Glycolipid core structure switching from globo- to lacto- and ganglio-series during retinoic acid-induced differentiation of TERA-2-derived human embryonal carcinoma cells

We have analyzed the glycolipid markers of a recently cloned human embryonal carcinoma (EC) cell line, NTERA-2, which differentiates extensively into a variety of somatic cell types when exposed to retinoic acid. These tumor cells provide a model system that can be used to study the ontogeny of glycolipid diversity during human embryonic development. Glycolipid antigens were identified by cell surface immunofluorescence and thin-layer chromatography immunostaining using a comprehensive set of anticarbohydrate monoclonal antibodies. Undifferentiated NTERA-2 cells were found to express predominantly globo-series glycolipids, including Gb3, Gb5 (IV3GalGb4), globo-ganglioside (IV3NeuAc alpha 2----3GalGb4), globo-H (IV3Fuc alpha 1----2GalGb4), and globo-A (IV3GalNAc alpha 1----3[Fuc alpha 1----2]GalGb4). When NTERA-2 cells were induced to differentiate by culturing in the presence of 10(-5) M retinoic acid, a remarkable shift of cellular glycolipids from globo-series to lacto- and ganglio-series was observed: Globo-series structures declined, particularly during the period 7-20 days after first exposure to retinoic acid, while lacto-series structures, including fucosyl alpha 1----3 type 2 chain (Lex) and sialosyl type 2 chain, and ganglio-series structures, including GM3, GD3, 9-O-acetyl-GD3, GM2, GD2, and GT3, increased. The presence of globo-A and globo-H as the major ABH blood group antigens in undifferentiated NTERA-2 cells suggests that globo-series blood group antigens are embryonic antigens, synthesis of which switches to lacto-series during human development. Two-color immunofluorescence analysis indicated preferential expression of several ganglio- and lacto-series antigens on different subsets of differentiated cells and permitted the relationship of these subsets to the development of neurons in NTERA-2 cultures to be determined. The results suggest that glycosyltransferase, particularly those involved in controlling glycoconjugate core structure assembly, are key enzymes regulated during the differentiation of human EC cells and, by implication, during human embryogenesis.     

Glycosphingolipids of human myometrium and endometrium and their changes during the menstrual cycle, pregnancy and ageing

The glycolipid composition of human myometrium and endometrium was examined at various stages of maturation and reproduction. The major neutral glycolipids of both myometrium and endometrium were identified by high-performance thin-layer chromatography as globo-series glycolipids, Gb3 and Gb4. The major acidic glycolipids (gangliosides) were identified similarly as GM3 and GD3, with lesser amounts of GM1, GD1a, and GT1b. During pregnancy, GD3 expression declined in both myometrium and endometrium, whereas GM3 expression increased. Reciprocal changes in GM3/GD3 expression were mirrored by appropriate changes in the glycosyltransferases required for their synthesis; alpha 2----3sialyltransferase activity increased approximately 3-fold during pregnancy, while alpha 2----8sialyltransferase activity declined to about 20%. The results focus attention on the glycolipids of uterine tissues, their regulation, and their possible role in reproduction and fertility.     

Stage-specific embryonic antigens (SSEA-3 and -4) are epitopes of a unique globo-series ganglioside isolated from human teratocarcinoma cells.

Two monoclonal antibodies (MC631 and MC813-70) raised against 4- to 8-cell stage mouse embryos and a human teratocarcinoma cell line, respectively, detect the stage-specific embryonic antigens, the previously defined SSEA-3 and SSEA-4, described herein. These antibodies were both reactive with a unique globo-series ganglioside with the structure shown below: (formula; see text) The antibodies were found to recognize sequential regions of this ganglioside, i.e., MC813-70 recognizes the terminal 'a' structure whereas antibody MC631 recognizes the internal 'b' structure. Thus, a set of two antibodies defines this unique embryonic antigen. During differentiation of human teratocarcinoma 2102Ep cells, the globo-series glycolipids defined by these antibodies decrease and the lacto-series glycolipids, reacting with the SSEA-1 antibody appear. This antigenic conversion suggests that a shift of glycolipid synthesis from globo-series to lacto-series glycolipids occurs during differentiation of human teratocarcinoma and perhaps of pre-implantation mouse embryos.     

Glycolipid glycosyltransferases in human embryonal carcinoma cells during retinoic acid induced differentiation

Retinoic acid induced differentiation of TERA-2-derived human embryonal carcinoma cells is accompanied by a dramatic reduction of extended globo-series glycolipids, including galactosyl globoside, sialylgalactosyl globoside, and globo-A antigen (each recognized by specific MoAbs). Associated with these glycolipid changes, the activities of two key enzymes, alpha 1----4 galactosyltransferase (for synthesis of globotriaosyl core structure) and beta 1----3 galactosyltransferase (for synthesis of galactosyl globoside), were found to be reduced 3- to 4-fold. The latter enzyme plays a key role in the synthesis of extended globo-series structures, and its characterization has not been reported previously. Therefore, its catalytic activity was studied in detail, including substrate specificity, detergent and phospholipid effects, pH and cation requirements, and apparent Km. During retinoic acid induced differentiation, a series of Lex glycolipid antigens (recognized by anti-SSEA-1 antibody) and their core structures (lacto-series type 2 chains) increase dramatically. In parallel with these changes in glycolipid expression, the activities of two key enzymes, beta 1----3 N-acetylglucosaminyltransferase (for extension of lacto-series type 2 chain) and alpha 1----3 fucosyltransferase (for synthesis of Lex structure), were found to increase by 4- and 2-fold, respectively. Similarly, an increase in the expression of several gangliosides (e.g., GD3 and GT3) during retinoic acid induced differentiation was mirrored by a 4-fold increase in the activity of alpha 2----3 sialyltransferase (for synthesis of ganglio core structure, GM3). The results suggest a coordinate regulation of key glycosyltransferases involved in core structure assembly and terminal chain modification.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycosphingolipids of rabbit endometrium and their changes during pregnancy.

The glycolipids of nonpregnant and pregnant rabbit endometrium were characterized using a combination of biochemical and immunochemical techniques. Quantitative analyses indicated a 70% decline in acidic glycolipid (ganglioside) content during early pregnancy (day 6), and a 2.5-fold increase in neutral glycolipid content during later pregnancy (day 26). The major gangliosides of rabbit endometrium were identified by thin-layer chromatography as GM3 and GD3, with minor amounts of GM1, GD1a and GT1b. The major neutral glycolipids were identified similarly as globo-series structures Gb3 and Gb4. Monoclonal antibodies (mAbs) directed to glycolipid antigens permitted the detection of additional glycolipid species, including sialylated, sulfated and fucosylated lacto-series structures. Difucosyl Ley structure (defined by mAb AH-6) and sulfated-galactosyl structure (defined by mAb VESP 6.2) were identified by indirect immunofluorescence along the luminal surface of the endometrium during the implantation period. Rapid changes in the glycolipid composition of endometrial cells during early pregnancy may facilitate embryo adhesion and trophectoderm outgrowth during implantation.     

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.     

Total metabolic flow of glycosphingolipid biosynthesis is regulated by UDP-GlcNAc:lactosylceramide beta 1-->3N-acetylglucosaminyltransferase and CMP-NeuAc:lactosylceramide alpha 2-->3 sialyltransferase in human hematopoietic cell line HL-60 during differentiation.

We have previously reported that ganglioside GM3 was remarkably increased during monocytoid differentiation of human myelogenous leukemia cell line HL-60 cells and that neolacto series gangliosides (NeuAc-nLc) were enriched during granulocytoid differentiation. In addition, HL-60 was differentiated into monocytic lineage by exogenous GM3 and into granulocytoid by NeuAc-nLc. In the present report, the enzymatic bases of glycosphingolipid biosynthesis in HL-60 during differentiation induced by 12-O-tetradecanoylphorbol-13-acetate and all-trans-retinoic acid were investigated. The following results were of particular interest. (i) Lactosylceramide alpha 2-->3 sialyltransferase (GM3 synthase) was remarkably up-regulated during monocyte differentiation, while the GM3 synthase level did not change in granulocytic differentiation. (ii) By contrast, lactosylceramide beta 1-->3N-acetylglucosaminyltransferase (Lc3Cer synthase) was down-regulated during monocytic differentiation, while the activity of Lc3Cer synthase was found to increase in granulocytic differentiation. (iii) The activities of four downstream glycosyltransferases (for synthesis of NeuAc-nLc) were found to increase or to remain unchanged during monocytic and granulocytic differentiation. These results strongly suggested the following. The dramatic GM3 increase and the decrease of NeuAc-nLc during monocytic differentiation are the consequences of the up-regulation of GM3 synthase and the down-regulation of Lc3Cer synthase, although the downstream enzymes are ready to catalyze their enzyme reactions. The notable increase of NeuAc-nLc and the relative decrease of GM3 during granulocytic differentiation are the results of the unchanged level of GM3 synthase and the up-regulation of Lc3Cer synthase together with the activation of the downstream glycosyltransferases. These results suggest that these two key upstream glycosyltransferases, GM3 synthase and Lc3Cer synthase, play critical roles in regulating the glycosphingolipid biosynthesis in HL-60 cells during differentiation. This switching mechanism of these two glycosyltransferases, together with our previous findings, might be one of the most important parts of the determining system of differentiation direction in human myeloid cells into monocytic or granulocytic lineages.     

Brefeldin A induced inhibition of de novo globo- and neolacto-series glycolipid core chain biosynthesis in human cells. Evidence for an effect on beta 1-->4galactosyltransferase activity.

De novo synthesis of neolacto-series glycolipids has been studied in human cell lines via metabolic labeling of ceramide with [3H]serine. Intense labeling of ceramide mono- and dihexoside glycolipids occurred with labeling of progressively longer chain derivatives with increasing time. Most of the label was recovered in neutral glycolipids with about 5% of the total labeling in the ganglioside fraction. Experiments done using cell treatment with 2.5 micrograms/ml brefeldin A resulted in a stimulation in the total amount of labeling, accumulation of a neutral glycolipid identified as Lc3 due to inhibited transfer of the neolacto-series core chain terminal beta-Gal residue, and a corresponding inhibition of labeling of longer chain neutral glycolipids in all cell lines. Brefeldin A also blocked synthesis of the globo-series precursor, Gb3, longer chain sialylated structures such as IV3NeuAcnLc4, but not de novo GM3 synthesis. Brefeldin A treatment had no effect on cellular beta 1-->3N-acetylglucosaminyl-, beta 1-->4galactosyl-, or alpha 1-->3fucosyltransferase specific activities, nor was it inhibitory in beta 1-->4galactosyltransferase assays in vitro. The results describe brefeldin A-induced blocks in globo- and neolacto-series glycolipid biosynthesis, consistent with differential localization of enzymes in intracellular membranes. In particular, the results suggest that the beta 1-->4galactosyltransferase in these cells is either not redistributed by brefeldin A or is otherwise rendered nonfunctional.     

Alteration of lacto-series glycolipid glycosyltransferase activities in human colonic adenocarcinoma DLD-1 cells after culture in N,N-dimethylformamide-containing medium

Human colonic adenocarcinoma DLD-1 cells were grown under conditions which induce characteristics of differentiated cells using medium containing 0.8% N,N-dimethylformamide in order to study alterations in glycosphingolipid glycosyltransferase activities during this process. Analysis of biosynthetic reactions involved in lacto-series antigen synthesis revealed no changes in the specific activities of either β1→4galactosyltransferase or α1→3/4fucosyltransferase with N,N-dimethylformamide treatment. However, a dramatic decrease of from 14- to 20-fold in the β1→3N-acetylglucosaminyltransferase activity was observed in the treated cells. This enzyme catalyzes the rate-limiting step in lacto-series core chain synthesis. This is consistent with the pattern of regulation of lacto-series antigen expression found to occur during oncogenesis in human colonic mucosa (Holmes EH, Hakomori S, Ostrander GK: J Biol Chem 262:15649, 1987). Total glycolipids from untreated and N,N-dimethylformamide-treated cells were isolated and subjected to TLC immunostain analysis and solid phase radioimmunoassay with a series of monoclonal antibodies specific for lacto-series-based carbohydrate antigens. A decrease of about 2-fold or less in the quantity of lacto-series antigens was observed as a consequence of N,N-dimethylformamide treatment in both neutral glycolipid and ganglioside fractions. The results suggest that only very low levels of β1→3N-acetylglucosaminyltransferase activity are required for the steady state expression of significant levels of lacto-series based glycolipids and that modulation of its activity levels by N,N-dimethylformamide treatment in DLD-1 cells represents a convenient in vitro system for studying aspects of regulation of lacto-series antigen expression.     

Changes of glycoconjugate expression profiles during early development

A variety of glycoconjugates, including glycosphingolipids (GSLs), expressed in mammalian tissues and cells were isolated and characterized in early biochemical studies. Later studies of virus-transformed fibroblasts demonstrated the association of GSL expression profiles with cell phenotypes. Changes of GSL expression profile were observed during mammalian embryogenesis. Cell surface molecules expressed on embryos in a stage-specific manner appeared to play key roles in regulation of cell-cell interaction and cell sorting during early development. Many mAbs showing stage-specific reactivity with mouse embryos were shown to recognize carbohydrate epitopes. Among various stage-specific embryonic antigens (SSEAs), SSEA-1 was found to react with neolacto-series GSL Le^x, while SSEA-3 and SSEA-4 reacted with globo-series Gb5 and monosialyl-Gb5, respectively. GSL expression during mouse early development was shown to shift rapidly from globo-series to neolacto/lacto-series, and then to ganglio-series. We found that multivalent Le^x caused decompaction of mouse embryos, indicating a functional role of Le^x epitope in the compaction process. Autoaggregation of mouse embryonal carcinoma (EC) F9 cells provided a useful model of the compaction process. We showed that Le^x-Le^x interaction, a novel type of molecular interavction termed carbohydrate-carbohydrate interaction (CCI), was involved in cell aggregation. Similar shifting of GSL expression profiles from globo-series and neolacto/lacto-series to ganglio-series was observed during differentiation of human EC cells and embryonic stem (ES) cells, reflecting the essential role of cell surface glycoconjugates in early development.     

Murine glycosyltransferases responsible for the expression of globo-series glycolipids: cDNA structures, mRNA expression, and distribution of their products

Biological functions of globo-series glycosphingolipids are not well understood. In this study, murine cDNAs of two glycosyltransferases responsible for the synthesis of globo-series glycolipids and mRNA expression of those genes were analyzed. Distribution of their products was also analyzed. Murine cDNAs for Gb3/CD77 synthase and Gb4 synthase predicted that both of them are type II membrane proteins with 348 and 331 amino acids, respectively. In northern blotting, Gb3/CD77 synthase gene was mainly expressed in kidney and lung but also detected in many other tissues. Gb4 synthase was expressed in brain, heart, kidney, liver, skin, and testis. In the immunohistological analysis, Gb3/CD77 was mainly expressed in the proximal tubules as revealed with coincidental expression with angiotensin-converting enzyme (ACE). In spleen, it was detected in pre-B cells in the peripheral region of the white pulp, as suggested with coincidental expression with CD10. It was also expressed on the endothelia of the alveolar capillaries in lung and on the sebaceous ducts aside of the hair follicles. Gb4 was also detected mainly on the proximal tubules in kidney and on the endothelia of the alveolar capillaries in lung as Gb3/CD77. But it was also detected on the epithelium of the bronchus, seminiferous tubules and tails of spermatozoa in testis, blood vessels of choroids plexus and endothelial cells in brain, and central and hepatoportal veins in liver. The expression patterns of two genes and their products almost corresponded with some exception. The results would provide essential information for the functional studies of globo-series glycolipids.     

Efficient generation of useful monoclonal antibodies reactive with globotriaosylceramide using knockout mice lacking Gb3/CD77 synthase

Efficient generation of useful monoclonal antibodies (mAbs) with high performance in cancer therapeutics has been expected. Generation of mAbs reactive with globotriaosylceramide (Gb3/CD77) was compared between A/J mice and Gb3/CD77 synthase-deficient (A4GalT-knockout) mice by immunizing Gb3-liposome. Specificity and functions of established antibodies were examined by ELISA, TLC- immunostaining, cytotoxicity of cancer cells and immunoblotting. Compared with results with conventional mice, better generation of mAbs with higher functions has been achieved with A4GalT-knockout mice, i.e. acquisition of IgG class antibodies, activities in antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and aggregation activity toward a Burkitt’s lymphoma line Ramos. Binding of mAb k52 induced tyrosine phosphorylation of several proteins in Ramos cells. One of the strongest phosphorylation bands turned out to be c-Cbl. Pretreatment of B cell lines with mAbs resulted in the attenuation of BCR-mimicking signaling. All these results suggested that A4GalT-knockout mice are very useful to generate mAbs against globo-series glycolipids, and that suppressive signaling pathway driven by endogenous Gb3-ligand molecules might be present in B cells.     

Cloning of a mouse beta 1,3 N-acetylglucosaminyltransferase GlcNAc(beta 1,3)Gal(beta 1,4)Glc-ceramide synthase gene encoding the key regulator of lacto-series glycolipid biosynthesis

The distinction between the different classes of glycolipids is conditioned by the action of specific core transferases. The entry point for lacto-series glycolipids is catalyzed by the beta1,3 N-acetylglucosaminyltransferase GlcNAc(beta1,3)Gal(beta1,4)Glc-ceramide (Lc3) synthase enzyme. The Lc3 synthase activity has been shown to be regulated during development, especially during brain morphogenesis. Here, we report the molecular cloning of a mouse gene encoding an Lc3 synthase enzyme. The mouse cDNA included an open reading frame of 1131 base pairs encoding a protein of 376 amino acids. The Lc3 synthase protein shared several structural motifs previously identified in the members of the beta1,3 glycosyltransferase superfamily. The Lc3 synthase enzyme efficiently utilized the lactosyl ceramide glycolipid acceptor. The identity of the reaction products of Lc3 synthase-transfected CHOP2/1 cells was confirmed by thin-layer chromatography immunostaining using antibodies TE-8 and 1B2 that recognize Lc3 and Gal(beta1,4)GlcNAc(beta1,3)Gal(beta1,4)Glc-ceramide (nLc4) structures, respectively. In addition to the initiating activity for lacto-chain synthesis, the Lc3 synthase could extend the terminal N-acetyllactosamine unit of nLc4 and also had a broad specificity for gangliosides GA1, GM1, and GD1b to generate neolacto-ganglio hybrid structures. The mouse Lc3 synthase gene was mainly expressed during embryonic development. In situ hybridization analysis revealed that that the Lc3 synthase was expressed in most tissues at embryonic day 11 with elevated expression in the developing central nervous system. Postnatally, the expression was restricted to splenic B-cells, the placenta, and cerebellar Purkinje cells where it colocalized with HNK-1 reactivity. These data support a key role for the Lc3 synthase in regulating neolacto-series glycolipid synthesis during embryonic development.     

Glycosphingolipid dynamics in human embryonic stem cell and cancer: their characterization and biomedical implications

Glycosphingolipids (GSLs) are composed of complex glycans linked to sphingosines and various fatty acid chains. Antibodies against several GSLs designated as stage-specific embryonic antigens (SSEAs), have been widely used to characterize differentiation of embryonic stem (ES) cells. In view of the cross-reactivities of these antibodies with multiple glycans, a few laboratories have employed advanced mass spectrometry (MS) technologies to define the dynamic changes of surface GSLs upon ES differentiation. However, the amphiphilic nature and heterogeneity of GSLs make them difficult to decipher. In our studies, systematic survey of GSL expression profiles in human ES cells and differentiated derivatives was conducted, primarily with matrix-assisted laser desorption/ionization MS (MALDI-MS) and MS/MS analyses. In addition to the well-known ES-specific markers, SSEA-3 and SSEA-4, several previously undisclosed globo- and lacto-series GSLs, including Gb4Cer, Lc4Cer, fucosyl Lc4Cer, Globo H, and disialyl Gb5Cer were identified in the undifferentiated human ES and induced pluripotent stem cells. Furthermore, during differentiation to embryoid body outgrowth, the core structures of GSLs switched from globo- and lacto- to ganglio-series. Lineage-specific differentiation was also marked by alterations of specific GSLs. During differentiation into neural progenitors, core structures shifted to primarily ganglio-series dominated by GD3. GSL patterns shifted to prominent expression of Gb4Cer with little SSEA-3 and-?4 or GD3 during endodermal differentiation. Several issues relevant to MS analysis and novel GSLs in ES cells were discussed. Finally, unique GSL signatures in ES and cancer cells are exploited in glycan-targeted anti-cancer immunotherapy and their mechanistic investigations were discussed using anti-GD2 mAb and Globo H as examples.     

Glycolipid glycosyltransferase activities during early development of Xenopus: effect of retinoic acid

Retinoic acid (RA) plays an important role in differentiation stage in which it also influences glycoconjugate metabolism. Previous work in our laboratory has shown that treatment with RA modifies glycolipid synthesis and distribution in total Xenopus embryos during development. In this study we have investigated the activity of the following anabolic enzymes involved in glycolipid biosynthesis: sialyltransferase-1 (SAT-1), GM3(beta1, 4)-N-acetylgalactosaminyltransferase (GalNAcT-1) and LacCer(beta1, 3)N-acetylglucosaminyltransferase (GlcNAcT-1). These enzymes are located at the branching point of lactosylceramide (Lc(2)) metabolism. Enzyme activities were assayed after treatment with different doses of RA added exogenously to the medium during the first 7 days of Xenopus embryo development. Our results show that RA activates GlcNAcT-1, the enzyme that drives Lc(2)to the glycolipids of the lacto-series, and SAT-1 that inserts Lc(2)in the ganglio-series pathway. These data support our previous analysis of glycolipid pattern in Xenopus embryos after RA treatment (Rizzo et al., 1995;Cell Biol Int19: 895-901) indicating a possible correlation between the distribution of glycolipids and the enzymes involved in their metabolism.     

Multiple phenotypic changes in mice after knockout of the <Emphasis Type="Italic">B3gnt5</Emphasis>gene,encoding Lc3 synthase—a key enzyme in lacto-neolacto ganglioside synthesis

Background  

Ganglioside biosynthesis occurs through a multi-enzymatic pathway which at the lactosylceramide step is branched into several biosynthetic series. Lc3 synthase utilizes a variety of galactose-terminated glycolipids as acceptors by establishing a glycosidic bond in the beta-1,3-linkage to GlcNaAc to extend the lacto- and neolacto-series gangliosides. In order to examine the lacto-series ganglioside functions in mice, we used gene knockout technology to generate Lc3 synthase gene B3gnt5-deficient mice by two different strategies and compared the phenotypes of the two null mouse groups with each other and with their wild-type counterparts.     

Immunization of A4galt-deficient mice with glycosphingolipids from renal cell cancers resulted in the generation of anti-sulfoglycolipid monoclonal antibodies

In this study, we immunized Gb3/CD77 synthase gene (A4galt) knockout (KO) mice with glycosphingolipids (GSLs) extracted from 3 renal cell cancer (RCC) cell lines to raise monoclonal antibodies (mAbs) reactive with globo-series GSLs specifically expressed in RCCs. Although a number of mAbs reactive with globo-series GSLs were generated, they reacted with both RCC cell lines and normal kidney cells. When we analyzed recognized antigens by mAbs that were specifically reactive with RCC, but not with normal kidney cells at least on the cell surface, many of them turned out to be reactive with sulfoglycolipids. Eight out of 11 RCC-specific mAbs were reactive with SM2 alone, and the other 3 mAbs were more broadly reactive with sulfated glycolipids, i.e. SM3 and SM4 as well as SM2. In the immunohistochemistry, these anti-sulfoglycolipids mAbs showed RCC-specific reaction, with no or minimal reaction with adjacent normal tissues. Thus, immunization of A4galt KO mice with RCC-derived GSLs resulted in the generation of anti sulfated GSL mAbs, and these mAbs may be applicable for the therapeutics for RCC patients.     

Glycolipid changes in murine myelogenous leukemias: neutral glycolipids as markers for specific populations of leukemias

We have studied the glycolipid composition of six different murine myelogenous leukemias as well as that of T-cell leukemias and normal spleen cells. Neutral and acidic lipid fractions were isolated by column chromatography on DEAE-Sephadex and analyzed by high-performance thin-layer chromatography (HPTLC) and an HPTLC overlay method. Murine myelogenous leukemias were found to contain globo- and ganglio-series neutral glycolipids, e.g., glucosylceramide (Glc-cer), lactosylceramide (Lac-cer), globotriaosylceramide (Gb3), globoside (Gb4), Forssman glycolipid (Gb5), and asialo-GM1 (GA1). Monoblastic leukemia cells contained increased proportions of Gb3, Gb4, Gb5, and GA1. Monocytic and myelomonocytic leukemia cells contained increased proportions of Glc-cer and Lac-cer. Especially, Glc-cer accounted for approximately 60% of the total neutral glycolipids in monocytic leukemia cells. Gb3 was the major neutral glycolipid in reticulum cell neoplasm type A, and it accounted for approximately 75% of the neutral glycolipids. GA1 was the major neutral glycolipid in myeloblastic and granulocytic leukemia cells as well as T-cell leukemias. Especially, granulocytic leukemia cells contained predominantly GA1, and it accounted for approximately 80% of the total neutral glycolipids. The pattern of gangliosides in myelogenous leukemias was more complex when compared with that of the neutral glycolipids; murine myelogenous leukemias contained at least 13 gangliosides, including such major gangliosides as GM1, GM1b containing N-acetyl neuraminic acid and N-glycolyl neuraminic acid, and Ga1NAc-GM1b. Alterations of glycolipid composition in murine myeloid leukemias may be associated with cellular differentiation and maturation, and therefore these characteristic glycolipid species may be regarded as markers for specific populations of leukemia cells.     

Molecular Species Analysis of 1,2-Diglycerides on Phorbol Ester Stimulation of LA-N-1 Neuroblastoma Cells During Proliferation and Differentiation

Abstract: The glycosphingolipid (GSL) composition of cells changes dramatically during cellular differentiation. Nerve growth factor (NGF) or forskolin (FRK) are known to induce cellular differentiation including process formation in PC12 pheochromocytoma cells. In this respect, we present the NGF/FRK-dependent regulation of glycosyltransferase activities and the corresponding GSL expression in PC12D cells. After treatment of PC12D cells with NGF or FRK, the cell processes, including varicoses and growth cones, became strongly immunoreactive with an antibody against a unique globo-series neutral GSL, Galα1-3Galα1-4Galβ1-4Glcβ1-1'Cer (GalGb3), and the activity of GalGb3-synthase increased significantly. Other glycosyltransferase activities, including GM1 containing blood group B determinant (BGM1)-, GM3-, GD1a-, and GM2-synthases, also increased significantly upon NGF treatment, but the immunoreactivity against BGM1 did not show any appreciable change. For the parent PC12 cells, NGF/FRK treatment significantly increased the percentage of anti-GalGb3 positive cells and induced some immunoreactive cell processes. Because the parent PC12 cells do not express appreciable amounts of GalGb3, and because PC12D cells are considered to be more differentiated than the parent PC12 cells, the expression of GalGb3 and the increase of GalGb3-synthase activity may be closely related to the cellular differentiation process in this cell line.     

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.