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

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Abstract
We report the identification, molecular cloning, and characterization of an endo-β-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-β-N-acetylglucosaminidase (Endo-M). Actually, the C. elegans extract contained endo-β-N-acetylglucosaminidase activity. The putative cDNA for the C. elegans endo-β-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. Man8GlcNAc2 was the best substrate for Endo-CE, and Man3GlcNAc2 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-β-N-acetylglucosaminidase from hen oviduct. Endo-CE was confirmed to exhibit transglycosylation activity, as seen for some microbial endo-β-N-acetylglucosaminidases. This is the first report of the molecular cloning of an endo-β-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.

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