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.     

A murine cytotoxic T lymphocyte cell line resistant to Vicia villosa lectin is deficient in UDP-GalNAc:beta-galactose beta 1,4-N-acetylgalactosaminyltransferase

We have reported the presence of N-acetylgalactosamine linked beta 1,4 to galactose on O-linked oligosaccharides of a cloned murine cytotoxic T cell line and the absence of these residues from the O-linked structures of a Vicia villosa lectin-resistant mutant line, VV6, derived from parental B6.1.SF.1 cells (Conzelmann, A., and Kornfeld, S. (1984) J. Biol. Chem. 259, 12528-12535). This study shows that B6.1.SF.1 cells contain an enzyme which transfers N-acetylgalactosamine from UDP-GalNAc onto the O-linked tetrasaccharides of human glycophorin A, giving rise to pentasaccharides which contain beta-glycosidically linked N-acetylgalactosamine. Desialylated glycophorin was inactive as an acceptor. The enzyme also transfers N-acetylgalactosamine to the N-linked oligosaccharides of the Tamm-Horsfall glycoprotein. This glycoprotein is known to contain N-linked oligosaccharides with beta-linked N-acetylgalactosamine residues which constitute the Sda blood group determinant. This N-acetylgalactosaminyltransferase could not be detected in VV6 cells which can account for the lack of beta-linked N-acetylgalactosamine residues on its O-linked oligosaccharides. The two cell lines have comparable levels of UDP-GalNAc:apomucin N-acetylgalactosaminyltransferase, demonstrating that the enzyme deficiency in VV6 cells is selective. Both cell lines have a similar glycolipid content, with the major component being asialo-GM1. Since this glycolipid contains N-acetylgalactosamine linked beta 1,4 to galactose, it would appear that the N-acetylgalactosyltransferase involved in the biosynthesis of glycolipids is different from the UDP-GalNAc:glycoprotein N-acetylgalactosaminyltransferase. An independently derived murine CTL line also contains the UDP-GalNAc:glycoprotein N-acetylgalactosaminyltransferase, suggesting that the expression of this enzyme is a common characteristic of this type of cell line.     

Molecular cloning and enzymatic characterization of a UDP-GalNAc:GlcNAc(beta)-R beta1,4-N-acetylgalactosaminyltransferase from Caenorhabditis elegans

A common terminal structure in glycans from animal glycoproteins and glycolipids is the lactosamine sequence Gal(beta)4GlcNAc-R (LacNAc or LN). An alternative sequence that occurs in vertebrate as well as in invertebrate glycoconjugates is GalNAc(beta)4GlcNAc-R (LacdiNAc or LDN). Whereas genes encoding beta4GalTs responsible for LN synthesis have been reported, the beta4GalNAcT(s) responsible for LDN synthesis has not been identified. Here we report the identification of a gene from Caenorhabditis elegans encoding a UDP-GalNAc:GlcNAc(beta)-R beta1,4-N-acetylgalactosaminyltransferase (Ce(beta)4GalNAcT) that synthesizes the LDN structure. Ce(beta)4GalNAcT is a member of the beta4GalT family, and its cDNA is predicted to encode a 383-amino acid type 2 membrane glycoprotein. A soluble, epitope-tagged recombinant form of Ce(beta)4GalNAcT expressed in CHO-Lec8 cells was active using UDP-GalNAc, but not UDP-Gal, as a donor toward a variety of acceptor substrates containing terminal beta-linked GlcNAc in both N- and O-glycan type structures. The LDN structure of the product was verified by co-chromatography with authentic standards and (1)H NMR spectroscopy. Moreover, Chinese hamster ovary CHO-Lec8 and CHO-Lec2 cells expressing Ce(beta)4GalNAcT acquired LDN determinants on endogenous glycoprotein N-glycans, demonstrating that the enzyme is active in mammalian cells as an authentic beta4GalNAcT. The identification and availability of this novel enzyme should enhance our understanding of the structure and function of LDN-containing glycoconjugates.     

Expression of E10 antigen on functionally distinct human T-cell subpopulations: comparison with 3A1 defined subsets

Using PWM-driven immunoglobulin synthesis, we studied the regulatory effects of the peripheral blood T-lymphocyte subpopulations defined by the E10 antigen. This previously described antigen (E10) is present on 60% of TPBL, of T4+, and of T8+ cells. The helper activity on PWM-driven B-cell differentiation appears to be highly increased in E10- T cells. This higher capacity does not apparently reflect a different susceptibility to suppressor influences as comparable results were obtained when such suppressor influences are minimized either by removal of T8+ cells from E10- and E10+ T cells, or by removal of monocytes from responding B-cell populations. In contrast, the relative function of T-cell subsets defined by the related antigen 3A1 are influenced by the presence of suppressor cells. It is only in the presence of both T8+ cells and monocytes that 3A1+ cells exhibit a higher inducer effect. Our results suggest that E10 and 3A1 antigens--although showing strong distribution homologies--define different regulatory T-cell populations.     

Comparison of two independent cDNA clones reported to encode beta 1,4 galactosyltransferase

The identity of cDNA encoding beta 1,4 galactosyltransferase (EC 2.4.1.38) has been controversial, since two independent and unrelated cDNAs have been cloned (GTcDNA-1 and -2), both of which are thought to encode beta 1,4 galactosyltransferase. We have resolved this issue by examining the expression of the corresponding mRNAs in tissues possessing varying levels of galactosyltransferase activity. The expression of GTcDNA-1 parallels the level of galactosyltransferase activity assayed enzymatically, while the expression of GTcDNA-2 is unrelated to the level of enzyme activity, being virtually undetectable in tissues with abundant galactosyltransferase activity. GTcDNA-2, therefore, does not likely encode beta 1,4 galactosyltransferase, but rather, encodes a product that indirectly influences enzyme activity following cellular transfection.     

Purification and characterization of UDP-N-acetylgalactosamine: globotriaosylceramide beta-3-N-acetylgalactosaminyltransferase, a synthase of human blood group P antigen, from canine spleen

A UDP-N-acetylgalactosamine:globotriaosylceramide beta-3-N-acetylgalactosaminyltransferase which catalyzes the conversion of human blood group Pk antigen into P antigen has been purified over 18,000-fold in 4% yield from a Triton X-100 extract of canine spleen microsomes by affinity chromatography on UDP-hexanolamine-Sepharose and globotriaosylceramide acid-Sepharose. The purified enzyme migrates as two major bands with apparent molecular weights of 64,000 and 57,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single band, with enzyme activity, was observed in nondenaturing acrylamide gels containing Triton X-100. Mn2+ was required for activity, and the pH optimum was 6.9. Km values for UDP-GalNAc and globotriaosylceramide were 14 and 2.5 microM, respectively. Studies on substrate specificities indicate that the preferred substrates have the general structure Gal alpha 1-4Gal-OR in which the nature of the R moiety has relatively little effect on activity. An antibody against the purified enzyme eliminated the activity of the enzyme, but did not neutralize the alpha-3-N-acetylgalactosaminyltransferase involved in the biosynthesis of Forssman glycolipid.     

Ig alpha and Ig beta are functionally homologous to the signaling proteins of the T-cell receptor.

Signal transduction by antigen receptors and some Fc receptors requires the activation of a family of receptor-associated transmembrane accessory proteins. One common feature of the cytoplasmic domains of these accessory molecules is the presence is at least two YXXA repeats that are potential sites for interaction with Src homology 2 domain-containing proteins. However, the degree of similarity between the different receptor-associated proteins varies from that of T-cell receptor (TCR) zeta and Fc receptor RIIIA gamma chains, which are homologous, to the distantly related Ig alpha and Ig beta proteins of the B-cell antigen receptor. To determine whether T- and B-cell antigen receptors are in fact functionally homologous, we have studied signal transduction by chimeric immunoglobulins bearing the Ig alpha or Ig beta cytoplasmic domain. We found that Ig alpha and Ig beta cytoplasmic domains were able to activate Ca2+ flux, interleukin-2 secretion, and phosphorylation of the same group of cellular substrates as the TCR in transfected T cells. Chimeric proteins were then used to examine the minimal requirements for activation of the Fyn, Lck, and ZAP kinases in T cells. Both Ig alpha and Ig beta were able to trigger Fyn, Lck, and ZAP directly without involvement of TCR components. Cytoplasmic tyrosine residues in Ig beta were required for recruitment and activation of ZAP-70, but these amino acids were not essential for the activation of Fyn and Lck. We conclude that Fyn and Lck are able to recognize a clustered nonphosphorylated immune recognition receptor, but activation of these kinases is not sufficient to induce cellular responses such as Ca2+ flux and interleukin-2 secretion. In addition, the molecular structures involved in antigen receptor signaling pathways are conserved between T and B cells.     

T-cell specific rearrangement of T-cell receptor beta transgenes in mice.

To study rearrangement of T cell receptor (TCR) genes, transgenic mice were generated with a TCR beta minilocus in germline configuration, containing three V beta, two D beta, fourteen J beta and two C beta gene segments and the TCR beta enhancer. Using the polymerase chain reaction as an analytical tool both partial DJ as well as complete VDJ rearrangements were seen, indicating that the minilocus contained all sequence elements required for rearrangment. Rearrangements of minilocus gene segments were restricted to T cells in the thymus and the periphery and did not occur in B cells. V beta 8.3 and V beta 5 sequences encoded by the minilocus were expressed on the surface of peripheral T cells at high frequencies. Transgenic mice with TCR minilocus genes will be a useful system to identify DNA sequence elements required for regulation of rearrangement in vivo.     

Identification and characterization of a novel UDP-GalNAc:GlcAbeta-R alpha1,4-N-acetylgalactosaminyltransferase from a human sarcoma cell line.

We recently discovered a novel alpha-N-acetylgalactosaminyltransferase in fetal bovine serum (Kitagawa et al., J. Biol. Chem., 270, 22190-22195, 1995) and also in mouse mast cytoma cells (Lidholt et al., Glycoconjugate J., 14, 737-742, 1997), which catalyzed the transfer of an alpha-GalNAc residue to the linkage tetrasaccharide-serine, GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser, derived from proteoglycans. In this study, we characterized this enzyme using a preparation obtained from the serum-free culture medium of a human sarcoma (malignant fibrous histiocytoma) cell line by phenyl-Sepharose chromatography. Structural characterization by1H NMR spectroscopy of the reaction product using the linkage tetrasaccharide-serine, GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser, as a substrate demonstrated that the enzyme was a UDP-GalNAc:GlcAbeta1-R alpha1,4-N -acetylgalactosaminyltransferase. This is the first identification of an alpha1,4-N-acetylgalactosaminyltransferase. Using N -acetylchondrosine GlcAbeta1-3GalNAc as an alternative substrate, the enzyme required divalent cations for the transferase reaction, with maximal activity at 20 mM Mn2+and exhibited a dual optimum at pH 6.5 and pH 7.4 depending upon the buffers used, with the highest activity in a 50 mM 2-( N -morpholino)ethanesulfonic acid buffer at pH 6.5. The apparent Km values obtained for N -acetylchondrosine, the linkage tetrasaccharide-serine, and UDP-GalNAc were 1060 microM, 188 microM, and 27 microM, respectively. This suggested that the linkage tetrasaccharide-serine was a good acceptor substrate for the enzyme. In addition, the enzyme utilized glucuronylneolactotetraosylceramide GlcAbeta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4G lcbeta1-1Cer but not sulfoglucuronylneolactotetraosylceramide GlcA(3-O -sulfate)beta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4Gl cbeta1-1Cer as acceptor substrates. The possibility of involvement of this enzyme in the biosynthesis of glycosaminoglycan as well as other GlcA-containing glycoconjugates is discussed.     

Expression of N-acetyllactosamine and beta1,4-galactosyltransferase (beta4GalT-I) during adenoma-carcinoma sequence in the human colorectum.

We set out to determine the expression profiles of glycoproteins possessing N-acetyllactosamine, a precursor carbohydrate of sialyl Le(x), during colorectal cancer development. We immunohistochemically analyzed the distribution of N-acetyllactosamine as well as of beta4GalT-I, a member of the beta1, 4-galactosyltransferase family responsible for N-acetyllactosamine biosynthesis, in normal mucosa and in adenoma and carcinoma of the human colorectum. Using monoclonal antibody H11, N-acetyllactosamine was barely detectable in the normal mucosa. In low-grade adenoma, however, N-acetyllactosamine was weakly but definitely expressed on the cell surface, and its expression level was moderately increased in high-grade adenoma and markedly increased in carcinoma in situ as well as in advanced carcinoma. To detect beta4GalT-I, we used a newly developed polyclonal antibody (designated A18G), which is specific for the stem region of human beta4GalT-I. Faint expression of beta4GalT-I was detectable in normal mucosa, and the expression level was moderately increased in low-grade adenoma and in high-grade adenoma and markedly increased in carcinoma in situ and advanced carcinoma. The expression of N-acetyllactosamine was highly correlated with the expression of beta4GalT-I in these tumor cells. These results indicate that the expression level of beta4GalT-I is apparently enhanced during tumorigenesis in the colorectum and that beta4GalT-I mostly directs the carcinoma-associated expression of N-acetyllactosamine on the colorectal tumor cell surface. (J Histochem Cytochem 47:1593-1601, 1999)     

UDP-GalNAc:NeuAc alpha 2,3Gal beta-R (GalNAc to Gal) beta 1,4-N-acetylgalactosaminyltransferase responsible for the Sda specificity in human colon carcinoma CaCo-2 cell line

The UDP-GalNAc:NeuAc alpha 2,3Gal beta-R (GalNAc to Gal) beta-1,4-N-acetylgalactosaminyltransferase (beta 1,4GalNAc-transferase) is the enzyme responsible for the addition of the immunodominant sugar of the Sda isto-blood group determinant. In humans the enzyme is mainly expressed in the large intestine. We screened nine human colorectal carcinoma cell lines (SW-948, SW-948 FL, SW-480, SW-48, SW-1417, COLO-205, LOVO, HT-29 and CaCo-2) in order to ascertain the occurrence of beta 1,4GalNAc-transferase. Only in CaCo-2 cells the glycosyltransferase was detected and the activity increased with the degree of enterocytic differentiation. Nevertheless, in highly differentiated CaCo-2 cells the activity was thirty times lower than that found in cells detached from normal colon mucosa. These results support the notion that the expression of beta 1,4GalNAc-transferase is a marker of the colonic cell maturation.     

Rearrangement and expression of T-cell antigen receptor genes in human T-lymphocyte tumor lines and normal human T-cell clones: evidence for allelic exclusion of Ti beta gene expression and preferential use of a J beta 2 gene segment.

The gene encoding the beta chain of the human T-cell receptor for antigen is composed of variable (V), diversity (D), joining (J), and constant (C) gene segments which undergo specific rearrangements during T-lymphocyte ontogeny. Southern blot analyses of seven human T-cell tumor lines and normal human T-lymphocyte clones revealed that most of these T-cell lines rearrange their Ti beta genes differently. The T-cell tumor line HPB-MLT rearranges and transcribes both of its Ti beta genes. Cloning and sequencing of the Ti beta cDNAs corresponding to these rearrangements revealed that one of the rearranged Ti beta genes is defective, while the other is functional and corresponds to the Ti beta protein expressed on the surface of these cells. Thus, this cell line displays a pattern of allelic exclusion of Ti beta gene expression. A comparison of four C beta 2-containing Ti beta cDNAs from three different cell lines revealed that three of the four utilize the same J beta 2.5 gene segment joined to different D beta and V beta genes, suggesting that there may be preferential use of this J gene during J beta 2 rearrangements. Hybridization analyses with probes for the alpha and beta genes of the T-cell receptor and the T-cell-specific T gamma gene revealed that HPB-MLT cells appear to express approximately equivalent amounts of RNA corresponding to each of the rearranged Ti alpha and Ti beta genes. However, they express a much lower level of T gamma RNA.     

Expression of functional G protein beta gamma dimers of defined subunit composition using a baculovirus expression system.

The 36-kDa beta 1, 35-kDa beta 2, and 6.5-kDa gamma 2 subunits of the heterotrimeric guanine nucleotide-binding proteins have been overexpressed in Sf9 cells using a baculovirus expression system. The gamma 2 subunit expressed in Sf9 cells incorporated label derived from [3H]mevalonate and is therefore likely to be isoprenylated, as is its mammalian counterpart. Extracts of Sf9 cells doubly infected with viruses encoding a beta subunit and viruses encoding a gamma subunit are active in promoting the pertussis toxin-catalyzed ADP-ribosylation of a G protein alpha subunit. However, extracts from Sf9 cells singly infected with viruses encoding either a beta or gamma subunit are not active in this assay. Results demonstrate utility of the insect/baculovirus system for expressing G protein beta gamma subunits of defined composition.     

Polyclonal T-cell activation protocol stimulates preferential expansion of EBV-specific T-cell clones in vitro

Purpose Allogeneic bone marrow transplantation (AlloBMT) can be curative for patients with leukemia. The most important anti-leukemic effect may be mediated by the T-cells contained within the graft; however, the allogeneic T-cells may also give rise to graft-vs-host disease (GVHD). One way to control GVHD might be to transduce the donor T-cells with a drug-inducible suicide gene. If a retrovirus vector is to be used for this transduction, activation of the T-cells is required for integration of the transgene to occur. The activation protocol should ensure expansion of a broad repertoire of donor T-cells. Notably, T-cells specific for herpes virus family antigens are important for adoptive immunoprotection.Methods To define optimal activation conditions for retrovirus-mediated suicide gene transduction of donor T-cells, we examined the repertoire of CD8+ T-cells in general, and Epstein-Barr virus (EBV) specific T-cells in particular, following two different activation and expansion procedures.Results We found that repeated CD3/CD28 stimulation resulted in a high level of activation-induced T-cell death, affecting in vivo expanded clones, some of which were specific for EBV, in particular. In contrast, initial CD3/CD28 activation followed by proliferation in interleukin-2 lead to expansion of EBV-specific clones over and above the expansion observed for CD8+ T-cells in general.Conclusion These results should impact on protocols for ex vivo activation of T-cells prior to suicide gene transduction.     

Dengue virus-specific, human CD4+ CD8- cytotoxic T-cell clones: multiple patterns of virus cross-reactivity recognized by NS3-specific T-cell clones.

Thirteen dengue virus-specific, cytotoxic CD4+ CD8- T-cell clones were established from a donor who was infected with dengue virus type 3. These clones were examined for virus specificity and human leukocyte antigen (HLA) restriction in cytotoxic assays. Six patterns of virus specificities were determined. Two serotype-specific clones recognized only dengue virus type 3. Two dengue virus subcomplex-specific clones recognized dengue virus types 2, 3, and 4, and one subcomplex-specific clone recognized dengue virus types 1, 2, and 3. Four dengue virus serotype-cross-reactive clones recognized dengue virus types 1, 2, 3, and 4. One flavivirus-cross-reactive clone recognized dengue virus types 1, 2, 3, and 4 and West Nile virus (WNV), but did not recognize yellow fever virus (YFV), whereas three flavivirus-cross-reactive clones recognized dengue virus types 1, 2, 3, and 4, WNV, and YFV. HLA restriction in the lysis by these T-cell clones was also heterogeneous. HLA-DP, HLA-DQ, and HLA-DR were used as restriction elements by various T-cell clones. We also examined the recognition of viral nonstructural protein NS3, purified from cells infected with dengue virus type 3 or WNV, by these T-cell clones. One serotype-specific clone, two dengue virus subcomplex-specific clones, and three dengue virus serotype-cross-reactive clones recognized NS3 of dengue virus type 3. One flavivirus-cross-reactive clone recognized NS3 of dengue virus type 3 and WNV. These results indicate that heterogeneous dengue virus-specific CD4+ cytotoxic T cells are stimulated in response to infection with a dengue virus and that a nonstructural protein, NS3, contains multiple dominant T-cell epitopes.     

Expression of specific clones during B cell development.

The expression of DNP- and TNP-specific B cells in spleens of neonatal BALB/c mice was analyzed by the in vitro splenic focus technique. B cells of these specificities were found to be present in slightly higher frequency in neonatal than in adult spleens. The parameters of stimulation of neonatal B cells were similar to those of adult B cells but the antibody-forming cell progeny of neonatal B cells produce predominantly gammaM rather than gammaG antibody and produce less antibody than the progeny of adult B cells. Isoelectric focusing analyses of monoclonal antibodies derived from neonatal B cells stimulated in vitro with DNP or TNP revealed that over 90 per cent of the antibodies could be identified as belonging to one of six predominant clonotypes, three specific for DNP and three for TNP. While individual neonates rarely expressed all of the predominant clonotypes, B cells of each of the six clonotypes were found in several donors. When B cells of a given predominant clonotype were present in an individual many such B cells could be found and in many cases the entire DNP- or TNP-specific B cell population of an individual could be accounted for by B cells of a single clonotype. These findings are discussed in terms of the diversity of clonotype specificities available in neonates, the kinetics of development of cells within a clonotype, and factors that may play a role in controlling the expression of B cell clones.