A direct interaction between EXT proteins and glycosyltransferases is defective in hereditary multiple exostoses.
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Hereditary multiple exostoses (HME) is an autosomal dominant condition in which bony outgrowths occur from the juxtaepiphyseal regions of the long bones. In a few percent of cases these exostoses undergo malignant transformation to chondrosarcomas. HME results from mutations in one of two homologous genes, EXT1 and EXT2. These are members of a new gene family that is conserved from Caenorhabditis elegans to higher vertebrates. In humans this family comprises five genes which are most conserved at their C-termini, but they do not contain any discernible functional motifs and their function(s) is unclear. Indirect evidence suggests that EXT proteins are involved in glycosaminoglycan synthesis, act as tumor suppressors and affect hedgehog signaling. One recent study has also reported that these proteins co-purify with glycosyltransferase (GlcA and GlcNAc transferase) activity and on that basis it has been postulated that they are themselves glycosyl-transferases. We performed two-hybrid screens with a fragment of EXT2 from the region that is most highly conserved in the gene family and identified two interacting proteins: the tumor necrosis factor type 1 associated protein and a novel UDP-GalNAc:poly-peptide N -acetylgalactosaminyltransferase. Significantly, both these interactions were abrogated by a disease-causing EXT mutation, indicating that they are important in the etiology of HME. The EXT2-GalNAc-T5 interaction provides the first direct physical link between EXT proteins and known components of glycosamino-glycan synthesis. (+info)
Expression of endoxyloglucan transferase genes in acaulis mutants of Arabidopsis.
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A mutant of Arabidopsis with reduced internodal cell length, acaulis5 (acl5), has recently been shown to have reduced transcript levels of a gene for endoxyloglucan transferase, EXGT-A1 (Y. Hanzawa, T. Takahashi, Y. Komeda [1997] Plant J 12: 863-874). In the present study, we cloned genomic fragments of five members of the EXGT gene family, EXGT-A1, EXGT-A3, EXGT-A4, XTR2, and XTR3, and examined their expression in the wild type and in a series of acl mutants. In wild-type plants, the EXGT-A3 gene showed higher expression in lower internodes (internodes between nodes bearing axillary shoots) than in upper and young internodes, in which EXGT-A1 was highly expressed. EXGT-A4 was preferentially expressed in roots and XTR3 in siliques. The XTR2 gene was constitutively expressed. In acl1, acl3, and acl4 mutants, which have a severe defect in leaf expansion as well as in internode elongation, the EXGT-A1 gene showed reduced levels of expression before bolting of plants. In contrast, XTR3 was increased in these mutant seedlings. Reduction of EXGT-A1 expression was also detected after bolting of all acl mutants except acl2, whose growth defect is restricted to lower internodes. These results suggest the involvement of each EXGT in different aspects of organ development. (+info)
Binding of calcium in the EF-hand of Escherichia coli lytic transglycosylase Slt35 is important for stability.
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The Escherichia coli lytic transglycosylase Slt35 contains a single metal ion-binding site that resembles EF-hand calcium-binding sites. The Slt35 EF-hand is only the second observation of such a domain in a prokaryotic protein. Two crystal structures at 2.1 A resolution show that both Ca2+ ions and Na+ ions can bind to the EF-hand domain, but in subtly different configurations. Heat-induced unfolding studies demonstrate that Ca2+ ions are preferentially bound, and that only Ca2+ ions significantly increase the melting temperature of Slt35. This shows that the EF-hand calcium-binding domain is important for the stability of Slt35. (+info)
The GPI biosynthetic pathway as a therapeutic target for African sleeping sickness.
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African sleeping sickness is a debilitating and often fatal disease caused by tsetse fly transmitted African trypanosomes. These extracellular protozoan parasites survive in the human bloodstream by virtue of a dense cell surface coat made of variant surface glycoprotein. The parasites have a repertoire of several hundred immunologically distinct variant surface glycoproteins and they evade the host immune response by antigenic variation. All variant surface glycoproteins are anchored to the plasma membrane via glycosylphosphatidylinositol membrane anchors and compounds that inhibit the assembly or transfer of these anchors could have trypanocidal potential. This article compares glycosylphosphatidylinositol biosynthesis in African trypanosomes and mammalian cells and identifies several steps that could be targets for the development of parasite-specific therapeutic agents. (+info)
Interference with murein turnover has no effect on growth but reduces beta-lactamase induction in Escherichia coli.
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Physiological studies of a mutant of Escherichia coli lacking the three lytic transglycosylases Slt70, MltA, and MltB revealed that interference with murein turnover can prevent AmpC beta-lactamase induction. The triple mutant, although growing normally, shows a dramatically reduced rate of murein turnover. Despite the reduction in the formation of low-molecular-weight murein turnover products, neither the rate of murein synthesis nor the amount of murein per cell was increased. This might be explained by assuming that during growth in the absence of the major lytic transglycosylases native murein strands are excised by the action of endopeptidases and directly reused without further breakdown to muropeptides. The reduced rate of murein turnover could be correlated with lowered cefoxitin-induced expression of beta-lactamase, present on a plasmid carrying the ampC and ampR genes from Enterobacter cloacae. Overproduction of MltB stimulated beta-lactamase induction, whereas specific inhibition of Slt70 by bulgecin repressed ampC expression. Thus, specific inhibitors of lytic transglycosylases can increase the potency of penicillins and cephalosporins against bacteria inducing AmpC-like beta-lactamases. (+info)
Regulation of natural killer cell-mediated swine endothelial cell lysis through genetic remodeling of a glycoantigen.
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The effect of remodeling of a glycoantigen such as the alpha-Gal epitope, Galalpha1,3Galbeta1,4GlcNAc-R, by the introduction of glycosyltransferase genes on natural killer (NK) cell-mediated direct cytotoxicity was investigated using human peripheral blood mononuclear cells (PBMC) or an NK-like cell line, YT cells, as an effector, and swine endothelial cells (SEC) as a target. Several SEC transfectants were established by transfection with the genes for beta1,4-N-acetylglucosaminyltransferase III, alpha2, 3-sialyltransferase and alpha1,2-fucosyltransferase. These transfections led to dramatic reductions in both direct and indirect NK cell-mediated cytotoxicity, by 72-94% in the case of PBMC and 27-72% in that of YT cells, in addition to an effective reduction in xenoantigenicity, which is substantially caused by the alpha-Gal epitope, to human natural antibodies. The NK cell-mediated direct cytotoxicity was remarkably blocked by an anti-alpha-Gal epitope monoclonal antibody or GSI lectin which preferentially binds to the epitope. Furthermore, treatment of the parental cells with alpha-galactosidase resulted in a significant reduction in cytotoxicity. These results suggest that the alpha-Gal epitope is involved not only in hyperacute rejection and acute vascular rejection, but also in NK cell-mediated direct cytotoxicity. Thus, the genetic remodeling of the alpha-Gal epitope and probably other glycoantigens as well can be expected to represent a new approach for overcoming not only indirect but also direct immunity to xenografts. (+info)
The remodeling of glycoconjugates in mice.
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A role for glycoconjugates in mediating cellular interactions is well established. To further understand the formation, function and regulation of various glycoconjugates in vivo, gene targeting approaches have been applied to glycosyltransferase and glycosidase enzymes involved in different biosynthetic pathways. The growing number of gene targeted mice generated have brought confirmations of the importance of both core and terminal glycosylation enzymes in normal development and physiology. Of particular interest has been the degree of cell and tissue specificity of phenotypes generated by systemic null mutations as well as the number of enzymes belonging to multigene families having overlapping activities. (+info)
Trafficking of oligomannosides released during N-glycosylation: a clearing mechanism of the rough endoplasmic reticulum.
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The main reaction of N-glycosylation of proteins is the transfer 'en bloc' of the oligosaccharide moieties of lipid intermediates to an asparagine residue of the nascent protein. For the past 15 years, a few laboratories including ours have shown that the process was accompanied by the release of oligosaccharide-phosphates and of neutral oligosaccharides possessing one GlcNAc (OS-Gn(1)) or two GlcNAc (OS-Gn(2)) at the reducing end. The aim of this review is to gather the evidence for the different origins of these soluble oligomannosides, to examine their subcellular location and intracellular trafficking. Furthermore, using Brefeldin A we demonstrated that this released oligomannoside material could be the substrate for the Golgi glycosidases and glycosyltransferases. Indeed, released oligomannoside never reach the Golgi vesicles either because they are directly produced in the cytosol as has been demonstrated for oligosaccharide-phosphates and for neutral oligosaccharides possessing one GlcNAc at the reducing end or because they are actively transported out of the rough endoplasmic reticulum to the cytosol. One of the functions of oligomannoside trafficking between rough endoplasmic reticulum, cytosol and lysosomes could be to prevent these oligosaccharides for competing with glycosylation in the Golgi. (+info)