Endo-beta-mannosidase, a plant enzyme acting on N-glycan: purification, molecular cloning, and characterization

The Journal of Biological Chemistry
Takeshi IshimizuS Hase

Abstract

Endo-beta-mannosidase is a novel endoglycosidase that hydrolyzes the Manbeta1-4GlcNAc 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-beta-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 Manalpha1-6Manbeta1-4Glc-NAcbeta1-4GlcNAc into Manalpha1-6Man and GlcNAcbeta1-4Glc-NAc-PA, showing that AtEBM is an endo-beta-mannosidase. AtEBM hydrolyzed Man(n)Manalpha1-6Manbeta1-4GlcNAcbeta1-4GlcNAc-PA (n = 0-2) but not PA-sugar chains contain...Continue Reading

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Citations

Aug 9, 2007·BMC Bioinformatics·Abdellali KelilAlain Fleury
May 1, 2010·Proceedings of the Japan Academy. Series B, Physical and Biological Sciences·Sumihiro Hase
Oct 24, 2013·Journal of Experimental Botany·Juan David RejónAntonio Jesús Castro
Jan 9, 2016·Critical Reviews in Biotechnology·Prakram Singh Chauhan, Naveen Gupta
Jun 22, 2006·Journal of Biochemistry·Takeshi IshimizuSumihiro Hase
Sep 18, 2013·The Journal of Biological Chemistry·Simon LadevèzeGabrielle Potocki-Véronèse
Jul 28, 2019·International Journal of Biological Macromolecules·Kavyashree Sakharayapatna RanganathaSiva Kumar Nadimpalli
Feb 10, 2009·Phytochemistry·Renaud LéonardFriedrich Altmann

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