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The exo-b-1,3-glucanase of Candida albicans (Exg) has a marked specificity for b-1,3-glucosidic linkages as judged by the kinetic constants for p-nitophenyl b-glucoside, b-linked disaccharides of glucose (laminaribiose, gentiobiose, and cellobiose), oligosaccharides of the laminari series, laminarin and pustulan. The k cat /K m ratios for a series of laminari oligosaccharides from -biose to -heptaose showed that Exg has an extended substratebinding site which contains at least five binding sites for sugar residues. Binding at position +2 (the third sugar residue) increases the k cat twofold while positions +3 and +4 lower the K m value further and thereby increase the catalytic efficiency. Exg catalyses an efficient transglucosylation reaction with high concentrations of laminarioligosaccharides which specifically form b-1,3 linkages and with yields up to 50%. The rate of the transglucosylation is concentration-dependent and can be more than 10 times faster than the hydrolytic reaction with excess donor substrates such as laminaritriose and laminarihexaose. The kinetics of Exg and the predicted substrate-binding site for up to five sugar residues are consistent with a recent structural analysis of the enzymebinding site.Keywords: Candida albicans; exo-b-1, 3-glucanase; glycosidase; transglucosylation.b2Glucan, a homopolymer of glucose containing b-1,3, b-1,6 and b-1,3,6 linkages is the main structural component of the cell walls of many yeasts and some filamentous fungi (for reviews, see [1,2]). Linear b-1,3-glucan is produced by a UDP-glucose-dependent synthetase located in the plasma membrane and delivered to the nascent cell wall in a vectorial process [3]. Other reactions involved in the assembly of b-glucan have not been identified but must include: the formation of b-1,6 linkages, b-1,3,6 branch points and crosslinkages to other wall constituents [2,4].Enzymes implicated in glucan catabolism have been studied in some detail. Endoglucanases and exoglucanases are secreted into the cell wall by many organisms and the putative roles for these include localized breakdown of b-glucan for wall expansion, mobilization of glucan for use as a fuel and the hydrolysis of exogenous material for uptake as a nutrient (for reviews see [5,6]). It has also been suggested that some glucanases may, within the wall, catalyze transglycosylation rather than hydrolytic reactions and thereby contribute to rearrangement and assembly of wall glucan [7]. For example, the BGL2 gene product of Candida albicans, Saccharomyces cerevisiae and Aspergillus fumigatus previously described as an endo-b-1,3-glucanase [8] preferentially catalyzes a glucanosyltransferase reaction in which new 1,6 linkages are introduced into 1,3 glucan [9±12].Exo-b-1,3-glucanase (Exg) has been purified from several sources. The genes for EXG1 of S. cerevisiae [13] and EXG of C. albicans [14] share 58% identity, and closely related genes have been detected in other Candida species [15]. The enzymes from S. cerevisiae and C. albicans are similar in many respects al...

Fluorescence energy transfer was used to study the conformation of each antenna of a complex biantennary oligosaccharide. A core fucosylated biantennary oligosaccharide was converted to a glycosylamine which allowed coupling of a naphthyl donor fluorophore directly to the reducing-end GlcNAc 1. After generating an aldehyde at C-6 of residue 6 or 6' using galactose oxidase, a dansyl ethylenediamine acceptor fluorophore was coupled to either antenna of the oligosaccharide resulting in two donor-acceptor pairs. [Formula: see text] The fluorescence properties of the naphthyl group allowed determination of the end-to-end donor-acceptor distance and antenna flexibility of each isomer by steady-state and time-resolved fluorescence energy transfer at temperatures ranging from 0 to 40 degrees C. Extended (20.6 A) and folded (11.4 A) donor-acceptor distance populations were identified for the isomer containing dansyl attached to Gal 6', whereas only a single extended population (19.7 A) was determined when dansyl was attached to Gal 6. The presence of Fuc 1' had a dramatic effect on the conformation of the 6' antenna. Temperature modulation failed to alter the ratio of extended/folded populations when fucose was present. However, following the removal of fucose, the ratio of the extended/folded populations for 6' exhibited a temperature dependent conformational equilibrium allowing calculation of the enthalpy and entropy of unfolding. These results established a unique conformational property for the 6' antenna of a biantennary oligosaccharide that is influenced by core fucosylation. Comparison of the results obtained for the 6 antenna of biantennary with previous fluorescence energy transfer studies on a triantennary glycopeptide also established conformational differences in this antenna which are dependent on oligosaccharide structure.

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The target site for N-linked biantennary and triantennary oligosaccharides containing multiple terminal Le X determinants was analyzed in mice. N-Linked oligosaccharides containing a single tert-butoxycarbonyl-tyrosine attached to the reducing end were used as synthons for human milk ␣-3/4-fucosyltransferase to prepare multivalent Le X (Gal␤1-4[Fuc␣1-3]GlcNAc) terminated tyrosinamide oligosaccharides. The oligosaccharides were radioiodinated and examined for their pharmacokinetics and biodistribution in mice. The liver was the major target site in mice at 30 min, which accumulated 18% of the dose for Le X biantennary compared with 6% for a nonfucosylated Gal biantennary. By comparison, Le X -and Gal-terminated triantennary accumulated in the liver with a targeting efficiency of 66 and 59%, respectively. The liver targeting of Le X biantennary was partially blocked by co-administration with either galactose or L-fucose whereas Le X triantennary targeting was only reduced by co-administration with galactose. In contrast to these results in mice, in vivo experiments performed in rats established that both Le X and Gal terminated biantennary target the liver with nearly identical efficiency (6 -7%). It is concluded that the asialoglycoprotein receptor in mice preferentially recognize Le X biantennary over Gal biantennary, whereas little or no differentiation exists in rats. Thereby, the mouse asialoglycoprotein receptor apparently possesses additional binding pockets that accommodate a fucose residue when presented as Le X .In mammals, carbohydrate/protein interactions often involve the binding of an oligosaccharide ligand to a cell surface receptor (1, 2). The ligands are most frequently N-or O-linked oligosaccharides that are covalently attached to a glycoprotein. N-Linked oligosaccharides possess a common pentasaccharide core structure which contains a branch point resulting in two or more nonreducing end sugar residues (3). It is often these terminal sugar residues on N-linked oligosaccharides which bind to spatially resolved binding sites on a lectin (4).The mammalian lectins discovered to date have been grouped into several subcategories (5). Several membrane spanning lectins are known to be C-type lectins which contain a carbohydrate recognition domain named for its calcium-dependent ligand binding. Of the C-type lectins, the asialoglycoprotein receptor (ASGP-R) 1 found on hepatocytes has been most thoroughly studied for its binding specificity and its intracellular routing of ligands (6 -8). N-Linked oligosaccharides containing multiple terminal Gal residues bind with high affinity, although GalNAc terminated N-linked oligosaccharides are much more potent ligands (9 -11). A biantennary oligosaccharide possessing only two terminal GalNAc residues is a superior ASGP-R ligand compared with a triantennary possessing three terminal Gal residues (12). In addition to the ASGP-R, several other mammalian lectins have been isolated and characterized in liver. Kupffer cells possess a C-type lectin that binds avidly to ...