Preparation and characterization of EGCase I, applicable to the comprehensive analysis of GSLs, using a rhodococcal expression system

Abstract

This article is available online at http://www.jlr.org Glycosphingolipids (GSLs), amphipathic compounds consisting of oligosaccharides and ceramide (Cer) moieties, are ubiquitous in the outer leafl et of the plasma membrane. More than 500 species of GSLs differing in oligosaccharides and Cer moieties have been characterized, some of which represent potential biomarkers for diseases and cell development. For instance, Forssman antigen is specifi cally detected in some gastric, colonic, and lung cancers ( 1 ), and stage-specifi c embryonic antigen (SSEA)-3 and SSEA-4, highly expressed at a stage of embryonic development, are utilized as a marker of embryonic stem cells ( 2 ) and induced pluripotent stem cells ( 3 ).The classifi cation of GSLs, into ganglio-series, globoseries and lacto-series GSLs, is entirely based on the structure of the saccharide moiety. However, the Cer moiety, composed of a sphingoid base and fatty acyl chain linked by an N -acyl linkage, shows heterogeneity in carbon chain length, and saturation and hydroxylation status, etc. The complexity of the Cer moiety prevents the comprehensive analysis of cellular GSLs by MS and/or LC-MS. Removal of the Cer moiety makes the analysis of GSLs much simpler and easier. For this purpose, chemical methods such as ozonolysis ( 4 ) and osmium-catalyzed periodate oxidation ( 5 ) have been developed. These chemical methods, however, require complicated and time-consuming procedures with relatively low yield ( 6 ). Alternatively, enzymes capable of detaching intact oligosaccharides from various GSLs Abstract Endoglycoceramidase (EGCase) is a glycosidase capable of hydrolyzing the ␤ -glycosidic linkage between the oligosaccharides and ceramides of glycosphingolipids (GSLs). Three molecular species of EGCase differing in specifi city were found in the culture fl uid of Rhodococcus equi (formerly Rhodococcus sp. M-750) and designated EGCase I, II, and III. This study describes the molecular cloning of EGCase I and characterization of the recombinant enzyme, which was highly expressed in a rhodococcal expression system using Rhodococcus erythropolis. Kinetic analysis revealed the turnover number (k cat ) ( k cat ) of the recombinant EGCase I to be 22-and 1,200-fold higher than that of EGCase II toward GM1a and Gb3Cer, respectively, although the K m of both enzymes was almost the same for these substrates.

Quantitative analysis of total serum glycome in human and mouse

“…All animal experiments were conducted under a protocol approved by the animal care committee of Hokkaido University, Japan. Rhodococcus endoglycoceramidase I (EGCase I) was prepared as described previously . BlotGlyco® beads and aminooxy‐WR (aoWR) reagent were from Sumitomo Bakelite Co. Ltd. (Tokyo, Japan).…”

New insights on glucosylated lipids: Metabolism and functions

“…Correspondingly, fungi possess a fungus-specific GCase, designated as EGCrP1 [119]. This enzyme is listed in several fungal genomic databases as a homolog of endoglycoceramidase, which cleaves the ceramide-glucosidic linkage of various GSLs to release free oligosaccharide and ceramide [120][121][122]. Recombinant EGCrP1 specifically hydrolyzes GlcCer but not other GSLs.…”

Quantitative GSL-glycome analysis of human whole serum based on an EGCase digestion and glycoblotting method

“…Rhodococcal EGCase I was prepared as described previously ( 20,21 ). Glucose oxidase (GOD) was from Wako Chemical Company, Ltd. (Osaka, Japan).…”