The Impact of β-1,4-Galactosyltransferase V on Microglial Function.
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ABSTRACT:
SUBMITTER: Wang X
PROVIDER: S-EPMC8446519 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Beta-1,4-galactosylransferase (beta4Gal-T1) participates in the synthesis of Galbeta1-4-GlcNAc-disaccharide unit of glycoconjugates. It is a trans-Golgi glycosyltransferase (Glyco-T) with a type II membrane protein topology, a short N-terminal cytoplasmic domain, a membrane-spanning region, as well as a stem and a C-terminal catalytic domain facing the trans-Golgi-lumen. Its hydrophobic membrane-spanning region, like that of other Glyco-T, has a shorter length compared to plasma membrane proteins, an important feature for its retention in the trans-Golgi. The catalytic domain has two flexible loops, a long and a small one. The primary metal binding site is located at the N-terminal hinge region of the long flexible loop. Upon binding of metal ion and sugar-nucleotide, the flexible loops undergo a marked conformational change, from an open to a closed conformation. Conformational change simultaneously creates at the C-terminal region of the flexible loop an oligosaccharide acceptor binding site that did not exist before. The loop acts as a lid covering the bound donor substrate. After completion of the transfer of the glycosyl unit to the acceptor, the saccharide product is ejected; the loop reverts to its native conformation to release the remaining nucleotide moiety. The conformational change in beta4Gal-T1 also creates the binding site for a mammary gland-specific protein, alpha-lactalbumin (LA), which changes the acceptor specificity of the enzyme toward glucose to synthesize lactose during lactation. The specificity of the sugar donor is generally determined by a few residues in the sugar-nucleotide binding pocket of Glyco-T, conserved among the family members from different species. Mutation of these residues has allowed us to design new and novel glycosyltransferases, with broader or requisite donor and acceptor specificities, and to synthesize specific complex carbohydrates as well as specific inhibitors for these enzymes.
| S-EPMC2365515 | biostudies-literature
Project description:A cDNA encoding a beta-1,4-galactosyltransferase named beta-1,4-GalT II was cloned from a cDNA library of the human breast tumor cell line, MRK-nu-1. Initially, a 860-bp PCR fragment was obtained from MRK-nu-1 mRNA by 3'-rapid amplification of cDNA ends by using two nested degenerate oligonucleotide primers based on a highly conserved amino acid sequence found in the catalytic domain of mammalian beta-1,4-galactosyltransferases and Lymnaea stagnalis beta-1,4-N-acetylglucosaminyltransferase (beta-1,4-GlcNAcT), both of which utilize the same sugar acceptor. This subsequently was used as a probe to isolate a 4.7-kb cDNA that contained an ORF of 1,164 bp predicting a polypeptide of 388 aa. Its deduced amino acid sequence shows an identity of 37% with that of the previously characterized human beta-1,4-galactosyltransferase (referred to as beta-1,4-GalT I) and of 28% with that of L. stagnalis beta-1,4-GlcNAcT. Study of the properties of the beta-1,4-GalT II fused to protein A expressed as a soluble form in COS-7 cells revealed that it is a genuine beta-1,4-GalT but has no lactose synthetase activity in the presence of alpha-lactalbumin. Northern blot analysis of 24 human tissues showed that they all express the beta-1,4-GalT II transcript, although the levels varied. These results indicate that human cells contain another beta-1,4-GalT.
| S-EPMC18444 | biostudies-literature
Project description:A bovine beta-1,4-galactosyltransferase (GT; EC 2.4.1.90) cDNA in an Okayama-Berg vector, pLsGT, was constructed from a partial cDNA clone and a genomic fragment. We report that the cDNA sequence of pLsGT, in a transient expression assay in COS-7 cells, codes for an enzymatically active GT protein. There is an approximately 12-fold increase in the GT activity in pLsGT-transfected cells compared to cells transfected with the antisense bovine GT construct, pLasGT, or pSV2Neo or mock-transfected cells. The increased activity is correlated with the increase in bovine GT mRNA, which is distinguishable from COS GT mRNA with a 3'-end-specific probe of pLsGT. The expressed GT activity is modulated by alpha-lactalbumin, which changes the acceptor specificity to glucose to synthesize lactose. Polyclonal antibody raised against SDS/PAGE-purified bovine milk GT and a monoclonal antibody (mAb 4-10) directed against a synthetic peptide corresponding to the amino-terminal region of the protein encoded by pLsGT bind the expressed protein, and the resulting immunoprecipitates exhibit GT enzymatic activity.
| S-EPMC297704 | biostudies-other
Project description:N-glycosylation of a mAb may have a major impact on its therapeutic merits. Here, we demonstrate that expression of a hybrid enzyme (called xylGalT), consisting of the N-terminal domain of Arabidopsis thaliana xylosyltransferase and the catalytic domain of human beta-1,4-galactosyltransferase I (GalT), in tobacco causes a sharp reduction of N-glycans with potentially immunogenic core-bound xylose (Xyl) and fucose (Fuc) residues as shown by Western blot and MALDI-TOF MS analysis. A radioallergosorbent test inhibition assay with proteins purified from leaves of WT and these transgenic tobacco plants using sera from allergic patients suggests a significant reduction of potential immunogenicity of xylGalT proteins. A mAb purified from leaves of plants expressing xylGalT displayed an N-glycan profile that featured high levels of galactose, undetectable xylose, and a trace of fucose. Hence, a transgenic plant expressing the hybrid GalT might yield more effective and safer monoclonals for therapeutic purposes than WT plants and even transgenic plants expressing the unchanged GalT.
| S-EPMC1472489 | biostudies-literature
Project description:Deficiency of the Golgi enzyme UDP-Gal:N-acetylglucosamine beta-1,4-galactosyltransferase I (beta4GalT I) (E.C.2.4.1.38) causes a new congenital disorder of glycosylation (CDG), designated type IId (CDG-IId), a severe neurologic disease characterized by a hydrocephalus, myopathy, and blood-clotting defects. Analysis of oligosaccharides from serum transferrin by HPLC, mass spectrometry, and lectin binding revealed the loss of sialic acid and galactose residues. In skin fibroblasts and leukocytes, galactosyltransferase activity was reduced to 5% that of controls. In fibroblasts, a truncated polypeptide was detected that was about 12 kDa smaller in size than wild-type beta4GalT I and that failed to localize to the Golgi apparatus. Sequencing of the beta4GalT I cDNA and gene revealed an insertion of a single nucleotide (1031-1032insC) leading to premature translation stop and loss of the C-terminal 50 amino acids of the enzyme. The patient was homozygous and his parents heterozygous for this mutation. Expression of a corresponding mutant cDNA in COS-7 cells led to the synthesis of a truncated, inactive polypeptide, which localized to the endoplasmic reticulum.
| S-EPMC150909 | biostudies-literature
Project description:Many glycosyltransferase inhibitors in the literature are structurally derived from the donor or acceptor substrate of the respective enzyme. A representative example is 2-naphthyl ?-d-GlcNAc, a synthetic GlcNAc glycoside that has been reported as a galactosyltransferase inhibitor. This GlcNAc derivative is attractive as a chemical tool compound for biological and biochemical studies because of its reported potency as an inhibitor, and its short and straightforward synthesis from readily available starting materials. We report that in our hands, 2-naphthyl ?-d-GlcNAc behaved, unexpectedly, as an acceptor substrate of the inverting ?-1,4-galactosyltransferase (?-1,4-GalT) from bovine milk. This substrate activity has not previously been described. We found that 2-naphthyl ?-d-GlcNAc can be an acceptor substrate both for recombinantly expressed ?-1,4-GalT, and for a commercial batch of the same enzyme, and both in the presence and absence of bovine serum albumin (BSA). As expected for a full acceptor substrate, this substrate activity was time- and concentration-dependent. Additional experiments show that the observed inhibitor/substrate switch is facilitated by a phosphatase that is an essential component of our enzyme-coupled glycosyltransferase assay. These findings suggest that the behaviour of 2-naphthyl ?-d-GlcNAc and related acceptor-based glycosyltransferase inhibitors is strongly dependent on the individual assay conditions. Our results therefore have important implications for the use of 2-naphthyl ?-d-GlcNAc and related glycosides as tool compounds in glycobiology and glycobiochemistry.
| S-EPMC5636615 | biostudies-literature
Project description:Glaucoma is a major cause of irreversible blindness worldwide. There is evidence showing that a subset of the disease is genetically determined. In this study, we screened for mutations in chromosome 1q-linked open-angle glaucoma (GLC1A) in a Chinese family with primary open-angle glaucoma (POAG).A total of 23 members from five generations of a family were enrolled and underwent thorough ophthalmologic examinations. In addition, 200 unrelated healthy Chinese controls were also recruited as normal control. GLC1A gene was amplified by polymerase chain reaction, and DNA sequencing was performed to screen for mutations.Six members were diagnosed as POAG, with severe clinical manifestations, and history of high intraocular pressures. The mean age of disease onset was 26.3 years. However, the others were asymptomatic. In six affected and three asymptomatic members, gene sequencing revealed a mutation c.C1456T in exon 3 of myocilin gene (MYOC). Furthermore, we also identified a novel mutation c.G322A in beta-1,4-galactosyltransferase 3 (B4GALT3) gene in all six affected and three asymptomatic members, which was not reported previously in POAG patients. The two newly identified variants were absent in other family members as well as controls.The mutations c.1456C < T (p.L486F) in MYOC and c.322G < A (p.V108I) in B4GALT3 are likely responsible for the pathogenesis of POAG in this family.
| S-EPMC5146788 | biostudies-literature
Project description:The expression of recombinant proteins in Escherichia coli often leads to inactive aggregated proteins known as the inclusion bodies. To date, the best available tool has been the use of fusion tags, including the carbohydrate-binding protein; e.g., the maltose-binding protein (MBP) that enhances the solubility of recombinant proteins. However, none of these fusion tags work universally with every partner protein. We hypothesized that galectins, which are also carbohydrate-binding proteins, may help as fusion partners in folding the mammalian proteins in E. coli. Here we show for the first time that a small soluble lectin, human galectin-1, one member of a large galectin family, can function as a fusion partner to produce soluble folded recombinant human glycosyltransferase, beta-1,4-galactosyltransferase-7 (beta4Gal-T7), in E. coli. The enzyme beta4Gal-T7 transfers galactose to xylose during the synthesis of the tetrasaccharide linker sequence attached to a Ser residue of proteoglycans. Without a fusion partner, beta4Gal-T7 is expressed in E. coli as inclusion bodies. We have designed a new vector construct, pLgals1, from pET-23a that includes the sequence for human galectin-1, followed by the Tev protease cleavage site, a 6x His-coding sequence, and a multi-cloning site where a cloned gene is inserted. After lactose affinity column purification of galectin-1-beta4Gal-T7 fusion protein, the unique protease cleavage site allows the protein beta4Gal-T7 to be cleaved from galectin-1 that binds and elutes from UDP-agarose column. The eluted protein is enzymatically active, and shows CD spectra comparable to the folded beta4Gal-T1. The engineered galectin-1 vector could prove to be a valuable tool for expressing other proteins in E. coli.
| S-EPMC2859968 | biostudies-literature
Project description:Alpha-Gal Syndrome (AGS) is an IgE-mediated delayed-type hypersensitivity reaction to the oligosaccharide galactose-α-1, 3-galactose (α-gal) injected into humans from the lone-star tick (Amblyomma americanum) bite. Indeed, α-gal is discovered in salivary glands of lone-star tick; however, the tick's specific intrinsic factors involved in endogenous α-gal production and presentation to host during hematophagy are poorly understood. This study aimed to investigate the functional role of two tick enzymes, α-D-galactosidase (ADGal) and β-1,4 galactosyltransferases (β-1,4GalT), in endogenous α-gal production, carbohydrate metabolism, and N-glycan profile in lone-star tick. The ADGal enzyme cleaves terminal α-galactose moieties from glycoproteins and glycolipids, whereas β-1,4GalT transfers α-galactose to a β1,4 terminal linkage acceptor sugars-GlcNAc, Glc, and Xyl-in various processes of glycoconjugate synthesis. An RNA interference approach was utilized to silence ADGal and β-1,4GalT in Am. americanum to examine their function in α-gal metabolism in tick and AGS onset. Silencing of ADGal led to the significant downregulation of genes involved in galactose metabolism and transport in Am. americanum. Immunoblot and N-glycan analysis of the Am. americanum salivary glands showed a significant reduction in α-gal levels in silenced tissues. However, there was no significant difference in the level of α-gal in β-1,4GalT-silenced tick salivary glands. A basophil-activation test showed a decrease in the frequency of activated basophil by ADGal-silenced salivary glands. These results provide an insight into the roles of ADGal and β-1,4GalT in α-gal production and presentation in ticks and the probable involvement in the onset of AGS.
| S-EPMC8671611 | biostudies-literature
Project description:All Haemophilus ducreyi strains examined contain a lipooligosaccharide (LOS) consisting of a single but variable branch oligosaccharide that emanates off the first heptose (Hep-I) of a conserved Hep(3)-phosphorylated 3-deoxy-D-manno-octulosonic acid-lipid A core. In a previous report, identification of tandem genes, lbgA and lbgB, that are involved in LOS biosynthesis was described (Stevens et al., Infect. Immun. 65:651-660, 1997). In a separate study, the same gene cluster was identified and the lbgB (losB) gene was found to be required for transfer of the second sugar, D-glycero-D-manno-heptose (DD-Hep), of the major branch structure (Gibson et al., J. Bacteriol. 179:5062-5071, 1997). In this study, we identified the function of the neighboring upstream gene, lbgA, and found that it is necessary for addition of the third sugar in the dominant oligosaccharide branch, a galactose-linked beta1-->4, to the DD-Hep. LOS from an lbgA mutant and an lbgAB double mutant were isolated and were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, carbohydrate analysis, mass spectrometry, and nuclear magnetic resonance spectroscopy. The results showed that the mutant strains synthesize truncated LOS glycoforms that terminate after addition of the first glucose (lbgAB) or the disaccharide DD-Hepalpha1-->6Glcbeta1 (lbgA) that is attached to the heptose core. Both mutants show a significant reduction in the ability to adhere to human keratinocytes. Although minor differences were observed after two-dimensional gel electrophoresis of total proteins from the wild-type and mutant strains, the expression levels of the vast majority of proteins were unchanged, suggesting that the differences in adherence and invasion are due to differences in LOS. These studies add to the mounting evidence for a role of full-length LOS structures in the pathophysiology of H. ducreyi infection.
| S-EPMC128009 | biostudies-literature