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J. Biol. Chem., Vol. 279, Issue 37, 38555-38562, September 10, 2004

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Endo--mannosidase, a Plant Enzyme Acting on N-Glycan

PURIFICATION, MOLECULAR CLONING, AND CHARACTERIZATION^*

Takeshi Ishimizu, Akiko Sasaki, Satoshi Okutani, Mami Maeda, Mai Yamagishi, and Sumihiro Hase

From the Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan

Endo--mannosidase is a novel endoglycosidase that hydrolyzes the Man1-4GlcNAc linkage in the trimannosyl core structure of N-glycans. This enzyme was partially purified and characterized in a previous report (Sasaki, A., Yamagishi, M., Mega, T., Norioka, S., Natsuka, S., and Hase, S. (1999) J. Biochem. 125, 363–367). Here we report the purification and molecular cloning of endo--mannosidase. The enzyme purified from lily flowers gave a single band on native-PAGE and three bands on SDS-PAGE with molecular masses of 42, 31, and 28 kDa. Amino acid sequence information from these three polypeptides allowed the cloning of a homologous gene, AtEBM, from Arabidopsis thaliana. AtEBM was engineered for expression in Escherichia coli, and the recombinant protein comprised a single polypeptide chain with a molecular mass of 112 kDa corresponding to the sum of molecular masses of three polypeptides of the lily enzyme. The recombinant protein hydrolyzed pyridylamino derivatives (PA) of Man1-6Man1-4Glc-NAc1-4GlcNAc into Man1-6Man and GlcNAc1-4Glc-NAc-PA, showing that AtEBM is an endo--mannosidase. AtEBM hydrolyzed Man_nMan1-6Man1-4GlcNAc1-4GlcNAc-PA (n = 0–2) but not PA-sugar chains containing Man1-3Man or Xylose1-2Man as for the lily endo--mannosidase. AtEBM belonged to the clan GH-A of glycosyl hydrolases. Site-directed mutagenesis experiments revealed that two glutamic acid residues (Glu-464 and Glu-549) conserved in this clan were critical for enzyme activity. The amino acid sequence of AtEBM has distinct differences from those of the bacterial, fungal, and animal exo-type -mannosidases. Indeed, AtEBM-like genes are only found in plants, indicating that endo--mannosidase is a plant-specific enzyme. The role of this enzyme in the processing and/or degradation of N-glycan will be discussed.

Received for publication, June 21, 2004

^* This work was supported in part in the 21st Century COE program (Creation of Integrated EcoChemistry), the Protein 3000 Program, and the Japan Health Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AB122060.

Present address: Major in Integrative Bioscience and Biomedical Engineering, Waseda University, 3-4-1 Okubo, Tokyo 169-8555, Japan.

To whom correspondence should be addressed. Tel.: 81-6-6850-5380; Fax: 81-6-6850-5383; E-mail: suhase{at}chem.sci.osaka-u.ac.jp.

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				T. Ishimizu, C. Hashimoto, R. Takeda, K. Fujii, and S. Hase A Novel {alpha}1,2-L-Fucosidase Acting on Xyloglucan Oligosaccharides is Associated with Endo- -Mannosidase J. Biochem., December 1, 2007; 142(6): 721 - 729. [Abstract] [Full Text] [PDF]

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