Man alpha1-2 Man alpha-OMe-concanavalin A complex reveals a balance of forces involved in carbohydrate recognition.
(25/2617)
We have determined the crystal structure of the methyl glycoside of Man alpha1-2 Man in complex with the carbohydrate binding legume lectin concanavalin A (Con A). Man alpha1-2 Man alpha-OMe binds more tightly to concanavalin A than do its alpha1-3 and alpha1-6 linked counterparts. There has been much speculation as to why this is so, including a suggestion of the presence of multiple binding sites for the alpha1-2 linked disaccharide. Crystals of the Man alpha1-2 Man alpha-OMe-Con A complex form in the space group P2(1)2(1)2(1) with cell dimensions a = 119.7 A, b = 119.7 A, c = 68.9 A and diffract to 2. 75A. The final model has good geometry and an R factor of 19.6% (Rfree= 22.8%). One tetramer is present in the asymmetric unit. In three of the four subunits, electron density for the disaccharide is visible. In the fourth only a monosaccharide is seen. In one subunit the reducing terminal sugar is recognized by the monosaccharide site; the nonreducing terminal sugar occupies a new site and the major solution conformation of the inter-sugar glycosidic linkage conformation is adopted. In contrast, in another subunit the non reducing terminal sugar sits in the so called monosaccharide binding site; the reducing terminal sugar adopts a different conformation about its inter-sugar glycosidic linkage in order for the methyl group to access a hydrophobic pocket. In the third subunit, electron density for both binding modes is observed. We demonstrate that an extended carbohydrate binding site is capable of binding the disaccharide in two distinct ways. These results provide an insight in to the balance of forces controlling protein carbohydrate interactions. (+info)
The acceptor substrate specificity of human beta4-galactosyltransferase V indicates its potential function in O-glycosylation.
(26/2617)
In order to assess the function of the different human UDP-Gal:GlcNAc beta4-galactosyltransferases, the cDNAs of two of them, beta4-GalT I and beta4-GalT V, were expressed in the baculovirus/insect cell expression system. The soluble recombinant enzymes produced were purified from the medium and used to determine their in vitro substrate specificities. The specific activity of the recombinant beta4-GalT V was more than 15 times lower than that of beta4-GalT I, using GlcNAc beta-S-pNP as an acceptor. Whereas beta4-GalT I efficiently acts on all substrates having a terminal beta-linked GlcNAc, beta4-GalT V appeared to be far more restricted in acceptor usage. Beta4-GalT V acts with high preference on acceptors that contain the GlcNAc beta1-->6GalNAc structural element, as found in O-linked core 2-, 4- and 6-based glycans, but not on substrates related to V-linked or blood group I-active oligosaccharides. These results suggest that beta4-GalT V may function in the synthesis of lacNAc units on O-linked chains, particularly in tissues which do not express beta4-GalT I, such as brain. (+info)
H (0) blood group determinant is present on soluble human L-selectin expressed in BHK-cells.
(27/2617)
In the present study we show that the H (0) blood group determinant Fuc alpha1-2Gal beta1-4GlcNAc beta1-R is present on N-linked glycans of soluble human L-selectin recombinantly expressed in baby hamster kidney (BHK) cells. The glycans were isolated using complementary HPLC techniques and characterized by a combination of exoglycosidase digestion and mass spectrometry. The linkage of the fucose residues was determined by incubation of the glycans with specific fucosidases. The H blood determinant Fuc alpha1-2Gal beta1-4GlcNAc beta1 was detected for bi-, 2,4 branched tri- and tetraantennary structures. To our knowledge, the proposed oligosaccharide structures represent a new glycosylation motif for recombinant glycoproteins expressed on BHK cells. (+info)
A predominantly hydrophobic recognition of H-antigenic sugars by winged bean acidic lectin: a thermodynamic study.
(28/2617)
The thermodynamics of binding of winged bean (Psophocarpus tetragonolobus) acidic agglutinin to the H-antigenic oligosaccharide (Fucalpha1-2Galbeta1-4GlcNAc-oMe) and its deoxy and methoxy congeners were determined by isothermal titration calorimetry. We report a relatively hydrophobically driven binding of winged bean acidic agglutinin to the congeners of the above sugar. This conclusion is arrived, from the binding parameters of the fucosyl congeners, the nature of the enthalpy-entropy compensation plots and the temperature dependence of binding enthalpies of some of the congeners. Thus, the binding site of winged bean acidic agglutinin must be quite extended to accommodate the trisaccharide, with non-polar loci that recognize the fucosyl moiety of the H-antigenic determinant. (+info)
NMR and molecular modeling studies on two glycopeptides from the carbohydrate-protein linkage region of connective tissue proteoglycans.
(29/2617)
Complete 1H and 13C NMR assignments are reported for two glycopeptides representing the carbohydrate-protein linkage region of connective tissue proteoglycans. These glycopeptides are the octasaccharide hexapeptide, Ser(GlcpAbeta(1-->3) Galpbeta(1-->3)Galpbeta(1-->4)Xylpbeta)-Gly-Ser-Gly-Se r (GlcpAbeta(1-->3)Galpbeta(1-->3)Galpbeta(1-->4)Xylp beta)-Gly (1), and the tetrasaccharide dipeptide, Ser(GlcpAbeta(1-->3)Galpbeta(1-->3)Galpbeta(1-->4)X ylpbeta)-Gly (2). The vicinal coupling constant data show that the monosaccharide residues adopt4 C 1 chair conformations. Distance geometry/simulated annealing calculations using 2D NOESY derived distance constraints yielded a single family of structures for the tetrasaccharide moiety, with well defined interglycosidic linkage conformations. The straight phi torsion angles of the glycosidic C1'-O1 bonds showed a strict preference for the -sc range whereas the psi torsion angles (O1-Cn) exhibited dependence upon the interglycosidic linkage position (-ac for beta(1-->3) linkage, +ac for beta(1-->4) linkage). The predominant conformation about the glycopeptide bond is straight phi = -sc and psi = +ac. The presence of strong daN (i, i+1) NOE contacts, and the general absence of dNN (i, i+1) contacts (except for a weak Ser-5/Gly-6 dNN contact) and the dbN (i, i+1) contacts (except for Ser-1/Gly-2) in the ROESY spectrum, suggest that the backbone for 1 is predominantly in an extended conformation. A comparison of the ROESY data for 1 with those obtained from the unglycosylated hexapeptide (3) of the same sequence suggests that glycosylation has only a marginal influence on the backbone conformation of the hexapeptide. (+info)
The O-linked fucose glycosylation pathway: identification and characterization of a uridine diphosphoglucose: fucose-beta1,3-glucosyltransferase activity from Chinese hamster ovary cells.
(30/2617)
O-Linked fucose is an unusual carbohydrate modification in which fucose is linked directly to the hydroxyl groups of serines or threonines. It has been found on the epidermal growth factor-like modules of several secreted proteins involved in blood coagulation and fibrinolysis. We have recently reported the existence of an elongated form of O-linked fucose in Chinese hamster ovary cells consisting of a glucose linked to the 3'-hydroxyl of fucose (Glcbeta1,3Fuc- O-Ser/Thr). This structure is highly unusual for two reasons. First, in mammalian systems fucose is usually a terminal modification of N - and O-linked oligosaccharides. Here the fucose is internal. Secondly, terminal beta-linked glucose is extremely rare on mammalian glycoconjugates. Thus, the Glcbeta1,3Fuc structure is a very unique mammalian carbohydrate structure. Here we report the identification and initial characterization of a novel enzyme activity capable of forming this unique linkage: UDP-glucose: O-linked fucose beta1,3 glucosyltransferase. The enzyme utilizes UDP-glucose as the high energy donor and transfers glucose to alpha-linked fucose residues. The activity is linearly dependent on time, enzyme, and substrate concentrations and is enhanced in the presence of manganese ions. Activity is present in extracts of cultured cells from a variety of species (hamster, human, mouse, rat, chicken) and is enriched in brain and spleen of a normal adult rat. Thus, while this glycosyltransferase appears to be widespread in biology, it forms a very unique linkage, and it represents the first mammalian enzyme identified capable of elongating fucose. (+info)
Structure of a plant cell wall fragment complexed to pectate lyase C.
(31/2617)
The three-dimensional structure of a complex between the pectate lyase C (PelC) R218K mutant and a plant cell wall fragment has been determined by x-ray diffraction techniques to a resolution of 2.2 A and refined to a crystallographic R factor of 18.6%. The oligosaccharide substrate, alpha-D-GalpA-([1-->4]-alpha-D-GalpA)3-(1-->4)-D-GalpA , is composed of five galacturonopyranose units (D-GalpA) linked by alpha-(1-->4) glycosidic bonds. PelC is secreted by the plant pathogen Erwinia chrysanthemi and degrades the pectate component of plant cell walls in soft rot diseases. The substrate has been trapped in crystals by using the inactive R218K mutant. Four of the five saccharide units of the substrate are well ordered and represent an atomic view of the pectate component in plant cell walls. The conformation of the pectate fragment is a mix of 21 and 31 right-handed helices. The substrate binds in a cleft, interacting primarily with positively charged groups: either lysine or arginine amino acids on PelC or the four Ca2+ ions found in the complex. The observed protein-oligosaccharide interactions provide a functional explanation for many of the invariant and conserved amino acids in the pectate lyase family of proteins. Because the R218K PelC-galacturonopentaose complex represents an intermediate in the reaction pathway, the structure also reveals important details regarding the enzymatic mechanism. Notably, the results suggest that an arginine, which is invariant in the pectate lyase superfamily, is the amino acid that initiates proton abstraction during the beta elimination cleavage of polygalacturonic acid. (+info)
Xyloglucan fucosyltransferase, an enzyme involved in plant cell wall biosynthesis.
(32/2617)
Cell walls are crucial for development, signal transduction, and disease resistance in plants. Cell walls are made of cellulose, hemicelluloses, and pectins. Xyloglucan (XG), the principal load-bearing hemicellulose of dicotyledonous plants, has a terminal fucosyl residue. A 60-kilodalton fucosyltransferase (FTase) that adds this residue was purified from pea epicotyls. Peptide sequence information from the pea FTase allowed the cloning of a homologous gene, AtFT1, from Arabidopsis. Antibodies raised against recombinant AtFTase immunoprecipitate FTase enzyme activity from solubilized Arabidopsis membrane proteins, and AtFT1 expressed in mammalian COS cells results in the presence of XG FTase activity in these cells. (+info)