Control of metastasis by Asn-linked, beta1-6 branched oligosaccharides in mouse mammary cancer cells. (1/1123)

Studies in cell lines and malignant human tissues have shown that increased cell-surface Asn-linked beta1-6(GlcNAcbeta1-6Man) branching is associated with increased tumorigenic and metastatic properties. In this study, three mouse mammary cancer cell lines were transfected with an expression vector containing the mouse cDNA for N-acetylglucosaminyltransferase V (GlcNAcT-V EC 2.4.1.155), the glycosyltransferase responsible for initiating beta1-6 branching on Asn-linked carbohydrates. The cell lines were screened for increased cytotoxicity to L-PHA, a lectin specific for beta1-6 branching structures. Cell lines exhibiting increased L-PHA cytotoxicity expressed increased levels of beta1-6 branching structures. Northern blots detected the presence of GlcNAcT-V transcribed from the expression vector in the L-PHA sensitive cell lines. After injection into the tail veins of mice, transfected cell lines with increased beta1-6 branching on the cell surface formed elevated levels of lung tumors relative to control transfected cell lines (P < 0.002). Western blots of membrane proteins from GlcNAcT-V transfected and control cells probed with the lectins DSA and WGA did not show an increase in polyN-acetyllactosamine and sialic acid content in the transfected cell lines. These results demonstrate that a specific increase in beta1-6 branching due to an elevation in GlcNAcT-V expression increases metastatic potential.  (+info)

Tissue specific expression and chromosomal mapping of a human UDP-N-acetylglucosamine: alpha1,3-d-mannoside beta1, 4-N-acetylglucosaminyltransferase. (2/1123)

A human cDNA for UDP- N -acetylglucosamine:alpha1,3-d-mannoside beta1,4- N- acetylglucosaminyltransferase (GnT-IV) was isolated from a liver cDNA library using a probe based on a partial cDNA sequence of the bovine GnT-IV. The cDNA encoded a complete sequence of a type II membrane protein of 535 amino acids which is 96% identical to the bovine GnT-IV. Transient expression of the human cDNA in COS7 cells increased total cellular GnT-IV activity 25-fold, demonstrating that this cDNA encodes a functional human GnT-IV. Northern blot analysis of normal tissues indicated that at least five different sizes of mRNA (9.7, 7.6, 5.1, 3.8, and 2.4 kb) forGnT-IV are expressed in vivo. Furthermore, these mRNAs are expressed at different levels between tissues. Large amounts of mRNA were detected in tissues harboring T lineage cells. Also, the promyelocytic leukemia cell line HL-60 and the lymphoblastic leukemia cell line MOLT-4 revealed abundant mRNA. Lastly, the gene was mapped at the locus on human chromosome 2, band q12 by fluorescent in situ hybridization.  (+info)

The genes for the Golgi apparatus N-acetylglucosaminyltransferase and the UDP-N-acetylglucosamine transporter are contiguous in Kluyveromyces lactis. (3/1123)

The mannan chains of Kluyveromyces lactis mannoproteins are similar to those of Saccharomyces cerevisiae except that they lack mannose phosphate and have terminal alpha(1-->2)-linked N-acetylglucosamine. Previously, Smith et al. (Smith, W. L. Nakajima, T., and Ballou, C. E. (1975) J. Biol. Chem. 250, 3426-3435) characterized two mutants, mnn2-1 and mnn2-2, which lacked terminal N-acetylglucosamine in their mannoproteins. The former mutant lacks the Golgi N-acetylglucosaminyltransferase activity, whereas the latter one was recently found to be deficient in the Golgi UDP-GlcNAc transporter activity. Analysis of extensive crossings between the two mutants led Ballou and co-workers (reference cited above) to conclude that these genes were allelic or tightly linked. We have now cloned the gene encoding the K. lactis Golgi membrane N-acetylglucosaminyltransferase by complementation of the mnn2-1 mutation and named it GNT1. The mnn2-1 mutant was transformed with a 9.5-kilobase (kb) genomic fragment previously shown to contain the gene encoding the UDP-GlcNAc transporter; transformants were isolated, and phenotypic correction was monitored after cell surface labeling with fluorescein isothiocyanate-conjugated Griffonia simplicifolia II lectin, which binds terminal N-acetylglucosamine, and a fluorescence-activated cell sorter. The above 9.5-kb DNA fragment restored the wild-type lectin binding phenotype of the transferase mutant; further subcloning of this fragment yielded a smaller one containing an opening reading frame of 1,383 bases encoding a protein of 460 amino acids with an estimated molecular mass of 53 kDa, which also restored the wild-type phenotype. Transformants had also regained the ability to transfer N-acetylglucosamine to 3-0-alpha-D-mannopyranosyl-D-mannopyranoside. The gene encoding the above transferase was found to be approximately 1 kb upstream from the previously characterized MNN2 gene encoding the UDP-GlcNAc Golgi transporter. Each gene can be transcribed independently by their own promoter. To our knowledge this is the first demonstration of two Golgi apparatus functionally related genes being contiguous in a genome.  (+info)

Core 2-containing O-glycans on CD43 are preferentially expressed in the memory subset of human CD4 T cells. (4/1123)

Human CD4 T cells can be divided into two functionally distinct subsets: a CD45RO+ memory subset and a CD45RA+ naive subset. In an attempt to identify novel cell surface molecules on these cells, we have developed a mAb, anti-1D4. The antigen defined by anti-1D4 was preferentially expressed on the memory subset of freshly isolated peripheral CD4 T cells and 1D4+ CD4 T cells functionally corresponded to memory T cells. Retrovirus-mediated expression cloning revealed that the 1 D4 antigen is human CD43. Transfection of CHO-leu cells, which stably express human CD43, with core 2 beta-1,6-N-acetylglucosaminyltransferase (C2GnT) conferred expression of the 1D4 antigen and mRNA of C2GnT was detected by RT-PCR only in 1D4+ T cells but not in 1D4- T cells, implying that the 1 D4 antigen is composed of core 2-containing O-glycans on CD43. Reactivity with anti-1 D4 was completely abolished when cells were treated with neuraminidase, while them remained weak binding of anti-T305, a previously described mAb which also reacts with CD43 modified with core 2-containing O-glycans. Moreover, anti-1D4 markedly reacted with NIH-3T3 cells expressing human CD43 and low levels of endogenous C2GnT, whereas anti-T305 reacted slightly. These results indicate that the 1D4 antigen is distinct from the epitope defined by anti-T305 and anti-1D4 is a more sensitive probe to detect core 2-containing O-glycans than anti-T305. Taken together, our results indicate that core 2-containing O-glycans, whose expression can easily be detected with anti-1D4, are preferentially expressed in the CD45RO+ memory subset of CD4 T cells.  (+info)

Enzymatic synthesis of natural and 13C enriched linear poly-N-acetyllactosamines as ligands for galectin-1. (5/1123)

As part of a study of protein-carbohydrate interactions, linear N-acetyl-polyllactosamines [Galbeta1,4GlcNAcbeta1,3]nwere synthesized at the 10-100 micromol scale using enzymatic methods. The methods described also provided specifically [1-13C]-galactose-labeled tetra- and hexasaccharides ([1-13C]-Galbeta1,4GlcNAcbeta1,3Galbeta1,4Glc and Galbeta1, 4GlcNAcbeta1,3[1-13C]Galbeta1,4GlcNAcbeta1,3Galbeta 1,4Glc) suitable for NMR studies. Two series of oligosaccharides were produced, with either glucose or N-acetlyglucosamine at the reducing end. In both cases, large amounts of starting primer were available from human milk oligosaccharides (trisaccharide primer GlcNAcbeta1,3Galbeta1, 4Glc) or via transglycosylation from N-acetyllactosamine. Partially purified and immobilized glycosyltransferases, such as bovine milk beta1,4 galactosyltransferase and human serum beta1,3 N- acetylglucosaminyltransferase, were used for the synthesis. All the oligo-saccharide products were characterized by1H and13C NMR spectroscopy and MALDI-TOF mass spectrometry. The target molecules were then used to study their interactions with recombinant galectin-1, and initial1H NMR spectroscopic results are presented to illustrate this approach. These results indicate that, for oligomers containing up to eight sugars, the principal interaction of the binding site of galectin-1 is with the terminal N-acetyllactosamine residues.  (+info)

Regulation of I-branched poly-N-acetyllactosamine synthesis. Concerted actions by I-extension enzyme, I-branching enzyme, and beta1,4-galactosyltransferase I. (6/1123)

I-branched poly-N-acetyllactosamine is a unique carbohydrate composed of N-acetyllactosamine branches attached to linear poly-N-acetyllactosamine, which is synthesized by I-branching beta1, 6-N-acetylglucosaminyltransferase. I-branched poly-N-acetyllactosamine can carry bivalent functional oligosaccharides such as sialyl Lewisx, which provide much better carbohydrate ligands than monovalent functional oligosaccharides. In the present study, we first demonstrate that I-branching beta1, 6-N-acetylglucosaminyltransferase cloned from human PA-1 embryonic carcinoma cells transfers beta1,6-linked GlcNAc preferentially to galactosyl residues of N-acetyllactosamine close to nonreducing terminals. We then demonstrate that among various beta1, 4-galactosyltransferases (beta4Gal-Ts), beta4Gal-TI is most efficient in adding a galactose to linear and branched poly-N-acetyllactosamines. When a beta1,6-GlcNAc branched poly-N-acetyllactosamine was incubated with a mixture of beta4Gal-TI and i-extension beta1,3-N-acetylglucosaminyltransferase, the major product was the oligosaccharide with one N-acetyllactosamine extension on the linear Galbeta1-->4GlcNAcbeta1-->3 side chain. Only a minor product contained galactosylated I-branch without N-acetyllactosamine extension. This finding was explained by the fact that beta4Gal-TI adds a galactose poorly to beta1,6-GlcNAc attached to linear poly-N-acetyllactosamines, while beta1, 3-N-acetylglucosaminyltransferase and beta4Gal-TI efficiently add N-acetyllactosamine to linear poly-N-acetyllactosamines. Together, these results strongly suggest that galactosylation of I-branch is a rate-limiting step in I-branched poly-N-acetyllactosamine synthesis, allowing poly-N-acetyllactosamine extension mostly along the linear poly-N-acetyllactosamine side chain. These findings are entirely consistent with previous findings that poly-N-acetyllactosamines in human erythrocytes, PA-1 embryonic carcinoma cells, and rabbit erythrocytes contain multiple, short I-branches.  (+info)

Expression of functional selectin ligands on Th cells is differentially regulated by IL-12 and IL-4. (7/1123)

Immune responses may be qualitatively distinct depending on whether Th1 or Th2 cells predominate at the site of Ag exposure. T cell subset-specific expression of ligands for vascular selectins may underlie the distinct patterns of recruitment of Th1 or Th2 cells to peripheral inflammatory sites. Here we examine the regulation of selectin ligand expression during murine T helper cell differentiation. Large numbers of Th1 cells interacted with E- and P-selectin under defined flow conditions, while few Th2 and no naive T cells interacted. Th1 cells also expressed more fucosyltransferase VII mRNA than naive or Th2 cells. IL-12 induced expression of P-selectin ligands on Ag-activated naive T cells, even in the presence of IL-4, and on established Th2 cells restimulated in the presence of IL-12 and IFN-gamma. In contrast, Ag stimulation alone induced only E-selectin ligand. Interestingly, restimulation of established Th2 cells in the presence of IL-12 and IFN-gamma induced expression of P-selectin ligands but not E-selectin ligands; IFN-gamma alone did not enhance expression of either selectin ligand. In summary, functional P- and E-selectin ligands are expressed on most Th1 cells, few Th2 cells, but not naive T cells. Furthermore, selectin ligand expression is regulated by the cytokine milieu during T cell differentiation. IL-12 induces P-selectin ligand, while IL-4 plays a dominant role in down-regulating E-selectin ligand.  (+info)

The tumor suppressor EXT-like gene EXTL2 encodes an alpha1, 4-N-acetylhexosaminyltransferase that transfers N-acetylgalactosamine and N-acetylglucosamine to the common glycosaminoglycan-protein linkage region. The key enzyme for the chain initiation of heparan sulfate. (8/1123)

We previously demonstrated a unique alpha-N-acetylgalactosaminyltransferase that transferred N-acetylgalactosamine (GalNAc) to the tetrasaccharide-serine, GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser (GlcA represents glucuronic acid), derived from the common glycosaminoglycan-protein linkage region, through an alpha1,4-linkage. In this study, we purified the enzyme from the serum-free culture medium of a human sarcoma cell line. Peptide sequence analysis of the purified enzyme revealed 100% identity to the multiple exostoses-like gene EXTL2/EXTR2, a member of the hereditary multiple exostoses (EXT) gene family of tumor suppressors. The expression of a soluble recombinant form of the protein produced an active enzyme, which transferred alpha-GalNAc from UDP-[3H]GalNAc to various acceptor substrates including GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser. Interestingly, the enzyme also catalyzed the transfer of N-acetylglucosamine (GlcNAc) from UDP-[3H]GlcNAc to GlcAbeta1-3Galbeta1-O-naphthalenemethanol, which was the acceptor substrate for the previously described GlcNAc transferase I involved in the biosynthetic initiation of heparan sulfate. The GlcNAc transferase reaction product was sensitive to the action of heparitinase I, establishing the identity of the enzyme to be alpha1, 4-GlcNAc transferase. These results altogether indicate that EXTL2/EXTR2 encodes the alpha1,4-N-acetylhexosaminyltransferase that transfers GalNAc/GlcNAc to the tetrasaccharide representing the common glycosaminoglycan-protein linkage region and that is most likely the critical enzyme that determines and initiates the heparin/heparan sulfate synthesis, separating it from the chondroitin sulfate/dermatan sulfate synthesis.  (+info)