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The Journal of Biological Chemistry Apr 2020Despite impressive progress made over the past 20 years in our understanding of mycolylarabinogalactan-peptidoglycan (mAGP) biogenesis, the mechanisms by which the...
Despite impressive progress made over the past 20 years in our understanding of mycolylarabinogalactan-peptidoglycan (mAGP) biogenesis, the mechanisms by which the tubercle bacillus adapts its cell wall structure and composition to various environmental conditions, especially during infection, remain poorly understood. Being the central portion of the mAGP complex, arabinogalactan (AG) is believed to be the constituent of the mycobacterial cell envelope that undergoes the least structural changes, but no reports exist supporting this assumption. Herein, using recombinantly expressed mycobacterial protein, bioinformatics analyses, and kinetic and biochemical assays, we demonstrate that the AG can be remodeled by a mycobacterial endogenous enzyme. In particular, we found that the mycobacterial GlfH1 (Rv3096) protein exhibits exo-β-d-galactofuranose hydrolase activity and is capable of hydrolyzing the galactan chain of AG by recurrent cleavage of the terminal β-(1,5) and β-(1,6)-Galf linkages. The characterization of this galactosidase represents a first step toward understanding the remodeling of mycobacterial AG.
Topics: Amino Acid Sequence; Amoeba; Galactans; Galactosyltransferases; Hydrolysis; Kinetics; Mycobacterium tuberculosis; Phylogeny; Sequence Homology
PubMed: 32107309
DOI: 10.1074/jbc.RA119.011817 -
The Journal of Biological Chemistry Jan 1991The region(s) of bovine galactosyltransferase that interacts with the lactose synthase regulatory protein alpha-lactalbumin was investigated using trace 3H acetylation...
The region(s) of bovine galactosyltransferase that interacts with the lactose synthase regulatory protein alpha-lactalbumin was investigated using trace 3H acetylation to probe the effects of alpha-lactalbumin on the reactivities of the individual amino groups of galactosyltransferase. In the presence of Mn2+, alpha-lactalbumin was found to reduce the reactivities of lysines 93 and 181 and to increase the reactivities of one or more of lysines 230, 237, and 241. The addition of N-acetylglucosamine (20 mM), which enhances complex formation between the two proteins, did not significantly alter the pattern of perturbation. These results indicate that the NH2-terminal region of the catalytic domain of galactosyltransferase, and possibly part of the proline-rich "stem" region, is affected by the association with alpha-lactalbumin and is therefore implicated in the binding of acceptor substrates. In a separate study only cysteines 176, 266, and 342 of galactosyltransferase were found to react with [3H]iodoacetic acid under denaturing conditions. From their lack of reactivity it is deduced that the remaining two cysteines, residues 134 and 247, are joined in a disulfide linkage. From these results and those of a previous study of UDP-galactose binding (Yadav, S., and Brew, K. (1990) J. Biol. Chem. 265, 14163-14169) it appears that the soluble form of galactosyltransferase is composed of two domains, the NH2-terminal 150 residues containing the Cys134-Cys247 disulfide bond, which functions in alpha-lactalbumin and acceptor binding, and the COOH-terminal region, which is involved in UDP-galactose binding.
Topics: Amino Acid Sequence; Amino Acids; Animals; Binding Sites; Cattle; Chromatography, High Pressure Liquid; Cysteine; Disulfides; Galactosyltransferases; Lactalbumin; Molecular Sequence Data; Structure-Activity Relationship; Sulfhydryl Compounds
PubMed: 1898734
DOI: No ID Found -
Journal of Biochemistry Jun 1998Galactosyltransferases are enzymes which transfer galactose from UDP-Gal to various acceptors with either retention of the anomeric configuration to form alpha1,2-,... (Review)
Review
Galactosyltransferases are enzymes which transfer galactose from UDP-Gal to various acceptors with either retention of the anomeric configuration to form alpha1,2-, alpha1,3-, alpha1,4-, and alpha1, 6-linkages, or inversion of the anomeric configuration to form beta1, 3-, beta1,4-, and beta1-ceramide linkages. During the last few years, several (c)DNA sequences coding for galactosyltransferases became available. We have retrieved these sequences and conducted sequence similarity studies. On the basis of both the nature of the reaction catalyzed and the protein sequence identity, these enzymes can be classified into twelve groups. Using a sensitive graphics method for protein comparison, conserved structural features were found in some of the galactosyltransferase groups, and other classes of glycosyltransferases, resulting in the definition of five families. The lengths and locations of the conserved regions as well as the invariant residues are described for each family. In addition, the DxD motif that may be important for substrate recognition and/or catalysis is demonstrated to occur in all families but one.
Topics: Amino Acid Sequence; Animals; Bacteria; DNA, Complementary; Galactosyltransferases; Humans; Molecular Sequence Data; Protein Conformation; Saccharomyces cerevisiae; Sequence Alignment; Sequence Analysis; Structure-Activity Relationship; Substrate Specificity
PubMed: 9603985
DOI: 10.1093/oxfordjournals.jbchem.a022035 -
Glycobiology Jun 2009Poly-N-acetyllactosamine (polyLacNAc) is a linear carbohydrate polymer composed of alternating N-acetylglucosamine and galactose residues involved in cellular functions...
Association of beta-1,3-N-acetylglucosaminyltransferase 1 and beta-1,4-galactosyltransferase 1, trans-Golgi enzymes involved in coupled poly-N-acetyllactosamine synthesis.
Poly-N-acetyllactosamine (polyLacNAc) is a linear carbohydrate polymer composed of alternating N-acetylglucosamine and galactose residues involved in cellular functions ranging from differentiation to metastasis. PolyLacNAc also serves as a scaffold on which other oligosaccharides such as sialyl Lewis X are displayed. The polymerization of the alternating N-acetylglucosamine and galactose residues is catalyzed by the successive action of UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 1 (B3GNT1) and UDP-Gal:betaGlcNAc beta-1,4-galactosyltransferase, polypeptide 1 (B4GALT1), respectively. The functional association between these two glycosyltransferases led us to investigate whether the enzymes also associate physically. We show that B3GNT1 and B4GALT1 colocalize by immunofluorescence microscopy, interact by coimmunoprecipitation, and affect each other's subcellular localization when one of the two proteins is artificially retained in the endoplasmic reticulum. These results demonstrate that B3GNT1 and B4GALT1 physically associate in vitro and in cultured cells, providing insight into possible mechanisms for regulation of polyLacNAc production.
Topics: Animals; Galactosyltransferases; HeLa Cells; Humans; N-Acetylglucosaminyltransferases; Polysaccharides; Protein Transport; trans-Golgi Network
PubMed: 19261593
DOI: 10.1093/glycob/cwp035 -
Frontiers in Cellular and Infection... 2021Alpha-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...
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 () 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 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 . Immunoblot and N-glycan analysis of the 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.
Topics: Amblyomma; Animals; Food Hypersensitivity; Galactosidases; Galactosyltransferases; Humans; Immunoglobulin E; alpha-Galactosidase
PubMed: 34926322
DOI: 10.3389/fcimb.2021.775371 -
Gastroenterology Nov 2018Pancreatic ductal adenocarcinomas (PDACs) produce higher levels of truncated O-glycan structures (such as Tn and sTn) than normal pancreata. Dysregulated activity of...
BACKGROUND & AIMS
Pancreatic ductal adenocarcinomas (PDACs) produce higher levels of truncated O-glycan structures (such as Tn and sTn) than normal pancreata. Dysregulated activity of core 1 synthase glycoprotein-N-acetylgalactosamine 3-β-galactosyltransferase 1 (C1GALT1) leads to increased expression of these truncated O-glycans. We investigated whether and how truncated O-glycans contributes to the development and progression of PDAC using mice with disruption of C1galt1.
METHODS
We crossed C1galt1 floxed mice (C1galt1) with Kras; Trp53; Pdx1-Cre (KPC) mice to create KPCC mice. Growth and progression of pancreatic tumors were compared between KPC and KPCC mice; pancreatic tissues were collected and analyzed by immunohistochemistry; immunofluorescence; and Sirius red, alcian blue, and lectin staining. We used the CRISPR/Cas9 system to disrupt C1GALT1 in human PDAC cells (T3M4 and CD18/HPAF) and levels of O-glycans were analyzed by lectin blotting, mass spectrometry, and lectin pulldown assay. Orthotopic studies and RNA sequencing analyses were performed with control and C1GALT1 knockout PDAC cells. C1GALT1 expression was analyzed in well-differentiated (n = 36) and poorly differentiated (n = 23) PDAC samples by immunohistochemistry.
RESULTS
KPCC mice had significantly shorter survival times (median 102 days) than KPC mice (median 200 days) and developed early pancreatic intraepithelial neoplasias at 3 weeks, PDAC at 5 weeks, and metastasis at 10 weeks compared with KPC mice. Pancreatic tumors that developed in KPCC mice were more aggressive (more invasive and metastases) than those in KPC mice, had a decreased amount of stroma, and had increased production of Tn. Poorly differentiated PDAC specimens had significantly lower levels of C1GALT1 than well-differentiated PDACs. Human PDAC cells with knockout of C1GALT1 had aberrant glycosylation of MUC16 compared with control cells and increased expression of genes that regulate tumorigenesis and metastasis.
CONCLUSIONS
In studies of KPC mice with disruption of C1galt1, we found that loss of C1galt1 promotes development of aggressive PDACs and increased metastasis. Knockout of C1galt1 leads to increased tumorigenicity and truncation of O-glycosylation on MUC16, which could contribute to increased aggressiveness.
Topics: Adenocarcinoma; Animals; CRISPR-Cas Systems; Carcinoma, Pancreatic Ductal; Cell Proliferation; Galactosyltransferases; Glycosylation; Humans; Mice; Mice, Inbred C57BL; Pancreatic Neoplasms
PubMed: 30086262
DOI: 10.1053/j.gastro.2018.08.007 -
Transplant International : Official... Feb 2007The production of alpha1,3-galactosyltransferase gene-knockout (GT-KO) pigs has overcome the barrier of preformed anti-Galalpha1,3Gal (Gal) antibodies that has inhibited... (Review)
Review
The production of alpha1,3-galactosyltransferase gene-knockout (GT-KO) pigs has overcome the barrier of preformed anti-Galalpha1,3Gal (Gal) antibodies that has inhibited progress in pig-to-primate organ xenotransplantation for many years. Survival of GT-KO pig organs in nonhuman primates is currently limited by the development of a thrombotic microangiopathy that results in increasing ischemic injury of the transplanted organ over weeks or months. Potential causative factors include vascular endothelial activation from preformed anti-nonGal antibodies or cells of the innate immune system that recognize nonGal pig antigens directly, and coagulation dysregulation associated with molecular incompatibilities between pig and primate. Carefully isolated pancreatic islets from wild-type (genetically unmodified) adult pigs express minimal Gal epitopes, allowing survival sometimes for weeks or months after transplantation into nonhuman primates receiving immunosuppression directed only at T-cell function. However, there is a considerable immediate loss of islets, probably related to activation of coagulation and complement cascades. Further genetic manipulation of organ-source pigs is therefore required to overcome these problems. GT-KO pigs expressing a human complement-regulatory protein, e.g. decay-accelerating factor, and/or an 'anti-coagulant' gene, e.g. human tissue factor pathway inhibitor, might prevent the change in vascular endothelium from an anti-coagulant to a procoagulant phenotype, and protect the islets from early loss.
Topics: Animals; Animals, Genetically Modified; Antibodies; Blood Coagulation Disorders; Galactosyltransferases; Genetic Engineering; Graft Rejection; Humans; Immunity, Innate; Islets of Langerhans Transplantation; Swine; Transplantation, Heterologous
PubMed: 17239018
DOI: 10.1111/j.1432-2277.2006.00398.x -
Molecular & Cellular Proteomics : MCP Aug 2023B4GALT1 encodes β-1,4-galactosyltransferase 1, an enzyme that plays a major role in glycan synthesis in the Golgi apparatus by catalyzing the addition of terminal...
B4GALT1 encodes β-1,4-galactosyltransferase 1, an enzyme that plays a major role in glycan synthesis in the Golgi apparatus by catalyzing the addition of terminal galactose. Studies increasingly suggest that B4GALT1 may be involved in the regulation of lipid metabolism pathways. Recently, we discovered a single-site missense variant Asn352Ser (N352S) in the functional domain of B4GALT1 in an Amish population, which decreases the level of LDL-cholesterol (LDL-c) as well as the protein levels of ApoB, fibrinogen, and IgG in the blood. To systematically evaluate the effects of this missense variant on protein glycosylation, expression, and secretion, we developed a nano-LC-MS/MS-based platform combined with TMT-labeling for in-depth quantitative proteomic and glycoproteomic analyses in the plasma of individuals homozygous for the B4GALT1 missense variant N352S versus non-carriers (n = 5 per genotype). A total of 488 secreted proteins in the plasma were identified and quantified, 34 of which showed significant fold changes in protein levels between N352S homozygotes and non-carriers. We determined N-glycosylation profiles from 370 glycosylation sites in 151 glycoproteins and identified ten proteins most significantly associated with decreased galactosylation and sialyation in B4GALT1 N352S homozygotes. These results further support that B4GALT1 N352S alters the glycosylation profiles of a variety of critical target proteins, thus governing the functions of these proteins in multiple pathways, such as those involved in lipid metabolism, coagulation, and the immune response.
Topics: Humans; Amish; Galactosyltransferases; Glycosylation; Proteomics; Tandem Mass Spectrometry
PubMed: 37328064
DOI: 10.1016/j.mcpro.2023.100595 -
Breast Cancer Research : BCR Jan 2021Existence of breast cancer stem cells (BCSCs) is implicated in disease relapse, metastasis, and resistance of treatment. β1,3-Galactosyltransferase 5 (B3GALT5) has been...
BACKGROUND
Existence of breast cancer stem cells (BCSCs) is implicated in disease relapse, metastasis, and resistance of treatment. β1,3-Galactosyltransferase 5 (B3GALT5) has been shown to be a pro-survival marker for BCSCs. However, little is known about the prognostic significance of B3GALT5 in breast cancer.
METHODS
Paired tissues (tumor part and adjacent non-tumor part) from a cohort of 202 women with breast cancer were used to determine the expression levels of B3GALT5 mRNA by qRT-PCR. Kaplan-Meier and multivariable Cox proportional hazard models were used to assess survival differences in terms of relapse-free survival (RFS) and overall survival (OS). Both breast cancer cells and cancer stem cells (BCSCs) were used to see the in vitro effects of knockdown or overexpression of B3GALT5 on cell migration, invasion, and epithelial-to-mesenchymal transition (EMT). A patient-derived xenograft (PDX) model was used to see the in vivo effects of knockdown of B3GALT5 in BCSCs on tumor growth and metastasis.
RESULTS
Higher expression of B3GALT5 in 202 breast cancer tissues, especially in adjacent non-tumor tissue, correlated with poor clinical outcomes including shorter OS and RFS in all patients, especially those with early stage breast cancer. In vitro studies showed B3GALT5 could enhance cell migration, invasion, mammosphere formation, and EMT. Of note, B3GALT5 upregulated the expression of β-catenin and EMT activator zinc finger E-box binding homeobox 1 (ZEB1) pathway in BCSCs. In vivo studies showed B3GALT5 expression in BCSCs is critical for not only tumor growth but also lymph node and lung metastasis in PDX mice.
CONCLUSION
Our results demonstrated the value of B3GALT5 as a prognostic marker of breast cancer, especially among the early stage patients, and its crucial roles in regulating EMT, cell migration, and stemness thereby promoting breast cancer progression.
Topics: Animals; Biomarkers, Tumor; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Disease Models, Animal; Disease Progression; Epithelial-Mesenchymal Transition; Female; Galactosyltransferases; Gene Expression; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Mice; Neoplasm Metastasis; Prognosis; Proportional Hazards Models; RNA Interference
PubMed: 33413566
DOI: 10.1186/s13058-020-01381-9 -
The Biochemical Journal Oct 1986Galactosyltransferase (EC 2.4.1.22) requires bivalent metal ions for its activity. However, preparations of this enzyme solubilized from Golgi membranes of lactating rat...
Galactosyltransferase (EC 2.4.1.22) requires bivalent metal ions for its activity. However, preparations of this enzyme solubilized from Golgi membranes of lactating rat mammary gland were shown to be activated not only by Mn2+, Ca2+ and Mg2+, but also by spermine, spermidine, lysyl-lysine, ethylenediamine and other diaminoalkanes, and by a range of basic proteins and peptides, including clupeine, histone, polylysine, ribonuclease, pancreatic trypsin inhibitor, cytochrome c, melittin, avidin and myelin basic protein. Both N-acetyl-lactosamine synthetase and lactose synthetase activities were enhanced. A basic protein fraction was isolated from bovine milk and shown to activate galactosyltransferase at low concentrations. The polyanions ATP, casein, chondroitin sulphate and heparin reversed the activation of galactosyltransferase by several of the above substances. Galactosyltransferase, assayed as a lactose synthetase, showed a 10-fold greater affinity for glucose when Mn2+ ions were replaced by clupeine or by ribonuclease as cationic activator. Evidence was obtained for the presence of an endogenous cationic activator in solubilized Golgi membrane preparations which evoked a similar low apparent Km,glucose. The findings are discussed in the light of cationic activations of glycosyltransferases generally, of the porous nature of the Golgi membrane, and of the unlikelihood of bivalent metal ions being the physiological activators of galactosyltransferase. It is suggested that the natural cationic activator of lactose synthetase may be a secretory protein acting in a manner analogous to the enzyme's activation by alpha-lactalbumin. A scheme is proposed for the two-stage synthesis of lactose and phosphorylation of casein within the cell, to accommodate the apparent incompatibility of these two processes.
Topics: Animals; Cations, Divalent; Enzyme Activation; Female; Galactosyltransferases; Glucose; Golgi Apparatus; Lactation; Lactose Synthase; Mammary Glands, Animal; Peptides; Polyamines; Pregnancy; Proteins; Rats; Rats, Inbred Strains
PubMed: 3101666
DOI: 10.1042/bj2390423