RESUMEN
Xyloside analogues with substitution of the endocyclic oxygen atom by sulfur or carbon were investigated as substrates for ß-1,4-galactosyltransferaseâ 7 (ß4GalT7), a key enzyme in the biosynthesis of glycosaminoglycan chains. The analogues with an endocyclic sulfur atom proved to be excellent substrates for ß4GalT7, and were galactosylated approximately fifteen times more efficiently than the corresponding xyloside. The 5a-carba-ß-xylopyranoside in the d-configuration proved to be a good substrate for ß4GalT7, whereas the enantiomer in the l-configuration showed no activity. Further investigations by X-ray crystallography, NMR spectroscopy, and molecular modeling provided a rationale for the pronounced activity of the sulfur analogues. Favorable π-π interactions between the 2-naphthyl moiety and a tyrosine side chain of the enzyme were observed for the thio analogues, which open up for the design of efficient GAG primers and inhibitors.
Asunto(s)
N-Acetil-Lactosamina Sintasa/metabolismo , Compuestos de Sulfhidrilo/química , Xilosa/análogos & derivados , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Cinética , Conformación Molecular , Simulación del Acoplamiento Molecular , N-Acetil-Lactosamina Sintasa/química , Resonancia Magnética Nuclear Biomolecular , Teoría Cuántica , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Especificidad por Sustrato , Compuestos de Sulfhidrilo/metabolismo , Xilosa/metabolismoRESUMEN
We show that the thermodynamics of metal ion-induced conformational changes aid to understand the functions of protein complexes. This is illustrated in the case of a metalloprotein, alpha-lactalbumin (aLA), a divalent metal ion binding protein. We use the histograms of dihedral angles of the protein, generated from all-atom molecular dynamics simulations, to calculate conformational thermodynamics. The thermodynamically destabilized and disordered residues in different conformational states of a protein are proposed to serve as binding sites for ligands. This is tested for ß-1,4-galactosyltransferase (ß4GalT) binding to the Ca(2+)-aLA complex, in which the binding residues are known. Among the binding residues, the C-terminal residues like aspartate (D) 116, glutamine (Q) 117, tryptophan (W) 118 and leucine (L) 119 are destabilized and disordered and can dock ß4GalT onto Ca(2+)-aLA. No such thermodynamically favourable binding residues can be identified in the case of the Mg(2+)-aLA complex. We apply similar analysis to oleic acid binding and predict that the Ca(2+)-aLA complex can bind to oleic acid through the basic histidine (H) 32 of the A2 helix and the hydrophobic residues, namely, isoleucine (I) 59, W60 and I95, of the interfacial cleft. However, the number of destabilized and disordered residues in Mg(2+)-aLA are few, and hence, the oleic acid binding to Mg(2+)-bound aLA is less stable than that to the Ca(2+)-aLA complex. Our analysis can be generalized to understand the functionality of other ligand bound proteins.
Asunto(s)
Lactalbúmina/química , Termodinámica , Animales , Sitios de Unión , Bovinos , Ácidos Grasos/química , Ligandos , Simulación de Dinámica Molecular , N-Acetil-Lactosamina Sintasa/química , Unión Proteica , Conformación ProteicaRESUMEN
The aim of this article is to present a unique in vitro folding technique for glycosyltransferases to generate active proteins that can be used for X-ray crystallographic and bioconjugation protocols. Although a number of in vitro refolding methods are available, ß1,4galactosyltransferases in large quantities can be made using the current protocol only. This technique is not only limited to glycosyltransferases alone but has been successfully used to refold single-chain antibodies and other molecules. Although this in vitro folding method is quite similar to other methods, it differs from them by the use of S-sulfonation of the inclusion bodies before setting up the in vitro refolding of the protein.
Asunto(s)
Cuerpos de Inclusión/química , N-Acetilgalactosaminiltransferasas/química , N-Acetil-Lactosamina Sintasa/química , Replegamiento Proteico , Animales , Electroforesis en Gel de Poliacrilamida/métodos , Humanos , N-Acetil-Lactosamina Sintasa/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Sulfonas/química , Polipéptido N-AcetilgalactosaminiltransferasaAsunto(s)
Fucosiltransferasas/química , N-Acetil-Lactosamina Sintasa/química , Péptidos/química , Secuencia de Aminoácidos , Membrana Celular/química , Membrana Celular/metabolismo , Dicroismo Circular , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Fucosiltransferasas/metabolismo , Helicobacter pylori/enzimología , Humanos , Datos de Secuencia Molecular , N-Acetil-Lactosamina Sintasa/genética , N-Acetil-Lactosamina Sintasa/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
A novel, inexpensive and versatile ionic-liquid-based catch and release mass spectrometry tag (I-Tag) that facilitates substrate purification, fast, robust and sensitive enzymatic reaction monitoring and quantitative kinetic analysis has been developed. The applicability of the system has been demonstrated in an enzymatic assay with ß-1,4-galactosyltransferase.
Asunto(s)
Líquidos Iónicos/química , N-Acetil-Lactosamina Sintasa/química , Cromatografía Liquida , Cinética , Espectrometría de Masas , N-Acetil-Lactosamina Sintasa/metabolismoRESUMEN
Cell surface glycans play important cellular functions and are synthesized by glycosyltransferases. Structure and function studies show that the donor sugar specificity of the invertebrate ß1,4-N-acetyl-glactosaminyltransferase (ß4GalNAc-T) and the vertebrate ß1,4-galactosyltransferase I (ß4Gal-T1) are related by a single amino acid residue change. Comparison of the catalytic domain crystal structures of the ß4Gal-T1 and the α-polypeptidyl-GalNAc-T (αppGalNAc-T) shows that their protein structure and sequences are similar. Therefore, it seems that the invertebrate ß4GalNAc-T and the catalytic domain of αppGalNAc-T might have emerged from a common primordial gene. When vertebrates emerged from invertebrates, the amino acid that determines the donor sugar specificity of the invertebrate ß4GalNAc-T might have mutated, thus converting the enzyme to a ß4Gal-T1 in vertebrates.
Asunto(s)
Evolución Molecular , Glicosiltransferasas/química , Glicosiltransferasas/metabolismo , Invertebrados/enzimología , Vertebrados , Secuencia de Aminoácidos , Animales , Glicosiltransferasas/genética , Humanos , Invertebrados/genética , Datos de Secuencia Molecular , N-Acetilgalactosaminiltransferasas/química , N-Acetilgalactosaminiltransferasas/genética , N-Acetilgalactosaminiltransferasas/metabolismo , N-Acetil-Lactosamina Sintasa/química , N-Acetil-Lactosamina Sintasa/genética , N-Acetil-Lactosamina Sintasa/metabolismo , Vertebrados/genética , Polipéptido N-AcetilgalactosaminiltransferasaRESUMEN
Two bacterial beta1-4-galactosyltransferases, NmLgtB and Hp1-4GalT, exhibit promiscuous and complementary acceptor substrate specificity. They have been used in an efficient one-pot multienzyme system to synthesize LacNAc, lactose, and their derivatives including those containing negatively charged 6-O-sulfated GlcNAc and C2-substituted GlcNAc or Glc, from monosaccharide derivatives and inexpensive Glc-1-P.
Asunto(s)
Galactósidos/síntesis química , Helicobacter pylori/enzimología , N-Acetil-Lactosamina Sintasa/química , Neisseria meningitidis/enzimología , Pasteurella multocida/enzimología , Proteínas Recombinantes de Fusión/química , Conformación de Carbohidratos , Secuencia de Carbohidratos , Clonación Molecular , Escherichia coli/genética , Galactósidos/química , N-Acetil-Lactosamina Sintasa/genética , Proteínas Recombinantes de Fusión/genética , Especificidad por SustratoRESUMEN
The beta1,4-galactosyltransferase-7 (beta4Gal-T7) enzyme, one of seven members of the beta4Gal-T family, transfers in the presence of manganese Gal from UDP-Gal to an acceptor sugar (xylose) that is attached to a side chain hydroxyl group of Ser/Thr residues of proteoglycan proteins. It exhibits the least protein sequence similarity with the other family members, including the well studied family member beta4Gal-T1, which, in the presence of manganese, transfers Gal from UDP-Gal to GlcNAc. We report here the crystal structure of the catalytic domain of beta4Gal-T7 from Drosophila in the presence of manganese and UDP at 1.81 A resolution. In the crystal structure, a new manganese ion-binding motif (HXH) has been observed. Superposition of the crystal structures of beta4Gal-T7 and beta4Gal-T1 shows that the catalytic pocket and the substrate-binding sites in these proteins are similar. Compared with GlcNAc, xylose has a hydroxyl group (instead of an N-acetyl group) at C2 and lacks the CH(2)OH group at C5; thus, these protein structures show significant differences in their acceptor-binding site. Modeling of xylose in the acceptor-binding site of the beta4Gal-T7 crystal structure shows that the aromatic side chain of Tyr(177) interacts strongly with the C5 atom of xylose, causing steric hindrance to any additional group at C5. Because Drosophila Cd7 has a 73% protein sequence similarity to human Cd7, the present crystal structure offers a structure-based explanation for the mutations in human Cd7 that have been linked to Ehlers-Danlos syndrome.
Asunto(s)
N-Acetil-Lactosamina Sintasa/química , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Cristalografía por Rayos X , Drosophila melanogaster , Humanos , Manganeso/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , N-Acetil-Lactosamina Sintasa/genética , N-Acetil-Lactosamina Sintasa/metabolismo , Unión Proteica , Conformación Proteica , Homología de Secuencia de Aminoácido , Uridina Difosfato/metabolismoRESUMEN
Recombinant glycosyltransferases are potential biocatalysts for the construction of a compound library of oligosaccharides, glycosphingolipids, glycopeptides, and various artificial glycoconjugates on the basis of combined chemical and enzymatic synthetic procedures. The structurally defined glycan-related compound library is a key resource both in the basic studies of their functional roles in various biological processes and in the discovery research of new diagnostic biomarkers and therapeutic reagents. Therefore, it is clear that the immobilization of extremely unstable membrane-bound glycosyltransferases on some suitable supporting materials should enhance the operational stability and activity of recombinant enzymes and makes facile separation of products and recycling use of enzymes possible. Until now, however, it seems that no standardized protocol preventing a significant loss of enzyme activity is available due to the lack of a general method of site-selective anchoring between glycosyltransferases and scaffold materials through a stable covalent bond. Here we communicate a versatile and efficient method for the immobilization of recombinant glycosyltransferases onto commercially available solid supports by means of transpeptidase reaction by Staphylococcus aureus sortase A. This protocol allowed for the first time highly specific conjugation at the designated C-terminal signal peptide moiety of recombinant human beta1,4-galactosyltransferase or recombinant Helicobacter pylori alpha1,3-fucosyltransferase with simple aliphatic amino groups displayed on the surface of solid materials. Site-specifically immobilized enzymes exhibited the desired sugar transfer activity, an improved stability, and a practical reusability required for rapid and large-scale synthesis of glycoconjugates. Considering that most mammalian enzymes responsible for the posttranslational modifications, including the protein kinase family, as well as glycosyltransferases are unstable and highly oriented membrane proteins, the merit of our strategy based on "site-specific" transpeptidation is evident because the reaction proceeds only at an engineered C-terminus without any conformational influence around the active sites of both enzymes as well as heptad repeats of rHFucT required to maintain native secondary and quaternary structures during the dimerization on cell surfaces.
Asunto(s)
Aminoaciltransferasas/metabolismo , Proteínas Bacterianas/metabolismo , Cisteína Endopeptidasas/metabolismo , Enzimas Inmovilizadas/metabolismo , Glicosiltransferasas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Recombinantes/metabolismo , Staphylococcus aureus/enzimología , Aminas/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Enzimas Inmovilizadas/química , Fucosiltransferasas/química , Fucosiltransferasas/metabolismo , Glicosiltransferasas/química , Helicobacter pylori/enzimología , Humanos , Antígeno Lewis X/biosíntesis , Antígeno Lewis X/química , Proteínas de la Membrana/química , Modelos Moleculares , N-Acetil-Lactosamina Sintasa/química , N-Acetil-Lactosamina Sintasa/metabolismo , Conformación Proteica , Estabilidad Proteica , Proteínas Recombinantes/química , Sefarosa/química , Sefarosa/metabolismo , Especificidad por SustratoRESUMEN
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.
Asunto(s)
N-Acetil-Lactosamina Sintasa/metabolismo , Conformación Proteica , Sitios de Unión , Humanos , Modelos Moleculares , Mutación , N-Acetil-Lactosamina Sintasa/químicaRESUMEN
Placing an 2-nitrobenzyl group on O-6 of the galactosyl residue in uridine-5'-diphosphogalactose (UDP-Gal) gives 6''-O-2-nitrobenzyl-UDP-Gal that is shown to be inactive as a donor substrate for beta-(1-->4)-galactosyltransferase (GalT). On irradiation at 365 nm, the nitrobenzyl group is completely removed yielding native UDP-Gal that then transfers normally to produce the expected betaGal-(1-->4)-betaGlcNAc disaccharidic linkage. 6''-O-2-Nitrobenzyl-UDP-Gal thus fulfils the minimum requirements of a 'caged' UDP-Gal for application in time-resolved crystallographic studies of beta-(1-->4)-GalT.
Asunto(s)
Nitrobencenos/química , Fotólisis , Uridina Difosfato Galactosa/química , Azúcares de Uridina Difosfato/química , Acetilglucosamina/química , Amino Azúcares/química , Estructura Molecular , N-Acetil-Lactosamina Sintasa/química , Nitrobencenos/síntesis química , Nitrobencenos/efectos de la radiación , Espectrometría de Masa por Ionización de Electrospray , Rayos Ultravioleta , Uridina Difosfato Galactosa/síntesis química , Uridina Difosfato Galactosa/efectos de la radiación , Azúcares de Uridina Difosfato/síntesis químicaRESUMEN
Glycosyltransferases are an enormous and diverse class of enzyme encompassing 1% of all sequenced genomes. They catalyze the transfer of a monosaccharide from an activated donor such as a sugar-nucleotide to an acceptor molecule. Though the primary sequences of glycosyltransferases have little homology, X-ray structural studies on glycosyltransferases have revealed that there are two main folds and that the orientation of the sugar donors with respect to the folds is highly conserved. It seems that glycosyltransferases have evolved diversified specificities toward donor sugars by changing the amino acids around the monosaccharide moiety without altering the orientation of the nucleotide moiety. In this study, we designed new glycosyltransferases with altered donor specificities by use of a novel empirical model called the Epimer Propensity Index (EPI). The EPI was constructed using 221 carbohydrate-protein complex structures in the Protein Data Bank with either galactose or glucose in the complex. The blood type B synthesizing glycosyltransferase GTB, a galactosyltransferase was our target enzyme. Two GTB mutants designed to exhibit enhanced glucosyltransferase activity were cloned, expressed and characterized experimentally. The predicted GTB mutants, Ser185Asn and Ser185Cys, exhibited 4.3- and 4.8-fold elevations in k(cat)/K(m) for UDP-Glc relative to that of wild-type enzyme.
Asunto(s)
Glucosa/metabolismo , N-Acetil-Lactosamina Sintasa/química , N-Acetil-Lactosamina Sintasa/metabolismo , Ingeniería de Proteínas/métodos , Sustitución de Aminoácidos , Conformación de Carbohidratos , Simulación por Computador , Bases de Datos de Proteínas , Glucosa/química , Humanos , Modelos Moleculares , N-Acetil-Lactosamina Sintasa/genética , Especificidad por SustratoRESUMEN
Our understanding of how complex carbohydrates function during embryonic development is still very limited, primarily due to the large number of glycosyltransferases now known to be involved in their synthesis. To overcome these limitations, we have taken advantage of the zebrafish system to analyze the function of complex carbohydrates during development by down-regulating the expression of specific glycosyltransferases. Herein, we report the identification of the zebrafish ortholog of mammalian beta1,4-galactosyltransferase I, beta4GalT1, and its requirement for proper convergent extension movements during gastrulation. beta4GalT1 is expressed in the oocyte and throughout the embryo during the first 24 h of development. Knockdown of zebrafish beta4GalT1 by two independent morpholino oligonucleotides results in embryos with a truncated anterior-posterior axis, as well as elongated somites and moderate defects in the patterning of the head mesenchyme. Co-injection of zebrafish beta4GalT1 mRNA returns galactosyltransferase activity to control levels and rescues the defects produced by morpholino oligonucleotides. In situ hybridizations of various molecular markers reveal that the axial mesoderm of epiboly stage embryos is abnormally widened in beta4GalT1 morphants, indicative of abnormal convergent extension. Consistent with this, the rate of anterior-posterior axis elongation is reduced relative to control-injected embryos, similar to that seen in known convergent extension mutants. Among the many potential substrates for beta4GalT1 is laminin, a principle component of the extracellular matrix that supports cell movements such as those that occur during convergent extension. Previous in vitro studies have shown that the galactosylation status of laminin directly influences its ability to support cell spreading and migration. In this regard, laminin isolated from beta4GalT1 morphant embryos is poorly galactosylated, which may contribute to defective cell migration during convergent extension movements. This work demonstrates that zebrafish can be used to identify critical developmental roles for specific glycosyltransferases that would not be obvious otherwise, such as an absolute requirement for beta4GalT1 during convergent extension movements.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , N-Acetil-Lactosamina Sintasa/fisiología , Secuencia de Aminoácidos , Animales , Matriz Extracelular/metabolismo , Galactosa/metabolismo , Humanos , Laminina/metabolismo , Modelos Genéticos , Datos de Secuencia Molecular , Mutación , N-Acetil-Lactosamina Sintasa/química , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Pez CebraRESUMEN
A simple and efficient assay for glycosyltransferase activity on gold colloidal nanoparticles (GCNPs) by using laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF MS) is demonstrated by the enzymatic synthesis of the Lewis X trisaccharide on GCNPs containing GlcNAc residues. GCNPs containing multivalent sugars were well dispersed in aqueous solution and proved to be excellent acceptor substrates for the glycosyltransferase reaction. Direct LDI-TOF MS analysis of these GCNPs provided the ion peaks of the sugar derivatives, chemisorbed through S--Au linkages onto the GCNPs, even in the presence of contaminants such as proteins and salts. Thus, it enabled the rapid and direct detection of the enzymatic reaction on the GCNPs by subjecting a small amount (0.15 muL) of the reaction mixture to MS analysis without purification. Subsequent MS/MS analyses (LDI-LIFT-TOF/TOF method) of the product-carrying GCNPs enabled the structures of the sugar derivatives that had been constructed on the GCNPs by enzymatic glycosylation to be determined. A quantitative inhibition assay for glycosyltransferase by using LDI-TOF MS analysis on the GCNPs was demonstrated by using uridine 5'-diphosphate (UDP) as the inhibitor. This simple assay was then applied to the detection of the enzymatic activity of a crude cell extract of Escherichia coli, which produces Neisseria meningitidis beta-1,4-galactosyltransferase (beta-1,4-GalT). In this case, the GCNPs were roughly purified by means of ultrafiltration to remove the buffer and detergents before MS analysis. That the GCNPs are dissolved in solution in the reaction medium but are solid in the purification process is greatly advantageous for the simple and efficient detection of enzymatic activity in crude biological samples. Thus, GCNPs containing a variety of biomolecules may become a versatile and efficient tool for the rapid and direct monitoring of metabolism (metabolomics) in living cells when combined with LDI-TOF MS analysis.
Asunto(s)
Glicosiltransferasas/metabolismo , Oro/química , Nanopartículas del Metal/química , Trisacáridos/biosíntesis , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Adsorción , Secuencia de Carbohidratos , Coloides , Fucosiltransferasas/química , Fucosiltransferasas/metabolismo , Glicosiltransferasas/química , Humanos , Antígeno Lewis X/análogos & derivados , Datos de Secuencia Molecular , N-Acetil-Lactosamina Sintasa/química , N-Acetil-Lactosamina Sintasa/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Trisacáridos/análisis , Trisacáridos/químicaRESUMEN
The MUC1 mucin is an important tumor-associated antigen that shows extensive glycosylation in vivo. The O-glycosylation of this molecule, which has been well characterized in many cell types and tissues, is important in conferring the unusual biochemical and biophysical properties on a mucin. N-Glycosylation is crucial to the folding, sorting, membrane trafficking, and secretion of many proteins. Here, we evaluated the N-glycosylation of MUC1 derived from two sources: endogenous MUC1 isolated from human milk and a recombinant epitope-tagged MUC1F overexpressed in Caco2 colon carcinoma cells. N-Glycans on purified MUC1F/MUC1 were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), gas chromatography-mass spectrometry (GC-MS), and CAD-ESI-MS/MS. The spectra indicate that MUC1F N-glycans have compositions consistent with high-mannose structures (Hex(5-9)HexNAc(2)) and complex/hybrid-type glycans (NeuAc(0-3)Fuc(0-3)Hex(3-8)HexNAc(3-7)). Many of the N-glycan structures are identical on MUC1F and native MUC1; however, a marked difference is seen between the N-glycans on membrane-bound and secreted forms of the native molecule.
Asunto(s)
Células Epiteliales/metabolismo , Mucina-1/química , Mucina-1/metabolismo , Polisacáridos/análisis , Células CACO-2 , Secuencia de Carbohidratos , Cromatografía de Gases y Espectrometría de Masas , Glicosilación , Humanos , Manosa/química , Leche Humana/química , Datos de Secuencia Molecular , Mucina-1/genética , N-Acetil-Lactosamina Sintasa/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , beta-Galactosidasa/químicaRESUMEN
During the catalytic cycle of beta1,4-galactosyltransferase-1 (Gal-T1), upon the binding of Mn(2+) followed by UDP-Gal, two flexible loops, a long and a short loop, change their conformation from open to closed. We have determined the crystal structures of a human M340H-Gal-T1 mutant in the open conformation (apo-enzyme), its Mn(2+) and Mn(2+)-UDP-Gal-bound complexes, and of a pentenary complex of bovine Gal-T1-Mn(2+)-UDP-GalNAc-Glc-alpha-lactalbumin. These studies show that during the conformational changes in Gal-T1, the coordination of Mn(2+) undergoes significant changes. It loses a coordination bond with a water molecule bound in the open conformation of Gal-T1 while forming a new coordination bond with another water molecule in the closed conformation, creating an active ground-state structure that facilitates enzyme catalysis. In the crystal structure of the pentenary complex, the N-acetylglucosamine (GlcNAc) moiety is found cleaved from UDP-GalNAc and is placed 2.7A away from the O4 oxygen atom of the acceptor Glc molecule, yet to form the product. The anomeric C1 atom of the cleaved GalNAc moiety has only two covalent bonds with its non-hydrogen atoms (O5 and C2 atoms), similar to either an oxocarbenium ion or N-acetylgalactal form, which are crystallographically indistinguishable at the present resolution. The structure also shows that the newly formed, metal-coordinating water molecule forms a hydrogen bond with the beta-phosphate group of the cleaved UDP moiety. This hydrogen bond formation results in the rotation of the beta-phosphate group of UDP away from the cleaved GalNAc moiety, thereby preventing the re-formation of the UDP-sugar during catalysis. Therefore, this water molecule plays an important role during catalysis in ensuring that the catalytic reaction proceeds in a forward direction.
Asunto(s)
N-Acetil-Lactosamina Sintasa/química , N-Acetil-Lactosamina Sintasa/metabolismo , Conformación Proteica , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Animales , Dominio Catalítico , Bovinos , Cristalografía por Rayos X , Galactosa/análisis , Humanos , Manganeso/química , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , N-Acetil-Lactosamina Sintasa/genética , Uridina Difosfato/análisis , Agua/químicaRESUMEN
Beta-1,4-galactosyltransferase-1, a housekeeping enzyme that functions in the synthesis of glycoconjugates, has two flexible loops, one short and one long. Upon binding a metal ion and UDP-galactose, the loops change from an open to a closed conformation, repositioning residues to lock the ligands in place. Residues at the N-terminal region of the long loop form the metal-binding site and those at the C-terminal region form a helix, which becomes part of the binding site for the oligosaccharide acceptor; the remaining residues cover the bound sugar-nucleotide. After binding of the oligosaccharide acceptor and transfer of the galactose moiety, the product disaccharide unit is ejected and the enzyme returns to the open conformation, repeating the catalytic cycle.
Asunto(s)
N-Acetil-Lactosamina Sintasa/metabolismo , Catálisis , Lactosa Sintasa/química , Lactosa Sintasa/metabolismo , Metales/metabolismo , Modelos Moleculares , Estructura Molecular , N-Acetil-Lactosamina Sintasa/química , Conformación ProteicaRESUMEN
Beta-1,4-galactosyltransferase (beta4Gal-T1) in the presence of manganese ion transfers galactose from UDP-galactose (UDP-Gal) to N-acetylglucosamine (GlcNAc) that is either free or linked to an oligosaccharide. Crystallographic studies on bovine beta4Gal-T1 have shown that the primary metal binding site is located in the hinge region of a long flexible loop, which upon Mn(2+) and UDP-Gal binding changes from an open to a closed conformation. This conformational change creates an oligosaccharide binding site in the enzyme. Neither UDP nor UDP analogues efficiently induce these conformational changes in the wild-type enzyme, thereby restricting the structural analysis of the acceptor binding site. The binding of Mn(2+) involves an uncommon coordination to the Sdelta atom of Met344; when it is mutated to His, the mutant M344H, in the presence of Mn(2+) and UDP-hexanolamine, readily changes to a closed conformation, facilitating the structural analysis of the enzyme bound with an oligosaccharide acceptor. Although the mutant M344H loses 98% of its Mn(2+)-dependent activity, it exhibits 25% of its activity in the presence of Mg(2+). The crystal structures of M344H-Gal-T1 in complex with either UDP-Gal.Mn(2+) or UDP-Gal.Mg(2+), determined at 2.3 A resolution, show that the mutant enzyme in these complexes is in a closed conformation, and the coordination stereochemistry of Mg(2+) is quite similar to that of Mn(2+). Although either Mn(2+) or Mg(2+), together with UDP-Gal, binds and changes the conformation of the M344H mutant to the closed one, it is the Mg(2+) complex that engages efficiently in catalyses. Thus, this property enabled us to crystallize the M344H mutant for the first time with the acceptor substrate chitobiose in the presence of UDP-hexanolamine and Mn(2+). The crystal structure determined at 2.3 A resolution reveals that the GlcNAc residue at the nonreducing end of chitobiose makes extensive hydrophobic interactions with the highly conserved Tyr286 residue.
Asunto(s)
Disacáridos/química , Histidina/genética , Metionina/genética , Mutagénesis Sitio-Dirigida , N-Acetil-Lactosamina Sintasa/química , N-Acetil-Lactosamina Sintasa/genética , Termodinámica , Acetilglucosamina/metabolismo , Animales , Conformación de Carbohidratos , Catálisis , Bovinos , Cristalización , Cristalografía por Rayos X , Disacáridos/metabolismo , Hexanos/química , Magnesio/química , Manganeso/química , Monosacáridos/química , N-Acetil-Lactosamina Sintasa/metabolismo , Conformación Proteica , Relación Estructura-Actividad , Tirosina/metabolismo , Uridina Difosfato Galactosa/químicaRESUMEN
Beta-1,4-galactosyltransferase (beta4Gal-T) (EC 2.4.1.38) plays a multifunctional role in many aspects of normal cell physiology. By now, several dozens of beta4Gal-T genes have been cloned, separated from mouse, chick, bovine, human, etc. This paper presents the cloning and GST-fused expression of mouse beta4Gal-T gene in Escherichia coli (E. coli). The target gene was cloned by PCR, followed by identification by DNA sequencing and expression in E.coli with isopropyl-beta-D-thiogalactoside (IPTG) gradient concentrations, products of which were separated on SDS-PAGE showing that the target protein had the same molecular weight as that of mouse beta4Gal-T. The transcriptional product of beta4Gal-T gene was proved by Western hybridization analysis to be due to GST-fusion.
Asunto(s)
Clonación Molecular/métodos , Escherichia coli/enzimología , Escherichia coli/genética , Glutatión Transferasa/biosíntesis , Glutatión Transferasa/genética , N-Acetil-Lactosamina Sintasa/biosíntesis , N-Acetil-Lactosamina Sintasa/genética , Secuencia de Aminoácidos , Animales , Regulación Bacteriana de la Expresión Génica/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Ratones , Datos de Secuencia Molecular , Peso Molecular , N-Acetil-Lactosamina Sintasa/química , Filogenia , Proteínas Recombinantes de Fusión/biosíntesis , Homología de Secuencia de Aminoácido , Transfección/métodosRESUMEN
We report a new chemoenzymatic strategy for the rapid and sensitive detection of O-GlcNAc posttranslational modifications. The approach exploits the ability of an engineered mutant of beta-1,4-galactosyltransferase to selectively transfer an unnatural ketone functionality onto O-GlcNAc glycosylated proteins. Once transferred, the ketone moiety serves as a versatile handle for the attachment of biotin, thereby enabling chemiluminescent detection of the modified protein. Importantly, this approach permits the rapid visualization of proteins that are at the limits of detection using traditional methods. Moreover, it bypasses the need for radioactive precursors and captures the glycosylated species without perturbing metabolic pathways. We anticipate that this general chemoenzymatic strategy will have broad application to the study of posttranslational modifications.