Os.

Dataset Information

Non-lysosomal Degradation of Singly Phosphorylated Oligosaccharides Initiated by the Action of a Cytosolic Endo-?-N-acetylglucosaminidase.

Publications

Non-lysosomal Degradation of Singly Phosphorylated Oligosaccharides Initiated by the Action of a Cytosolic Endo-β-N-acetylglucosaminidase.

Similar Datasets

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