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1.
Deglycosylation of proteins for crystallization using recombinant fusion protein glycosidases. 总被引:4,自引:0,他引:4
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F. Grueninger-Leitch A. D'Arcy B. D'Arcy C. Chne 《Protein science : a publication of the Protein Society》1996,5(12):2617-2622
Obtaining high quality protein crystals remains a rate-limiting step in the determination of three-dimensional X-ray structures. A frequently encountered problem in this respect is the high or heterogeneous carbohydrate content of many eukaryotic proteins. A number of reports have demonstrated the use of enzymatic deglycosylation in the crystallization of certain glycoproteins. Although this is an attractive tool, there are some problems that hinder the more widespread use of glycosidases in crystallization. First, commercially available glycosidases are relatively expensive, which virtually prohibits their use on a large scale. Second, the glycosidase must be removed from the glycoprotein of interest following deglycosylation, which is not always straightforward. To circumvent these problems we have cloned the two most generally useful glycosidases, peptide-N-glycosidase F and endoglycosidase F1 from Flavobacterium meningosepticum, as fusion proteins with glutathione S-transferase. The fusion not only allows rapid purification of these enzymes from Escherichia coli cell extracts, but also permits rapid removal from target proteins following deglycosylation. We have used these enzymes to obtain crystals of phytase from Aspergillus ficuum and acid phosphatase from Aspergillus niger and to obtain a new crystal form of recombinant human renin. 相似文献
2.
Use of exoglycosidases from Mercenaria mercenaria (hard shelled clam) as a tool for structural studies of glycosphingolipids and glycoproteins. 总被引:1,自引:0,他引:1
S Ghosh S Lee T A Brown M Basu J W Hawes D Davidson S Basu 《Analytical biochemistry》1991,196(2):252-261
The hepatopancreatic extract of M. mercenaria (hard shelled clam) was found to be a rich source for at least 16 different glycosidases. These glycosidases were successfully employed for the degradation of oligosaccharides, glycolipids, and glycoproteins at analytical as well as preparative levels. The identified glycosidases differ considerably in their stability profiles with respect to time and temperature of storage and presence of glycerol. However, most of the enzymes show higher activity at pH 4.5 than at pH 7.0, and could be bound on a DEAE CL-6B Sepharose anion-exchange column suggesting similar charge characteristics on the protein surface. A Gal beta 1, 3R linkage-specific beta-galactosidase activity has also been detected in the glycosidase-enriched fraction and has been utilized to obtain quantitative conversion of the ganglioside GM1 to GM2 on a preparative scale. The glycosidase-rich extract does not have detectable protease activity at the pH of optimal glycosidase activity (pH 4.5) and, hence, can be safely used for specific hydrolysis of carbohydrate moieties of glycoproteins and glycopeptides. This is the first report to characterize a repertoire of glycosidases from an inexpensive, dependable and convenient source that can be easily employed for compositional studies involving glycoconjugates. 相似文献
3.
Glycosidase mechanisms 总被引:2,自引:0,他引:2
Insights into glycosidase mechanisms have come from X-ray crystallographic studies on complexes with substrate analogs and inhibitors, representing all the intermediate species along the reaction coordinate. Site-directed mutagenesis continues to play a significant role in understanding mechanisms, but is also proving important in generating glycosidases of modified mechanism or specificity. 相似文献
4.
Glycosidase susceptibility: a probe for the distribution of glycoprotein oligosaccharides in Sindbis virus.
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Intact Sindbis virus and Triton-solubilized viral glycoprotein were treated with alpha-mannosidase and with a preparation of mixed glycosidases from Diplococcus pneumoniae to probe the accesibility of carbohydrate units on the viral surface. The products of glycosidase attack on Triton-solubilized virus showed that mose carbohydrate units of the glycoproteins are good substrates for these enzymes. The relative resistance of most of the viral oligosaccharides in intact virus particles showed that much of the carbohydrate is not accessible to glycosidases, probably because it is not exposed at the viral surface. The only completely accessible carbohydrate units on Sindbis glycoproteins were the type A oligosaccharides of E2. This differential accessibility of Sindbis oligosaccharides is discussed in relation to the organization of the viral surface. 相似文献
5.
6.
Kleiger G Panina EM Mallick P Eisenberg D 《Protein science : a publication of the Protein Society》2004,13(1):221-229
The identification of the enzymes involved in the metabolism of simple and complex carbohydrates presents one bioinformatic challenge in the post-genomic era. Here, we present the PFIT and PFRIT algorithms for identifying those proteins adopting the alpha/beta barrel fold that function as glycosidases. These algorithms are based on the observation that proteins adopting the alpha/beta barrel fold share positions in their tertiary structures having equivalent sets of atomic interactions. These are conserved tertiary interaction positions, which have been implicated in both structure and function. Glycosidases adopting the alpha/beta barrel fold share more conserved tertiary interactions than alpha/beta barrel proteins having other functions. The enrichment pattern of conserved tertiary interactions in the glycosidases is the information that PFIT and PFRIT use to predict whether any given alpha/beta barrel will function as a glycosidase or not. Using as a test set a database of 19 glycosidase and 45 nonglycosidase alpha/beta barrel proteins with low sequence similarity, PFIT and PFRIT can correctly predict glycosidase function for 84% of the proteins known to function as glycosidases. PFIT and PFRIT incorrectly predict glycosidase function for 25% of the nonglycosidases. The program PSI-BLAST can also correctly identify 84% of the 19 glycosidases, however, it incorrectly predicts glycosidase function for 50% of the nonglycosidases (twofold greater than PFIT and PFRIT). Overall, we demonstrate that the structure-based PFIT and PFRIT algorithms are both more selective and sensitive for predicting glycosidase function than the sequence-based PSI-BLAST algorithm. 相似文献
7.
Deschamp J Mondon M Nakagawa S Kato A Alonzi DS Butters TD Zhang Y Sollogoub M Blériot Y 《Bioorganic & medicinal chemistry》2012,20(2):641-649
Noeuromycin is a highly potent albeit unstable glycosidase inhibitor due to its hemiaminal function. While stable D-gluco-like analogs have been reported, no data are available for D-manno-like structures. A series of tri- and tetrahydroxylated seven-membered iminosugars displaying either a D-manno-or a L-gulo-like configuration, were synthesized from methyl α-D-mannopyranoside using a reductive amination-mediated ring expansion as the key step. Screening towards a range of commercial glycosidases demonstrated their potency as competitive glycosidase inhibitors while cellular assay showed selective albeit weak glycoprotein processing mannosidase inactivation. 相似文献
8.
Treatment of antigen prepared from asexual blood stages of the human malarial parasite Plasmodium falciparum with a mixture of glycosidases resulted in a reduction in the ability of the antigen to bind antibodies from immune human and monkey sera in an ELISA assay. Some of the epitopes in the parasite material were heat stable, protease resistant, and sensitive to glycosidases. Proteins of Mr 110,000 and 65,000 from parasitized RBC were shown to have reduced antigenicity in Western blots after glycosidase treatment. The carbohydrate side chains of parasite glycoproteins therefore make a contribution to the total antigenicity of the parasite. 相似文献
9.
Gramer MJ 《Molecular biotechnology》2000,15(1):69-75
Heterogeneity of cell culture-produced glycoproteins often results from the presence or absence of a few sugars found on the
terminus of glycoprotein oligosaccharides. Variability in bioprocess factors can potentially lead to variability in this oligosaccharide
heterogeneity (1). Although stochastic events in the intracellular biosynthetic process have long been recognized as a cause of oligosaccharide
heterogeneity (2), more recent data has demonstrated that extracellular degradation by glycosidases can also contribute to oligosaccharide
heterogeneity (3,4). The purpose of this chapter is to introduce the concept and consequence of glycosidase degradation, to discuss methods
for evaluating whether glycosidase degradation is significant for a particular process, and to provide some potential remedies
to alleviate undesirable degradation. 相似文献
10.
Asano N 《Journal of enzyme inhibition》2000,15(3):215-234
Alkaloids mimicking the structures of sugars inhibit glycosidases because of a structural resemblance to the sugar moiety of the natural substrate. Glycosidases are involved in a wide range of important biological processes, such as intestinal digestion, post-translational processing of glycoproteins and the lysosomal catabolism of glycoconjugates. The realization that alkaloidal sugar mimics might have enormous therapeutic potential in many diseases such as viral infection, cancer and diabetes led to increasing interest and demand for these compounds. In this review, the structural basis of the specificity of alkaloidal sugar mimics and their current and potential applications to biomedical problems are reviewed. 相似文献
11.
Sørensen JF Kragh KM Sibbesen O Delcour J Goesaert H Svensson B Tahir TA Brufau J Perez-Vendrell AM Bellincampi D D'Ovidio R Camardella L Giovane A Bonnin E Juge N 《Biochimica et biophysica acta》2004,1696(2):275-287
The nutrient content of food and animal feed may be improved through new knowledge about enzymatic changes in complex carbohydrates. Enzymatic hydrolysis of complex carbohydrates containing alpha or beta glycosidic bonds is very important in nutrition and in several technological processes. These enzymes are called glycosidases (Enzyme Class 3.2.1) and include amylases, pectinases and xylanases. They are present in many foods such as cereals, but their microbial analogues are often produced and added in many food processes, for instance to improve the shelf-life of bakery products, clear beer, produce glucose, fructose or dextrins, hydrolyse lactose, modify food pectins, or improve processes. However, many plant foods also contain endogenous inhibitors, which reduce the activity of glycosidases, in particular, proteins, peptides, complexing agents and phenolic compounds. The plant proteinaceous inhibitors of glycosidases are in focus in this review whose objective is to report the effect and implications of these inhibitors in industrial processes and applications. These studies will contribute to the optimisation of industrial processes by using modified enzymes not influenced by the natural inhibitors. They will also allow careful selection of raw material and reaction conditions, and future development of new genetic varieties low in inhibitors. These are all new and very promising concepts for the food and feed sector. 相似文献
12.
《Journal of enzyme inhibition and medicinal chemistry》2013,28(3):215-234
AbstractAlkaloids mimicking the structures of sugars inhibit glycosidases because of a structural resemblance to the sugar moiety of the natural substrate. Glycosidases are involved in a wide range of important biological processes, such as intestinal digestion, post-translational processing of glycoproteins and the lysosomal catabolism of glycoconjugates. The realization that alkaloidal sugar mimics might have enormous therapeutic potential in many diseases such as viral infection, cancer and diabetes led to increasing interest and demand for these compounds. In this review, the structural basis of the specificity of alkaloidal sugar mimics and their current and potential applications to biomedical problems are reviewed. 相似文献
13.
Glycosidase inhibitors as antiviral and/or antitumor agents. 总被引:5,自引:0,他引:5
A D Elbein 《Seminars in cell biology》1991,2(5):309-317
Glycoprotein processing inhibitors prevent the normal processing of N-linked glycoproteins by inhibiting specific glycosidases involved in these reactions. Thus, a number of compounds are now known that inhibit alpha-glucosidase I and alpha-glucosidase II and therefore prevent the removal of glucoses from the high-mannose chains. Some of these compounds are more potent inhibitors of one or the other of these glucosidases. There are also a number of inhibitors that affect one of the processing alpha-mannosidases (i.e. mannosidase I or mannosidase II). These compounds; especially the glucosidase inhibitors, have been valuable tools to help us understand the role of carbohydrate in viral envelope glycoprotein function. Such processing inhibitors have also been used with various tumorigenic cell lines to determine the function of N-linked glycoproteins in cancer. 相似文献
14.
Ball AL Chambers KA Hewinson M Navaratnarajah S Samrin L Thomas N Tyler AE Wall AJ Lloyd MD 《Journal of enzyme inhibition and medicinal chemistry》2008,23(1):131-135
Glycosidases perform a wide range of functions in physiology and pathology, and are potential targets for the treatment of diseases such as influenza, cancer, AIDS and diabetes. This paper reports a convenient discontinuous colourimetric assay for the measurement of glycosidase activity. The assay utilises 4-nitrophenyl- substrates and quantities of product are determined by measuring absorbance at 405 nm. This assay is performed in a 96 well microtitre plate and has been used to characterise the properties of seven different glycosidases from bacteria, yeast and higher eukaryotes and their kinetic parameters determined. Assays in the presence of known inhibitors showed that inhibition modes can be determined, and IC(50) and K(i) values calculated. This assay appears to be of widely applicable and of general utility for the measurement of glycosidase activity and the evaluation of inhibitors. 相似文献
15.
Robert A. Field Alan H. Haines Ewan J. T. Chrystal 《Bioorganic & medicinal chemistry letters》1991,1(12):667-672
A range of 1,4- and 1,5-anhydroalditols have been synthesized and assessed for their ability to inhibit glycosidases. Observed inhibition indicates that aglycone-enzyme interactions contribute significantly to both the affinity and the stereoselectivity of substrate binding. Such interactions may also contribute to enzyme-transition state interactions. Implications for the design of potent glycosidase inhibitors are discussed. 相似文献
16.
2-Deoxy-2-fluoro-D-glycosyl fluorides. A new class of specific mechanism-based glycosidase inhibitors 总被引:1,自引:0,他引:1
Mechanism-based glycosidase inhibitors are of considerable use in studies of enzyme mechanism, in studies of glycoprotein processing, and possibly therapeutically in control of sugar uptake. This paper describes a new general approach to mechanism-based inactivation of glycosidases which involves trapping a covalent glycosyl enzyme intermediate. This is achieved by use of 2-deoxy-2-fluoro-D-glycosyl fluorides, for which the rate of hydrolysis of the fluoroglycosyl enzyme intermediate is extremely slow, resulting in accumulation of the intermediate. Eleven different glycosidases were tested with their corresponding 2-deoxy-2-fluoro-D-glycosyl fluorides. Eight of the eleven were inactivated, four of them according to pseudo first-order kinetics and four according to a more complex kinetic scheme. The specificity of these inhibitors was investigated by assaying for inhibition of one enzyme with four different 2-deoxy-2-fluoro-D-glycosyl fluorides. Large differences in inactivation rate were observed which paralleled previously observed substrate specificities. 相似文献
17.
Andrea L. Ball Kirsty A. Chambers Meera Hewinson Sambavi Navaratnarajah Lamia Samrin Nesta Thomas 《Journal of enzyme inhibition and medicinal chemistry》2013,28(1):131-135
Glycosidases perform a wide range of functions in physiology and pathology, and are potential targets for the treatment of diseases such as influenza, cancer, AIDS and diabetes. This paper reports a convenient discontinuous colourimetric assay for the measurement of glycosidase activity. The assay utilises 4-nitrophenyl- substrates and quantities of product are determined by measuring absorbance at 405 nm. This assay is performed in a 96 well microtitre plate and has been used to characterise the properties of seven different glycosidases from bacteria, yeast and higher eukaryotes and their kinetic parameters determined. Assays in the presence of known inhibitors showed that inhibition modes can be determined, and IC50 and Ki values calculated. This assay appears to be of widely applicable and of general utility for the measurement of glycosidase activity and the evaluation of inhibitors. 相似文献
18.
Kondo KI Adachi H Shitara E Kojima F Nishimura Y 《Bioorganic & medicinal chemistry》2001,9(5):1091-1095
The relationships between structures and inhibitory activities of glycosidase inhibitors of gem-diamine 1-N-iminosugars in media of enzyme assays have been investigated. It has been proved that gem-diamine 1-N-iminosugar smoothly undergoes a structural change to a hydrated ketone or its derivative via a hemiaminal in the media (pH 5.0-6.3), and that the products generated in the media as well as the parent gem-diamine 1-N-iminosugars potently inhibit glycosidases. 相似文献
19.
Li Liu Tharmala Tharmalingam Eva Maischberger Simone Albrecht Mary E. Gallagher Raúl Miranda-CasoLuengo Wim G. Meijer Pauline M. Rudd Jane A. Irwin 《Glycoconjugate journal》2013,30(8):791-800
Many disorders are characterised by changes in O-glycosylation, but analysis of O-glycosylation has been limited by the availability of specific endo- and exo-glycosidases. As a result chemical methods are employed. However, these may give rise to glycan degradation, so therefore novel O-glycosidases are needed. Artificial substrates do not always identify every glycosidase activity present in an extract. To overcome this, an HPLC-based protocol for glycosidase identification from microbial culture was developed using natural O-glycans and O-glycosylated glycoproteins (porcine stomach mucin and fetuin) as substrates. O-glycans were released by ammonia-based β-elimination for use as substrates, and the bacterial culture supernatants were subjected to ultrafiltration to separate the proteins from glycans and low molecular size molecules. Two bacterial cultures, the psychrotroph Arthrobacter C1-1 and a Corynebacterium isolate, were examined as potential sources of novel glycosidases. Arthrobacter C1-1 culture contained a β-galactosidase and N-acetyl-β-glucosaminidase when assayed using 4-methylumbelliferyl substrates, but when defucosylated O-glycans from porcine stomach mucin were used as substrate, the extract did not cleave β-linked galactose or N-acetylglucosamine. Sialidase activity was identified in Corynebacterium culture supernatant, which hydrolysed sialic acid from fetuin glycans. When both culture supernatants were assayed using the glycoproteins as substrate, neither contained endoglycosidase activity. This method may be applied to investigate a microbial or other extract for glycosidase activity, and has potential for scale-up on high-throughput platforms. 相似文献
20.
In the lysosome, glycosidases degrade glycolipids, glycoproteins, and oligosaccharides. Mutations in glycosidases cause disorders characterized by the deposition of undegraded carbohydrates. Schindler and Fabry diseases are caused by the incomplete degradation of carbohydrates with terminal alpha-N-acetylgalactosamine and alpha-galactose, respectively. Here we present the X-ray structure of alpha-N-acetylgalactosaminidase (alpha-NAGAL), the glycosidase that removes alpha-N-acetylgalactosamine, and the structure with bound ligand. The active site residues of alpha-NAGAL are conserved in the closely related enzyme a-galactosidase A (alpha-GAL). The structure demonstrates the catalytic mechanisms of both enzymes and reveals the structural basis of mutations causing Schindler and Fabry diseases. As alpha-NAGAL and alpha-GAL produce type O "universal donor" blood from type A and type B blood, the alpha-NAGAL structure will aid in the engineering of improved enzymes for blood conversion. 相似文献