Substrate Specificity Of Glycosyltransferases Research Articles

An Update on Saccharide Arrays

Saccharide arrays are powerful tools to investigate the saccharide-mediated recognition events in highthroughput mode. Many methods have been used to immobilize the saccharides on support surface in the array format. The array-based technologies were widely utilized to analyze the characters of saccharide-binding lectins and antibodies, quantitatively determine saccharide-protein interactions, detect cells and pathogens, identify disease-related antisaccharide antibodies for diagnosis, and fast assess substrate specificities of glycosyltransferases. This review has mainly summarized the fabrication methods of saccharide arrays and their applications in recent years.

Synthesis of phosphorylated 3,4-branched trisaccharides corresponding to LPS inner core structures of Neisseria meningitidis and Haemophilus influenzae

2-( N-Benzyloxycarbonyl)aminoethyl 7- O-acetyl-6- O-allyl-2- O-benzoyl-4- O-benzyl-3- O-chloroacetyl- l- glycero-α- d- manno-heptopyranosyl-(1→3)-[2,3,4,6-tetra- O-benzoyl-β- d-glucopyranosyl-(1→4)]-6,7-di- O-acetyl-2- O-benzyl- l- glycero-α- d- manno-heptopyranoside, a spacer-equipped protected derivative of the common 3,4-branched diheptoside trisaccharide structure of the lipopolysaccharide core of Neisseria meningitidis and Haemophilus influenzae has been synthesized. The protecting group pattern installed allows regioselective introduction of phosphoethanolamine residues in the 3- and 6-position of the second heptose unit in accordance with native structures. From this intermediate the 3-and 6-monophosphoethanolamine as well as the non-phosphorylated deprotected trisaccharides have been synthesized to be used in evaluation of antibody binding specificity and in investigation of the substrate specificity of glycosyl transferases involved in the biosynthesis of LPS core structures.

Reconstitution of antibiotics glycosylation by domain exchanged chimeric glycosyltransferase

Glycosyltransferase (GT), which catalyzes the transfer of nucleotide-activated sugar to aglycone, is the key enzyme for the preparation of a variety of glycosylated natural products. In this study, we report that the substrate specificity of GTs can be altered via domain exchanging accompanied by rationally designed peptide linkers. Comprehensive similarity searches were first conducted based upon the amino acid sequences of antibiotic GTs. The phylogenetic tree displays a distinct separation of 25 antibiotic GTs into three groups according to their aglycone substrates. Two different groups of GTs, KanF (aminoglycoside) and GtfE (glycopeptide) were employed for domain exchanging of GT-B fold enzyme. In order to determine linker peptide sequences, the secondary structures of KanF and GtfE were carefully evaluated via 3D-PSSM. The N-terminal domain of KanF and the C-terminal domain of GtfE were fused with rationally designed linker sequences. The hybrid GT, referred to as KE chimera, showed hybrid activity toward dTDP- d-glucose and 2-deoxystreptamine. Moreover, the key catalytic amino acid residues of KE chimera were investigated via protein modeling and docking simulations.

S1.6 Substrate specificities of glycosyltransferases involved in the formation of heparin and ofE. coli K5 capsular polysaccharide

S1.6 Substrate specificities of glycosyltransferases involved in the formation of heparin and ofE. coli K5 capsular polysaccharide

Structural heterogeneity of sugar chains in immunoglobulin G. Conformation of immunoglobulin G molecule and substrate specificities of glycosyltransferases.

The heterogeneous asparagine-linked sugar chains of bovine and human immunoglobulins G were separated into 12 components by reversed-phase high performance liquid chromatography, and their structures were determined by 1H NMR spectroscopy. Both immunoglobulin (Ig) G sources contained eight non-bisected biantennary complexes and four bisected biantennary complexes. In the non-bisected sugar chains of bovine IgG, galactosylation of the Man alpha 1-3 branch predominated over that of the Man alpha 1-6, whereas in the bisected complexes galactosylation of the Man alpha 1-6 branch predominated. This difference can be explained by the substrate specificities of the galactosyl-transferases and of the N-acetylglucosaminyltransferase III involved in their synthesis. The sugar chains of human IgG1 differs in the distribution of its galactose residues from bovine IgG and human IgG2. The Man alpha 1-6 branch of all IgG1s was more highly galactosylated than the Man alpha 1-3 branch even in the non-bisected complexes. Such findings are in conflict with the substrate specificities of galactosyltransferases. Whereas these enzymes derivatized more of the Man alpha 1-6 branch of native human IgG1, in denatured protein more of the Man alpha 1-3 branch was galactosylated. Thus, protein conformation may influence the structure of its sugar chains.