Expression cloning of human globoside synthase cDNAs. Identification of beta 3Gal-T3 as UDP-N-acetylgalactosamine:globotriaosylceramide beta 1,3-N-acetylgalactosaminyltransferase

By using a eukaryocytic cell expression cloning system, we have isolated cDNAs of the globoside synthase (beta1, 3-N-acetylgalactosaminyltransferase) gene. Mouse fibroblast L cells transfected with SV40 large T antigen and previously cloned Gb3/CD77 synthase cDNAs were co-transfected with a cDNA library prepared from mRNA from human kidney together with Forssman synthase cDNA, and Forssman antigen-positive cells were panned using an anti-Forssman monoclonal antibody. The isolated cDNAs contained a single open reading frame predicting a type II membrane protein with 351 amino acids. Surprisingly, the cDNA clones turned out to be identical with previously reported beta3Gal-T3, which had been cloned by sequence homology with other galactosyltransferases. Substrate specificity analysis with extracts from cDNA-transfected L cells confirmed that the gene product was actually beta1, 3-N-acetylgalactosaminyltransferase that specifically catalyzes the transfer of N-acetylgalactosamine onto globotriaosylceramide. Results of TLC immunostaining of neutral glycolipids from the cDNA-transfected cells also supported the identity of the newly synthesized component as globoside. The results show that glycosyltransferases apparently belonging to a single glycosyltransferase family do not necessarily catalyze reactions utilizing the same acceptor or even the same sugar donor. The globoside synthase gene was expressed in many tissues, such as heart, brain, testis, etc. We propose the designation beta3GalNAc-T1 for the cloned globoside synthase gene.     

Decreased expression of alpha2,8 sialyltransferase and increased expression of beta1,4 N-acetylgalactosaminyltransferase in gastrointestinal cancers

Gangliosides such as GD3, GM2, and GD2 are abundantly expressed on the cell surfaces of various malignant cells, suggesting the potential for anti-ganglioside antibody therapy for tumors. Anti-ganglioside GD2 antibody treatment is currently undergoing clinical trials for melanoma and neuroblastoma. We previously reported high in vivo antitumor effects of anti-GM2 ganglioside antibody against lung cancer. To determine whether anti-GM2 antibody may be clinically indicated for gastrointestinal cancers, we evaluated the mRNA expression of alpha2,8 sialyltransferase, a GD3 synthase, and beta1,4 N-acetylgalactosaminyltransferase (beta1,4 GalNAc-T), a GM2/GD2 synthase, in gastrointestinal cancers. We performed modified semi-quantitative RT-PCR, which reduces complexity incidental to radiolabeling on samples taken from small surgically removed clinical specimens. Stomach (19/22) and colorectal (21/30) cancers showed decreased expression of alpha2,8 sialyltransferase as compared with respective normal tissues (P < 0.05). In contrast, increased expression of beta1,4 GalNAc-T was detected in both types of tumors. Clinicopathological analysis revealed significantly higher expression level of alpha2,8 sialyltransferase in the poorly differentiated than in the well-differentiated stomach cancer group (P < 0.05). Furthermore, the expression level of alpha2,8 sialyltransferase was significantly decreased in male as compared with female colorectal cancer patients (P < 0.05). These results suggest that expression level of GM2 ganglioside is elevated in gastrointestinal cancer, and that anti-GM2 antibody may be applicable to its treatment.     

Molecular cloning of the human beta1,4 N-acetylgalactosaminyltransferase responsible for the biosynthesis of the Sd(a) histo-blood group antigen: the sequence predicts a very long cytoplasmic domain

The Sd(a) antigen is a carbohydrate determinant expressed on erythrocytes, the colonic mucosa and other tissues. This epitope, whose structure is Siaalpha2,3[GalNAcbeta1,4]Gal beta1,4GlcNAc, is synthesized by a beta1,4 N-acetylgalactosaminyltransferase (beta4GalNAc-T) that transfers a beta1,4-linked GalNAc to the galactose residue of an alpha2,3-sialylated chain. We have cloned from human colon carcinoma Caco2 cells a cDNA whose transfection in COS cells induces a GalNAc-T active on sialylated but not on asialylated fetuin and putatively represents the human Sd(a) beta4GalNAc-T. The cDNA predicts a 566 aa protein showing 66.6% and 39% identity with mouse CT beta4GalNAc-T and human GM2/GD2 synthase, respectively, with a typical type II glycosyltransferase organization, no potential N-glycosylation sites and a 67 aa cytoplasmic tail, which is probably the longest among the glycosyltransferases cloned to date. The gene maps in chromosome 17q23, and is composed of at least 11 exons. Exons 2-11 are homologous to exons 2-11 of the previously cloned CT beta4GalNAc-T from murine cytotoxic T lymphocytes while exons 1 of the two enzymes are totally different. The mRNA is expressed at a high level in differentiated Caco2 cells and in colonic mucosa and at a much lower level in lymphocytes and other colon cancer cell lines.     

Enhanced activity of recombinant beta-secretase from Drosophila melanogaster S2 cells transformed with cDNAs encoding human beta1,4-galactosyltransferase and Galbeta1,4-GlcNAc alpha2,6-sialyltransferase

beta-Secretase (betaSEC) was expressed in Drososphila melanogaster Schneider 2 (S2) cells transformed with cDNAs encoding beta1,4-galactosyltransferase (GalT) and Galbeta1,4-GlcNAc alpha2,6-sialyltransferase (ST). The apparent molecular weight of recombinant beta-secretase was increased from 56kDa to 61kDa. A lectin blot analysis indicated that recombinant beta-secretase from S2betaSEC/GalT-ST cells (S2 cells co-transformed with cDNAs encoding beta-secretase, glycosyltransferases, GalT, and ST) contained the glycan residues of beta1,4-linked galactose and alpha2,6-linked sialic acid. Two dimensional electrophoresis revealed that recombinant beta-secretase from S2betaSEC/GalT-ST cells had a lower isoelectric point compared to beta-secretase from control S2betaSEC cells (S2 cells transformed only with beta-secretase cDNA). Recombinant beta-secretase from transformed S2 cells was also present as heterogeneous forms. The enzyme activity of recombinant beta-secretase from S2betaSEC/GalT-ST cells was enhanced up to 260% compared to control S2betaSEC cells. We have shown that an exogeneous human glycosyltransferases cDNA can be introduced into S2 cells to extend the N-glycan processing capabilities of the insect cell line, and that the extended glycosylation improves the activity of recombinant beta-secretase.     

Down-regulation of GD3 ganglioside and its O-acetylated derivative by stable transfection with antisense vector against GD3-synthase gene expression in hamster melanoma cells: effects on cellular growth, melanogenesis, and dendricity

The expression of gangliosides in hamster melanoma cells is closely related to cellular growth and degree of differentiation, with slow-growing, highly differentiated melanotic melanoma cells expressing GM3 and fast-growing, undifferentiated amelanotic Ab melanoma cells having a preponderance of GD3 and O-acetyl-GD3. We recently showed that down-regulation of O-acetyl-GD3 expression in hamster melanoma cells by introducing the influenza C virus O-acetylesterase cDNA into the cells resulted in induction of dendricity, with a concomitant increased expression of GD3. To examine the effect of the increased GD3 expression in the plasma membrane on the dendricity of the AbC-1 cells, we first established the cDNA coding for hamster GD3-synthase. We then targeted the sialyltransferase gene expression by the antisense knockdown experiment, and the results showed that inhibition of the expression of gangliosides GD3 and O-acetyl-GD3 induced dendricity in the hamster melanoma AbC-1 cell line. These GD3- and O-acetyl-GD3-depleted cells also demonstrated a decreased rate of cell growth, but their melanogenic potential was not affected. These results rule out the possibility that GD3 may serve as an active molecule for dendrite outgrowth in this cell line and suggest that the enhanced expression of O-acetyl-GD3 ganglioside may stimulate cellular growth and suppress certain differentiated phenotypes such as dendrite formation, but not melanogenesis, in our system.     

Monoclonal antibody Leo Mel 3, which inhibits killing of human melanoma cells by anomalous killer cells, binds to a sugar sequence in GD2 (II3(NeuAc)2-GgOse3Cer) and several other gangliosides

Human anomalous killer (AK) cells lyse freshly isolated human melanoma cells which are insensitive to human natural killer cell-mediated lysis. Monoclonal antibody Leo Mel 3, an IgM (k), produced by a hybridoma obtained from a mouse immunized with human melanoma cells, binds to melanoma cells and inhibits their conjugate formation with AK cells as well as their AK cell-mediated lysis. Other IgM antibodies from the same fusion that bind melanoma cells do not inhibit (Werkmeister, J. A., Triglia, T., Andrews, P., and Burns, G. F. (1985) J. Immunol. 135, 689-695). Leo Mel 3 binds several different gangliosides from melanoma cells, as determined by immunostaining thin layer chromatograms. Binding is abolished by treatment of the gangliosides with neuraminidase. In solid-phase radioimmunoassay, Leo Mel 3 binds strongly to ganglioside GD2 and less strongly to gangliosides GT3, GD3, and GQ1b. It does not bind to other gangliosides including GM1, GM2, GM3, GD1a, GD1b, and GT1b. Thus, the epitope recognized by antibody Leo Mel 3 is found in the sugar sequence of ganglioside GD2, GalNAc beta 1-4[NeuAc alpha 2-8NeuAc alpha 2-3]Gal beta 1-4Glc beta 1 .... This sequence may contain a target in melanoma cells recognized by AK cells.     

Gangliosides from human melanoma immunomodulate response of T cells to interleukin-2

The gangliosides expressed by normal melanocytes are predominantly GM3 (greater than 90%) and GD3 (less than 5%). Malignant melanoma can express several other types of gangliosides in significant quantities, including GM2 and GD2. Melanoma patients can develop an immune response against some of these ganglioside antigens on autologous melanoma cells. The four major gangliosides expressed by human melanoma cells (GM3, GD3, GM2, and GD2) were examined for their immunomodulatory effect on lymph node lymphocytes from melanoma patients. Gangliosides were added exogenously to lymphocytes grown in the presence of IL-2. Preferential interactions of specific melanoma gangliosides on IL-2 stimulation were found. While GM2 and GD2 enhanced the lymphocyte response to IL-2, GM3 and GD3 significantly inhibited this response. GM2 and GD2 differ from GM3 and GD3 by the presence of a terminal N-acetylgalactosamine. Since different gangliosides can up-regulate and down-regulate lymphocyte responses to IL-2, the ganglioside phenotype of melanoma cells may play a major role in determining whether an individual tumor causes immune stimulation or suppression.     

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.     

Beta1,4-N-acetylgalactosaminyltransferase--GM2/GD2 synthase: a key enzyme to control the synthesis of brain-enriched complex gangliosides

Beta1,4-N-acetylgalactosaminyltransferase (GM2/GD2 synthase) is a key enzyme which catalyzes the conversion of GM3, GD3 and lactosylceramide (LacCer) to GM2, GD2 and asialo-GM2 (GA2), respectively. This step is critical for the synthesis of all complex gangliosides enriched in the nervous system of vertebrates. Following the cloning of cDNAs encoding GM2/GD2 synthase by an expression cloning approach, substantial evidence for the roles of complex gangliosides have been obtained. Above all, knock-out mice lacking all complex gangliosides revealed important roles of complex gangliosides in vivo, i.e., in the maintenance and repair of nervous tissues, in the intact differentiation of spermatocytes via the transport of testosterone, and in the regulation of interleukin-2 receptor complex. Molecular mechanisms for these functions of complex gangliosides in vivo remain to be clarified.     

Isolation of 10 differentially expressed cDNAs in differentiated Neuro2a cells induced through controlled expression of the GD3 synthase gene

Recently, we showed that transfection of GD3 synthase cDNA into Neuro2a cells, a mouse neuroblastoma cell line, causes cell differentiation with neurite sprouting. In a search for the genes involved in this ganglioside-induced Neuro2a differentiation, we used a tetracycline-regulated GD3 synthase cDNA expression system combined with differential display PCRs to identify mRNAs that were differentially expressed at four representative time points during the process. We report here the identification of 10 mRNAs that are expressed highly at the Neuro2a differentiated stage. These cDNAs were named GDAP1-GDAP10 for (ganglioside-induced differentiation-associated protein) cDNAs. It is interesting that in retinoic acid-induced neural differentiated mouse embryonic carcinoma P19 cells, GDAP mRNA expression levels were also up-regulated (except that of GDAP3), ranging from three to >10 times compared with nondifferentiated P19 cells. All the GDAP genes (except that of GDAP3) were developmentally regulated. The GDAP1, 2, 6, 8, and 10 mRNAs were expressed highly in the adult mouse brain, whereas all the other GDAP mRNAs were expressed in most tissues. Our results suggested that these GDAP genes might be involved in the signal transduction pathway that is triggered through the expression of a single sialyltransferase gene to induce neurite-like differentiation of Neuro2a cells.     

Human homolog of Caenorhabditis elegans sqv-3 gene is galactosyltransferase I involved in the biosynthesis of the glycosaminoglycan-protein linkage region of proteoglycans.

A cDNA encoding a novel galactosyltransferase was identified based on BLAST analysis of expressed sequence tags, and the cDNA clones were isolated from a human melanoma line library. The new cDNA sequence encoded a type II membrane protein with 327 amino acid sequence and showed 38% homology to the Caenorhabditis elegans sqv-3 gene involved in the vulval invagination and oocyte development. Extracts from L cells transfected with the galactosyltransferase cDNA in an expression vector and a fusion protein with protein A exhibited marked galactosyltransferase activity specific for p-nitrophenyl-beta-D-xylopyranoside. Moreover, transfection with the cloned cDNA restored glycosaminoglycan synthesis of galactosyltransferase I-deficient Chinese hamster ovary mutant pgsB-761 cells. Analysis of the enzyme product by beta-galactosidase digestion, mass spectroscopy, and NMR spectroscopy revealed that the reaction product was formed via beta-1,4 linkage, indicating that the enzyme is galactosyltransferase I (UDP-galactose:O-beta-D-xylosylprotein 4-beta-D-galactosyltransferase, EC 2.4.1.133) involved in the synthesis of the glycosaminoglycan-protein linkage region of proteoglycans.     

Suppression subtractive hybridization identifies differentially expressed genes in Brassica napus chlorophyll-reduced mutant

Suppressive subtraction hybridization (SSH) was used to identify differentially expressed genes caused by a chlorophyll-reduced mutation in B. napus. The cDNA fragments, derived from SSH positive subtractive library (tester: normal wild type, driver: mutant) were cloned into pMD18-T vector. Two hundred SSH cDNA clones were screened by dot blot array, and 151 clones were identified as differentially expressed cDNA fragments in Cr3529 line. Thirty-six positive clones which showed marked expression differences were selected and sequenced. After redundant cDNAs were removed, 33 differentially expressed unique cDNA section clones were obtained. Among the 33 clones, two clones possess different parts of the cDNA sequence of the same gene coding geranylgeranyl reductase, four clones belong to unknown proteins, and the rest share homology to genes of diverse class. Sequence analysis showed that at least 12 genes were discovered to be related to the photosynthesis, seven of them coded the proteins which belong to the subunit of photosystem 2. RNA gel blot analysis showed that compared with 3529, the gene expression of the chlorophyll a/b-binding protein Lhcb2 in photosystem 2 declined markedly in the cotyledons and seedling leaves of Cr3529, indicating that the reduced light-harvesting complex 2 accumulation in thylakoid membrane of Cr3529 was due to the decrease of the related gene mRNA level for translation.     

cDNA cloning of IL-1 alpha and IL-1 beta from mRNA of U937 cell line

Clones of cDNAs encoding growth inhibitory factors for human melanoma cell line A375 were isolated from cDNA library prepared by using mRNA derived from human histiocytic lymphoma cell line U937 induced with PMA and further stimulated with LPS. Cloning was achieved using Okayama-Berg cDNA expression vector system that permits expression of the inserted cDNA segments in mammalian cells. By assaying the transfected COS-1 cells supernatants and cell extracts, we isolated two distinct cDNA clones encoding growth inhibitory factors. It was determined by the nucleotide sequences of the inserts, the cDNAs corresponded to IL-1 alpha and -1 beta. Our results indicate U937 cells can be induced to produce both interleukin-1s.     

Genetic mechanisms for the synthesis of fucosyl GM1 in small cell lung cancer cell lines

Fucosyl GM1 has been reported to be specifically expressed in small cell lung cancer (SCLC) cells. However, the genetic basis for the synthesis of fucosyl GM1 has not been investigated. We analyzed the glycosyltransferases responsible for the synthesis of fucosyl GM1 in SCLC cell lines. In four SCLC cell lines expressing fucosyl GM1, both FUT1 and FUT2 mRNAs were detected, indicating that either one or both of alpha1,2-fucosyltransferases may be involved in the expression of fucosyl GM1. However, three of these four lines contained function-loss mutations in the FUT2 coding region, suggesting that FUT1 is mainly involved in the alpha1,2-fucosylation of GM1. The expression levels of the GM1 synthase gene showed no correlation with those of fucosyl GM1, whereas the co-transfection of GM1 synthase cDNA with FUT1 or FUT2 into SK-LC-17 clearly enhanced the neo-expression of fucosyl GM1, indicating its essential role. In contrast, the co-transfection of GD3 synthase cDNA reduced the expression levels of fucosyl GM1 with FUT1 or FUT2. Consequently, FUT1 seems to mainly contribute to the expression of fucosyl GM1, although both FUT1 and FUT2 are capable of generating the antigen. These results should promote the functional analysis of fucosyl GM1 leading to the development of novel therapies for SCLC.     

Developmental changes of glycosphingolipids and expression of glycogenes in mouse brains

Glycosphingolipids (GSLs) and their sialic acid-containing derivatives, gangliosides, are important cellular components and are abundant in the nervous system. They are known to undergo dramatic changes during brain development. However, knowledge on the mechanisms underlying their qualitative and qualitative changes is still fragmentary. In this investigation, we have provided a detailed study on the developmental changes of the expression patterns of GSLs, GM3, GM1, GD3, GD1a, GD2, GD1b, GT1b, GQ1b, A2B5 antigens (c-series gangliosides such as GT3 and GQ1c), Chol-1alpha (GT1aalpha and GQ1balpha), glucosylceramide, galactosylceramide (O1 antigen), sulfatide (O4 antigen), stage-specific embryonic antigen-1 (Lewis x) glycolipids, and human natural killer-1 glycolipid (sulfoglucuronosyl paragloboside) in developing mouse brains [embryonic day 12 (E12) to adult]. In E12-E14 brains, GD3 was a predominant ganglioside. After E16, the concentrations of GD3 and GM3 markedly decreased, and the concentrations of a-series gangliosides, such as GD1a, increased. GT3, glucosylceramide, and stage-specific embryonic antigen-1 were expressed in embryonic brains. Human natural killer-1 glycolipid was expressed transiently in embryonic brains. On the other hand, Chol-1alpha, galactosylceramide, and sulfatide were exclusively found after birth. To provide a better understanding of the metabolic basis for these changes, we analyzed glycogene expression patterns in the developing brains and found that GSL expression is regulated primarily by glycosyltransferases, and not by glycosidases. In parallel studies using primary neural precursor cells in culture as a tool for studying developmental events, dramatic changes in ganglioside and glycosyltransferase gene expression were also detected in neurons induced to differentiate from neural precursor cells, including the expression of GD3, followed by up-regulation of complex a- and b-series gangliosides. These changes in cell culture systems resemble that occurring in brain. We conclude that the dramatic changes in GSL pattern and content can serve as useful markers in neural development and that these changes are regulated primarily at the level of glycosyltransferase gene expression.