Expression And Characterization Of Endo-?-N-acetylglucosaminidase From Trypanosoma Brucei

Endo-?-N-acetylglucosaminidases(ENGases)belonging to the glycoside hydrolase family 85(GH85)hydrolyze the glycosidic bond of the N,N'-diacetylchitobiose moieties of N-glycan cores.Some of the ENGases have been reported to show another activity that transfers chemically synthesized glycans onto glycoproteins.This activity is a valuable tool for the chemo-enzymatic synthesis of proteins with a defined N-glycan structure.In the present study,we charectertized new ENGase from Trypanosoma brucei.The homolog(Endo Tb)was searched by BLAST program using ENGase sequence from Ogataea minuta(Endo-Om),and we found that Endo Tb possessed peptidase C97 and PUB domains at N-terminus of GH85 catalytic domain.The sequence similarity and identitiy between GH85 domain of Endo Tb and Endo-Om were 41.0% and 28.8%,respectively.Three types of recombinant Endo Tb,such as full length and those lacking peptidase C97 domain(D1)or both peptidase C97 and PUB domains(D2)were successfully expressed as hexa-histidine and Nus tags fused proteins in Escherichia coli and exhibited a hydrolytic activity toward 9-fluorenyl-methyloxycarbonyl-labeled asparagine-linked agalacto-biantennary oligosaccharide.Site-directed mutagenesis of highly conserved residues amoung GH85 family proteins resulted in the critical reduction of Endo Tb activity.These results suggested that Endo Tb is a GH85 enzyme and indicated that peptidase C97 and PUB domains are not essential for its enzymatic activity.Substrate specifity of this enzyme was measured with pyridylamino oligosaccharides and glycoproteins modified with defined N-glycans.Unlikely bacterial ENGases,such from Arthrobacter protophormiae and Bacillus halodurans,Endo Tb hydrolyzed high-mannose and complex oligosaccharides.We also detected the glycosyltransferase activity of Endo Tb by fluorescence-based method developed by Hauser et al.(Biochem.Biophys.Res.Commun.,2005),which 4-methylumbelliferyl N-acetylglucosaminide and sialylated glycopeptide were used as acceptor and donor substrates,respectively.Taken together,our biochemical studies conclude that Endo Tb is a new ENGase that will be applied to chemo-enzymatic synthesis of proteins with a defined N-glycan,such as pharmaceutical glycoproteins.