Cloning, Sequencing, and Expression ofArthrobacter protophormiaeEndo-β-N-acetylglucosaminidase inEscherichia coli

Takegawa, Kaoru; Yamabe, Kayo; Fujita, Kiyotaka; Tabuchi, Mitsuaki; Mita, Masanori; Izu, Hiroyuki; Watanabe, Akira; Asada, Yasuhiko; Sano, Mutsumi; Kondo, Akihiro; Kato, Ikunoshin; Iwahara, Shojiro

doi:10.1006/abbi.1996.9803

Cited by 60 publications

(44 citation statements)

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“…31 and 32) or Endo-A (U59168; ref. 34). These fungal͞bacterial ENGases are secreted enzymes (31,33) and are apparently different from the cytosolic ENGases in terms of physiological functions.…”

Section: Discussionmentioning

confidence: 99%

“…Taken together, these results suggested the wide distribution of this enzyme in eukaryotes. Interestingly, when a nonredundant sequence database was surveyed, previously characterized bacterial͞microbial ENGases, such as Endo-M from Mucor hiemalis (31,32) or Endo-A from Arthrobacter protophormiae (33,34), were also found to have less, but significant, homology with human ENGase (data not shown).…”

Section: (Sus Scrofa Bos Taurus Mus Musculus and Rattus Norvegicusmentioning

confidence: 99%

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Endo-β- N -acetylglucosaminidase, an enzyme involved in processing of free oligosaccharides in the cytosol

Suzuki

Yano

Sugimoto

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

125

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Formation of oligosaccharides occurs both in the cytosol and in the lumen of the endoplasmic reticulum (ER). Luminal oligosaccharides are transported into the cytosol to ensure that they do not interfere with proper functioning of the glycan-dependent quality control machinery in the lumen of the ER for newly synthesized glycoproteins. Once in the cytosol, free oligosaccharides are catabolized, possibly to maximize the reutilization of the component sugars. An endo-␤-N-acetylglucosaminidase (ENGase) is a key enzyme involved in the processing of free oligosaccharides in the cytosol. This enzyme activity has been widely described in animal cells, but the gene encoding this enzyme activity has not been reported. Here, we report the identification of the gene encoding human cytosolic ENGase. After 11 steps, the enzyme was purified 150,000-fold to homogeneity from hen oviduct, and several internal amino acid sequences were analyzed. Based on the internal sequence and examination of expressed sequence tag (EST) databases, we identified the human orthologue of the purified protein.The human protein consists of 743 aa and has no apparent signal sequence, supporting the idea that this enzyme is localized in the cytosol. By expressing the cDNA of the putative human ENGase in COS-7 cells, the enzyme activity in the soluble fraction was enhanced 100-fold over the basal level, confirming that the human gene identified indeed encodes for ENGase. Careful gene database surveys revealed the occurrence of ENGase homologues in Drosophila melanogaster, Caenorhabditis elegans, and Arabidopsis thaliana, indicating the broad occurrence of ENGase in higher eukaryotes. This gene was expressed in a variety of human tissues, suggesting that this enzyme is involved in basic biological processes in eukaryotic cells.

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Section: Discussionmentioning

confidence: 99%

Section: (Sus Scrofa Bos Taurus Mus Musculus and Rattus Norvegicusmentioning

confidence: 99%

Endo-β- N -acetylglucosaminidase, an enzyme involved in processing of free oligosaccharides in the cytosol

Suzuki

Yano

Sugimoto

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

125

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“…It was also noticed, at that moment, that one interesting structural feature of the cytosolic ENGase is that this protein shares reduced, but significant, homology with ENGases such as Endo M [111,112] or Endo A [81]. These ENGases (Endo M, A, and cytosolic ENGases), which the authors [110] proposed should be called class II ENGases are structurally distant from class I ENGase (Endo H and its structurally related enzymes), for which extensive structural studies have been carried out.…”

Section: Animal N-glycan-engasesmentioning

confidence: 99%

“…The enzyme induction seems to have a close relation to its substrate specificity, very similar to that of Endo CII. The gene encoding Endo A was cloned later [81]. The primary structure of Endo A, composed of a 24 amino acid signal peptide and of a mature 621 amino acid protein, exhibited significant homology with a gene product from Caenorhabditis elegans, of unknown function at that moment, but later characterised [82], and no significant homology with any previously reported ENGases.…”

Section: Bacterial N-glycan-engasesmentioning

confidence: 99%

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Endo-N-Acetyl-β-D-Glucosaminidases and Peptide-N4-(N-acetyl-β-D-Glucosaminyl) Asparagine Amidases: More Than Just Tools

Karamanos¹

2013

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Abstract:Since the discovery of endo-N-acetyl-β-D-glucosaminidases (ENGase) and peptide-N4-(N-acetyl-β-D-glucosaminyl) asparagine amidases (PNGase) most of the published work described their use for structural studies. Less attention was given to the potential roles of those enzymes in the physiology of the cells/organisms they produced them. The scope of this review is firstly to analyse the data on the occurrence and characteristics of murein-, chitin-, and N-glycan-ENGases acting on GlcNAc-containing polymers in three structural families, namely murein, chitin, and N-glycosylproteins, and of PNGases, only acting on N-glycosylproteins, and secondly to discuss the biological roles of the enzymes in the producing cells. The analysis demonstrates the remarkable diversity of the enzymes, and simultaneously the interest of studying their substrate specificity and their structural features. Many examples illustrate the importance of the structure/function relationships studies. Diverse biological roles were anticipated, e.g. they are useful for feeding purposes, are implicated in pathogenesis processes, modulate the activity of macromolecules, and help in the destruction of misfolded proteins. Their effect can be direct or indirect, through the reaction products. Current knowledge only partially explains the biological roles of ENGases and PNGases, thus further studies are expected for determining novel possibilities and elucidating other cell pathways.

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Unusual Transglycosylation Activity of Flavobacterium meningosepticum Endoglycosidases Enables Convergent Chemoenzymatic Synthesis of Core Fucosylated Complex N‐Glycopeptides

Huang

Wang

2011

ChemBioChem

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Structurally well-defined, homogeneous glycopeptides and glycoproteins are indispensable tools for functional glycomics studies. By screening of various endo-β-N-acetylglucosaminidases using an appropriate synthetic donor and acceptor substrates, we have found that the Flavobacterium meningosepticum endo-β-N-acetylglucosaminidases glycosidases (GH family 18), including Endo-F2 and Endo-F3, were able to glycosylate α-1,6-fucosylated GlcNAc derivative to provide natural, core-fucosylated complex type N-glycopeptides. The Endo-F2 and Endo-F3 were efficient for transferring both sialylated and asialylated glycans and were highly specific for an α-1,6-fucosyllated GlcNAc-peptide as acceptor for transglycosylation, as they showed only marginal activity on non-fucosylated GlcNAc-peptide. In contrast, we found that the commonly used endoglycosidases such as Endo-A and Endo-M, which belong to GH family 85, were unable to take α-1,6-fucosyl-GlcNAc derivative as acceptor for transglycosylation. The novel activity of Endo-F2 and Endo-F3 was successfully applied for a highly convergent chemoenzymatic synthesis of a full-size CD52 glycopeptide antigen carrying both terminal sialic acid and core fucose. This is the first report on endoglycosidases that are able to glycosylate α-1,6-fucosylated GlcNAc derivatives to form natural core-fucosylated glycopeptides.

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Cloning, Sequencing, and Expression ofArthrobacter protophormiaeEndo-β-N-acetylglucosaminidase inEscherichia coli

Cited by 60 publications

References 0 publications

Endo-β- N -acetylglucosaminidase, an enzyme involved in processing of free oligosaccharides in the cytosol

Endo-β- N -acetylglucosaminidase, an enzyme involved in processing of free oligosaccharides in the cytosol

Endo-N-Acetyl-β-D-Glucosaminidases and Peptide-N4-(N-acetyl-β-D-Glucosaminyl) Asparagine Amidases: More Than Just Tools

Unusual Transglycosylation Activity of Flavobacterium meningosepticum Endoglycosidases Enables Convergent Chemoenzymatic Synthesis of Core Fucosylated Complex N‐Glycopeptides

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