Identification of an endo-beta-N-acetylglucosaminidase gene in Caenorhabditis elegans and its expression in Escherichia coli.
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.
Kato
Toshihiko
T
Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Fujita
Kiyotaka
K
Takeuchi
Makoto
M
Kobayashi
Kazuo
K
Natsuka
Shunji
S
Ikura
Koji
K
Kumagai
Hidehiko
H
Yamamoto
Kenji
K
D000118:Acetylglucosaminidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000235:genetics*; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics*; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D029742:Caenorhabditis elegans Proteins; D018076:DNA, Complementary; D011994:Recombinant Proteins; D000118:Acetylglucosaminidase; C473069:eng-1 protein, C elegans