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

Endoglycoceramidase (EGCase) is a glycosidase capable of hydrolyzing the β -glycosidic linkage between the oligosaccharides and ceramides of glycosphingolipids (GSLs). Three molecular species of EGCase differing in specificity were found in the culture fluid 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. Comparison of the three-dimensional structure of EGCase I (model) and EGCase II (crystal) indicated that a flexible loop hangs over the catalytic cleft of EGCase II but not EGCase I. Deletion of the loop from EGCase II increased the k(cat) of the mutant enzyme, suggesting that the loop is a critical factor affecting the turnover of substrates and products in the catalytic region. Recombinant EGCase I exhibited broad specificity and good reaction efficiency compared with EGCase II, making EGCase I well-suited to a comprehensive analysis of GSLs.     

Establishment of cells exhibiting mutated glycolipid synthesis from mouse thymus by immortalization with SV-40 virus

Immortalization with simian virus-40 and cloning of immortalized cells from mouse thymus were performed to establish cell lines for characterization of the mode of glycolipid expression in the thymic cells. Among the 25 cell lines obtained, three lines with different morphologies were established, that is, epithelial (IMTH-E), fibroblastic (IMTH-F), and asterisk-like (IMTH-I) cells, and their glycolipids, together with those in the thymus, were determined systematically. The major glycolipids in mouse thymus were the globo- and ganglio-series, both of which, were co-expressed in the three cell lines established. However, the mode of modification of the globo- and ganglio-series was distinct for each cell line. As to the globo-series, the structures with the longest carbohydrate chain for IMTH-E, -F, and -I cells were Gb₃Cer, Gb₄Cer, and Forssman antigen, respectively, having stepwise shorter carbohydrates at the nonreducing termini. Although the acidic glycolipids in IMTH-E cells comprised GM3 and GM2, and their sulfated isomers, IMTH-F and -I cells expressed GMlb and GDlc for the α-pathway, and up to GDI a for the a-pathway of ganglio-series glycolipids. GMlb-GalNAc present in the thymus was not detected in IMTH-F and -I cells, probably due to the lower synthetic activity for the metabolic intermediate Gg₄Cer. The results indicate that the immortalization technique is useful for obtaining individual cells having unique glycolipid profiles for analysis of the functional significance and metabolism of glycolipids in the thymus. Abbreviations: Glycolipids are abbreviated according to the recommendations of the IUPAC-IUBMB Commission on Biochemical Nomenclature. Eur J Biochem 79, 11-21 (1977). The ganglioside nomenclature of Svennerholm [1] is employed throughout, except that GMlb, GMlb-GalNAc, GMlb-GaINAc-Gal, GDlc, and GDI α denote IV³ NeuAcα-Gg₄Cer, IV⁴GalNAcβ, IV³NeuAcα-Gg₄Cer, IV⁴(Galβ-3GalNAcβ),IV³NeuAcα-Gg₄Cer, IV³NeuAcα₂-Gg₄Cer, and III⁶ NeuAcα, IV³NeuAcα-Gg₄Cer, respectively. BSA, bovine serum albumin; PBS, phosphate-buffered saline; FCS, fetal calf serum; SV, simian virus; FABMS, fast atom bombardment mass spectrometry; NeuAc, N-acetylneuraminic acid; NeuGc, N-glycolylneuraminic acid; Hex, hexose: HexNAc, N-acetylhexosamine.     

The Role of Globo-Series Glycolipids in Neuronal Cell Differentiation—A Review

Alterations in glycolipid composition as well as glycosyltransferase activities during cellular differentiation and growth have been well documented. However, the underlying mechanisms for the regulation of glycolipid expression remain obscure. One of the major obstacles has been the lack of a well defined model system for studying these phenomena. We have chosen PC 12 pheochrom-ocytoma cells as a model because (a) the properties of these cells have been well characterized, and (b) they respond to nerve growth factor (NGF) by differentiating into sympathetic-like neurons and are amenable to well-controlled experimentation. Thus, PC12 cells represent a suitable model for studying changes in glycolipid metabolism in relation to cellular differentiation. We have previously shown that subcloned PC12 cells accumulate a unique series of globo-series neutral glycolipids which are not expressed in parental PC12 cells. This unusual change in glycolipid distribution is accompanied by changes in the activities of specific glycosyltransferases involved in their synthesis and is correlated with neuritogenesis and/or cellular differentiation in this cell line. We have further demonstrated that changes in the glycosyltransferase activities may be modulated by the phosphorylation states of the cells via protein kinase systems. We conclude that these unique globo-series glycolipids may play a functional role in the initiation and/or maintenance of neurite outgrowth in PC12 cells.     

Monoclonal antibody (5F3) defining renal cell carcinoma-associated antigen disialosyl globopentaosylceramide (V3NeuAcIV6NeuAcGb5), and distribution pattern of the antigen in tumor and normal tissues

Renal cell carcinoma (RCC) has been characterized by high expression of three types of disialogangliosides: two based on lacto-series type 1 structure (disialosyl Lc₄, GalNAc disialosyl Lc₄), the other based on globo-series structure (disialosyl globopentaosylceramide; disialosyl Gb5). The present study established a mAb, 5F3, directed to disialosyl Gb5. 5F3 was established after immunization with RCC cell line ACHN. The major disialoganglioside antigen isolated from ACHN cells, showing specific reactivity with 5F3, was characterized unequivocally as disialosyl Gb5 (V³NeuAcIV⁶NeuAcGb5) by identification of the core structure as globopentaosylceramide (Gb5) after enzymatic and acid hydrolysis, and by 2-dimensional ¹H-NMR spectroscopy. 5F3 does not react with monosialosyl Gb5 (V³NeuAcGb5), Gb5, or any lacto-series structures. 5F3 strongly stained 19 of 41 cases of primary RCC tissue. It reacted with proximal tubules (but not distal tubules) of kidney, microglial cells of cerebrum and cerebellum, goblet cells of stomach and intestine, smooth muscle of various organs. It did not react with parenchymatous cells of various organs, except for kidney epithelia and prostate stroma. Immunostaining of RCC tissue by mAb 5F3, in combination with staining by other antibodies directed to globo-series and lacto-series structures, has prognostic significance in defining metastatic potential of RCC.