Subcellular localization of glycosidases and glycosyltransferases involved in the processing of N-linked oligosaccharides

Using isopycnic sucrose gradients, we have ascertained the subcellular location of several enzymes involved in the processing of the N-linked oligosaccharides of glycoproteins in developing cotyledons of the common bean, Phaseolus vulgaris. All are localized in the endoplasmic reticulum (ER) or Golgi complex as determined by co-sedimentation with the ER marker, NADH-cytochrome c reductase, or the Golgi marker, glucan synthase I. Glucosidase activity, which removes glucose residues from Glc₃Man₉(GlcNAc)₂, was found exclusively in the ER. All other processing enzymes, which act subsequent to the glucose trimming steps, are associated with the Golgi. These include mannosidase I (removes 1-2 mannose residues from Man_6-9[GlcNAc]₂), mannosidase II (removes mannose residues from GlcNAcMan₅[GlcNAc]₂), and fucosyltransferase (transfers a fucose residue to the Asn-linked GlcNAc of appropriate glycans). We have previously reported the localization of two other glycan modifying enzymes (GlcNAc-transferase and xylosyltransferase activities) in the Golgi complex. Attempts at subfractionation of the Golgi fraction on shallow sucrose gradients yielded similar patterns of distribution for all the Golgi processing enzymes. Subfractionation on Percoll gradients resulted in two peaks of the Golgi marker enzyme inosine diphosphatase, whereas the glycan processing enzymes were all enriched in the peak of lower density. These results do not lend support to the hypothesis that N-linked oligosaccharide processing enzymes are associated with Golgi cisternae of different densities.     

The use of HPLC-pulsed amperometry for the characterization and assay of glycosidases and glycosyltransferases.

A sensitive and reproducible high performance chromatographic procedure is described for the assay of jack bean beta-galactosidase in which the reaction products are separated on a Dionex AS6 ion exchange column under alkaline conditions and detected by triple-pulsed amperometry. Quantition of the enzyme-released galactose is accomplished by using either fucose or lactose, the substrate, as an internal standard. The validity of the procedure as a general method for the assay and kinetic characterization of exoglycosidases was demonstrated by performing parallel measurements of galactose using an established coupled-enzyme assay, and using these values to calculate Km and Vmax values against lactose. Additional data are presented which establish the applicability of using a similar HPLC approach for the assay of glycosyltransferases.     

Engineering flavonoid glycosyltransferases for enhanced catalytic efficiency and extended sugar-donor selectivity

Flavonoids are predominantly found as glycosides in plants. The glycosylation of flavonoids is mediated by uridine diphosphate-dependent glycosyltransferases (UGT). UGTs attach various sugars, including arabinose, glucose, galactose, xylose, and glucuronic acid, to flavonoid aglycones. Two UGTs isolated from Arabidopsis thaliana, AtUGT78D2 and AtUGT78D3, showed 89 % amino acid sequence similarity (75 % amino acid sequence identity) and both attached a sugar to the 3-hydroxyl group of flavonols using a UDP-sugar. The two enzymes used UDP-glucose and UDP-arabinose, respectively, and AtUGT78D2 was approximately 90-fold more efficient than AtUGT78D3 when judged by the k _cat/K _m value. Domain exchanges between AtUGT78D2 and AtUGT78D3 were carried out to find UGTs with better catalytic efficiency for UDP-arabinose and exhibiting dual sugar selectivity. Among 19 fusion proteins examined, three showed dual sugar selectivity, and one fusion protein had better catalytic efficiency for UDP-arabinose compared with AtUGT78D3. Using molecular modeling, the changes in enzymatic properties in the chimeric proteins were elucidated. To the best of our knowledge, this is the first report on the construction of fusion proteins with expanded sugar-donor range and enhanced catalytic efficiencies for sugar donors.     

Role of prolactin on epididymal glycoprotein metabolism in matured monkeys, Macaca radiata: specific activities of glycosyltransferases and glycosidases.

Impact of altered serum prolactin status on enzymes involved in glycoprotein metabolism in epididymal tissue of matured monkeys was studied. Hyperprolactinemia (ovine prolactin-250 micrograms/kg body weight/day for 30 days) significantly inhibited the specific activities of dolichylphosphate mannosyl transferase, dolichylphosphate glucosyl transferase and galactosyl transferase, in the epididymal tissues. However, it had an enhanced effect on epididymal glycosidases such as beta-galactosidase, beta-N-acetyl glucosaminidase, beta-N-acetyl galactosaminidase, alpha-mannosidase and alpha-L-fucosidase. Hypoprolactinemia (bromocriptine mesylate-1-mg/kg body weight/day for 30 days) on other hand had no significant effect on the specific activities of both, glycosyltransferases and glycosidases, in the epididymal tissues. The results suggest that hyperprolactinemia inhibits epididymal glycoprotein metabolism by impairing the incorporation of oligosaccharide units into proteins with enhanced degradation. This may have adverse effect on events leading to sperm maturation in epididymal environment.     

Lysosomal glycosidases and glycoproteinoses

The development of chemicall, physical and enzymatic methods lead to the determination of numerous structures of glycoprotein glycans and allowed to classify them into "structural families". On the basis of this knowledge, it has been possible, 1) to demonstrate that the oligosaccharides and glycoasparagines accumulating in tissues and urines of patients with diseases characterized by a lack in lysosomal glycosidases originate from glycoprotein glycans incompletely catabolized; 2) to propose a scheme for the normal and pathological catabolism of glycoproteins and 3) to elucidate the problem of the origin of lysosomal glycosidases. These latter are internalized into the lysosomes either through a mechanism of secretion-reuptake, or by following an intracellular traffic, or via the cell plasma membrane. In al cases, membrane receptors intervene which specifically recognize phosphorylated oligomannosidic structures carried by the acidic hydrolases.     

Glycoconjugate glycosyltransferases

Assay methods for representative glycosyltransferases were described, covering those involved in the synthesis of glycosphingolipids, N-glycans, O-glycans, and proteoglycan glycosaminoglycans. In addition, intracellular localization of glycosyltransferase was comprehensively summarized. Lastly, complex formation of glycosyltransferase proteins with related molecules including subunits, chaperones, and enzyme regulators, that have been recently reported was also summarized.     

Plant glycosyltransferases

Glycosyltransferases are involved in the biosyntheses of cell-wall polysaccharides, the addition of N-linked glycans to glycoproteins, and the attachment of sugar moieties to various small molecules such as hormones and flavonoids. In the past two years, substantial progress has been made in the identification and cloning of genes that encode glycosyltransferases. Moreover, analysis of the recently completed Arabidopsis genome sequence indicates the existence of several hundred additional genes encoding putative glycosyltransferases.     

Rat-urine glycosidases and kidney damage

1. The activities of beta-galactosidase, beta-glucosidase, beta-glucuronidase and N-acetyl, beta-glucosaminidase were estimated in normal and pathological rat urine, with 4-methylumbelliferyl glycosides as substrates. 2. Kidney damage induced by injections of uranium nitrate, mercuric chloride, potassium dichromate or 4-nitrophenylarsonic acid causes a marked increase in the urinary excretion of all four enzymes. 3. The rise in beta-glucosidase activity was associated with the appearance of a new urinary enzyme species, which was examined by starch-gel electrophoresis, DEAE-cellulose chromatography and filtration on Sephadex G-75 and G-200. 4. This enzyme appears to be identical with its counterpart in the kidney, and it is suggested that it arises in the urine as a result of renal tubular breakdown. 5. The other glycosidases examined also show some physical similarities to the corresponding enzymes of the rat kidney.     

Structures and mechanisms of glycosyltransferases

Glycosyltransferases (GTs) catalyze the transfer of a sugar moiety from an activated donor sugar onto saccharide and nonsaccharide acceptors. A sequence-based classification spreads GTs in many families thus reflecting the variety of molecules that can be used as acceptors. In contrast, this enzyme family is characterized by a more conserved three-dimensional architecture. Until recently, only two different folds (GT-A and GT-B) have been identified for solved crystal structures. The recent report of a structure for a bacterial sialyltransferase allows the definition of a new fold family. Progress in the elucidation of the structures and mechanisms of GTs are discussed in this review. To accommodate the growing number of crystal structures, we created the 3D-Glycosyltransferase database to gather structural information concerning this class of enzymes.     

Microbial glycosaminoglycan glycosyltransferases

Glycosaminoglycans, a class of linear polysaccharides composed of repeating disaccharide units containing a hexosamine, are important carbohydrates found in many organisms. Vertebrates utilize glycosaminoglycans in structural, recognition, adhesion, and signaling roles. Certain pathogenic bacteria produce extracellular capsules composed of glycosaminoglycans or glycosaminoglycan-like polymers that enhance the microbes' ability to infect or to colonize the host. In the period from 1993 to 2001, bacterial enzymes were discovered that catalyze the polymerization of the repeating unit of hyaluronan, chondroitin, or N-acetylheparosan (unsulfated, unepimerized heparin). Depending on the specific carbohydrate and the microorganism, either a dual-action enzyme (synthase) that transfers two distinct monosaccharides or a pair of single-action transferases are utilized to synthesize the glycosaminoglycan polymer. Current views on the enzymology, structures, potential evolution, and the roles of the known glycosyltransferases from Streptococcus, Pasteurella, and Escherichia are discussed.     

Comparative aspects of glycosyltransferases

Glycosyltransferases, the enzymes that build oligosaccharides and glycoconjugates, have received much interest in recent years owing to their biological functions and their potential uses in biotechnology. Despite the fact that many glycosyltransferases recognize similar donor or acceptor substrates, there is surprisingly limited sequence identity between different classes. On the one hand, the glycosyltransferases are found in a large number of families, by sequence-based classification. On the other hand, only two structural folds have been identified among the fewer than one dozen glycosyltransferases that have been crystallized at present. Detection of conserved motifs that have a direct role in the functional aspects of glycosyltransferases is one approach for identifying remote similarity. With the availability of more crystal structures, the use of the fold-recognition approach is also very promising.     

Bacterial toxin and effector glycosyltransferases

Clostridial glucosylating cytotoxins, including Clostridium difficile toxins A and B, Clostridium novyi α-toxin, and Clostridium sordellii lethal toxin, are major virulence factors and causative agents of human diseases. These toxins mono-O-glucosylate (or mono-O-GlcNAcylate) a specific threonine residue of Rho/Ras-proteins, which is essential for the function of the molecular switches. Recently, a related group of glucosyltransferases from Legionella pneumophila has been identified. These Legionella glucosyltransferases modify the large GTPase elongation factor eEF1A at a serine residue by mono-O-glucosylation, thereby inhibiting protein synthesis of target cells. Recent results on structures, functions and biological roles of both groups of bacterial toxin glucosyltransferases will be discussed.     

Higher plant glycosyltransferases

Uridine diphosphate (UDP) glycosyltransferases (UGTs) mediate the transfer of glycosyl residues from activated nucleotide sugars to acceptor molecules (aglycones), thus regulating properties of the acceptors such as their bioactivity, solubility and transport within the cell and throughout the organism. A superfamily of over 100 genes encoding UGTs, each containing a 42 amino acid consensus sequence, has been identified in the model plant Arabidopsis thaliana. A phylogenetic analysis of the conserved amino acids encoded by these Arabidopsis genes reveals the presence of 14 distinct groups of UGTs in this organism. Genes encoding UGTs have also been identified in several other higher plant species. Very little is yet known about the regulation of plant UGT genes or the localization of the enzymes they encode at the cellular and subcellular levels. The substrate specificities of these UGTs are now beginning to be established and will provide a foundation for further analysis of this large enzyme superfamily as well as a platform for future biotechnological applications.     

The glycosidases of Cellulomonas

Summary A Cellulomonas strain, with potential for saccharification of sugar cane bagasse, has been found to possess a range of glycosidase activities which could be necessary for effective hydrolysis of the hemicellulose fraction of the bagasse.     

Automated assay of glycosidases

The Technicon Basic AutoAnalyzer sampler system was modified for simultaneous sampling of glycosidase(s) and substrate-buffer solutions. The inexpensive modification allows performance of automated enzyme analyses and enzyme kinetic studies with minimal consumption of substrate and/or enzyme.     

Structural and functional features of glycosyltransferases

Most of the glycosylation reactions that generate the great diversity of oligosaccharide structures of eukaryotic cells occur in the Golgi apparatus. This review deals with the most recent data that provide insight into the functional organization of Golgi-resident glycosyltransferases. We also focus on the recent successes in X-ray crystal structure determination of glycosyltransferases. These new structures begin to shed light on the molecular bases accounting for donor and acceptor substrate specificities as well as catalysis.     

Flexibility and mutagenic resiliency of glycosyltransferases

The human blood group A and B antigens are synthesized by two highly homologous enzymes, glycosyltransferase A (GTA) and glycosyltransferase B (GTB), respectively. These enzymes catalyze the transfer of either GalNAc or Gal from their corresponding UDP-donors to αFuc1–2βGal-R terminating acceptors. GTA and GTB differ at only four of 354 amino acids (R176G, G235S, L266M, G268A), which alter the donor specificity from UDP-GalNAc to UDP-Gal. Blood type O individuals synthesize truncated or non-functional enzymes. The cloning, crystallization and X-ray structure elucidations for GTA and GTB have revealed key residues responsible for donor discrimination and acceptor binding. Structural studies suggest that numerous conformational changes occur during the catalytic cycle. Over 300 ABO alleles are tabulated in the blood group antigen mutation database (BGMUT) that provides a framework for structure-function studies. Natural mutations are found in all regions of GTA and GTB from the active site, flexible loops, stem region and surfaces remote from the active site. Our characterizations of natural mutants near a flexible loop (V175M), on a remote surface site (P156L), in the metal binding motif (M212V) and near the acceptor binding site (L232P) demonstrate the resiliency of GTA and GTB to mutagenesis.