Mutsumi Sano

Two-dimensional mapping by high-performance liquid chromatography of pyridylamino oligosaccharides from various glycosphingolipids

A method to map sugars two-dimensionally for the analysis of the structures of oligosaccharides from glycosphingolipids is described. Nine neutral and ten acidic oligosaccharides were obtained from glycosphingolipids by endoglycoceramidase digestion and labelled with 2-aminopyridine. The pyridylamino oligosaccharides were clearly separated by high-performance liquid chromatography on commercially available C18-silica and amide-silica column. All compounds tested were mapped without any overlapping. The separation of the pyridylamino oligosaccharides on the C18-silica column depended on the numbers and positions of sialic acid and N-acetylhexosamine residues; on the amide-silica column, the separation depended on the total number of sugar residues.

Molecular Cloning, Expression, and Sequence Analysis of the Endoglycoceramidase II Gene from Rhodococcus Species Strain M-777

Endoglycoceramidase (EGCase (EC 3.2.1.123)) is a hydrolase that hydrolyzes the linkage between the oligosaccharide and ceramide of various glycosphingolipids. This paper describes the molecular cloning and expression of EGCase II, one of the isoforms of EGCases. The gene encoding EGCase II was obtained by screening of a genomic DNA library from Rhodococcus sp. strain M-777 constructed in pUC19 with oligonucleotide probes deduced from a partial amino acid sequence of the enzyme protein. RecombinantEscherichia coli cells in which the EGCase II gene was expressed produced 14 units of the enzyme per liter of culture medium but did not produce sphingomyelinase. Recombinant EGCase II was a functioning enzyme with substrate specificity identical to that of the wild-type enzyme. Sequence analysis showed the presence of an open reading frame of 1470 base pairs encoding 490 amino acids. The N-terminal region of the deduced amino acid sequence had the general pattern of signal peptides of secreted prokaryotic proteins. Interestingly, the consensus sequence in the active site region of the endo-1,4-β-glucanase family A was found in the amino acid sequence of EGCase II.

Transfer of high-mannose-type oligosaccharides to disaccharides by endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae

Endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) has transglycosylation activity, and high-mannose-type oligosaccharides are transferred to suitable glycosides as acceptor substrates. The acceptor specificity of Endo-A-catalyzed transglycosylation toward various disaccharides was investigated. To identify an effective acceptor for the transglycosylation by Endo-A, the reaction was carried out using various disaccharides. Endo-A transferred high-mannose-type oligosaccharides more efficiently to beta-linked disaccharides (cellobiose, gentiobiose, sophorose, and laminaribiose) than to alpha-linked disaccharides (isomaltose, maltose, nigerose, kojibiose, and trehalose) as acceptor substrates. The transglycosylation products, (Man)6GlcNAc-Glc-beta-Glc, were more rapidly hydrolyzed than (Man)6GlcNAc-Glc-alpha-Glc. These results indicate that Endo-A recognizes the anomeric configuration of the acceptor substrates, and beta-linked glycosides are suitable for the synthesis of transglycosylation products.