Identification of an endo- -N-acetylglucosaminidase gene in Caenorhabditis elegans and its expression in Escherichia coli

Abstract

We report the identification, molecular cloning, and characterization of an endo-beta-N-acetylglucosaminidase from the nematode Caenorhabditis elegans. A search of the C. elegans genome database revealed the existence of a gene exhibiting 34% identity to Mucor hiemalis (a fungus) endo-beta-N-acetylglucosaminidase (Endo-M). Actually, the C. elegans extract contained endo-beta-N-acetylglucosaminidase activity. The putative cDNA for the C. elegans endo-beta-N-acetylglucosaminidase (Endo-CE) was amplified by polymerase chain reaction from the Uni-ZAP XR library, cloned, and sequenced. The recombinant Endo-CE expressed in Escherichia coli exhibited substrate specificity mainly for high-mannose type oligosaccharides. Man(8)GlcNAc(2) was the best substrate for Endo-CE, and Man(3)GlcNAc(2) was also hydrolyzed. Biantennary complex type oligosaccharides were poor substrates, and triantennary complex substrates were not hydrolyzed. Its substrate specificity was similar to those of Endo-M and endo-beta-N-acetylglucosaminidase from hen oviduct. Endo-CE was confirmed to exhibit transglycosylation activity, as seen for some microbial endo-beta-N-acetylglucosaminidases. This is the first report of the molecular cloning of an endo-beta-N-acetylglucosaminidase gene from a multicellular organism, which shows the possibility of using this well-characterized nematode as a model system for elucidating the role of this enzyme.

Synthesis and inhibitory activity of oligosaccharide thiazolines as a class of mechanism-based inhibitors for endo-β-N-acetylglucosaminidases

“…For Endo-A and Endo-M, Man9GlcNAc2Asn was used as the substrate and the hydrolytic activity of the enzymes in the presence or absence of the respective inhibitors were measured by quantifying the hydrolytic product Man9GlcNAc using the HPAEC-PED method. 25 It was found that all the three oligosaccharide thiazolines (2)(3)(4) showed inhibitory activities against Endo-A. But the larger oligosaccharide derivative 4 was much more potent in inhibiting Endo-A activity than the tetrasaccharide and disaccharide derivatives 2 and 3, respectively ( Figure 2A).…”

Enzymes Involved in Generation and Degradation of the Free Oligosaccharides in the Cytosol of Caenorhabditis elegans

“…On the basis of sequence homology with Endo M, we cloned the ENG 1 cDNA from C. elegans, which is encoded by F01F1.10. 29) ENG 1 is a 433 amino acid polypeptide and exhibited 34% identity with Endo M. The human gene encoding ENGase was also reported around the same time. 30) ENGases from various animals including vertebrates and invertebrates belong to GH85 and the putative homologues are found from several plants, protozoa and fungi, but not from yeast Saccharomyces cerevisiae and Schizosaccharomyces pombe.…”

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

“…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.…”

“…Caenorabditis elegans Endo CE [82] Homo sapiens Engase 1, ENGase [110] Plant N-glycan-ENGases Arabidopsis thaliana ENGase [183] Solanum lycopersicum Endo LE [121] Fungal N-glycan-ENGases…”