Exploration of the active site of β4GalT7: modifications of the aglycon of aromatic xylosides

Abstract

Proteoglycans (PGs) are macromolecules that consist of long linear polysaccharides, glycosaminoglycan (GAG) chains, covalently attached to a core protein by the carbohydrate xylose. The biosynthesis of GAG chains is initiated by xylosylation of the core protein followed by galactosylation by the galactosyltransferase β4GalT7. Some β-d-xylosides, such as 2-naphthyl β-d-xylopyranoside, can induce GAG synthesis by serving as acceptor substrates for β4GalT7 and by that also compete with the GAG synthesis on core proteins. Here we present structure-activity relationships for β4GalT7 and xylosides with modifications of the aromatic aglycon, using enzymatic assays, cell studies, and molecular docking simulations. The results show that the aglycons reside on the outside of the active site of the enzyme and that quite bulky aglycons are accepted. By separating the aromatic aglycon from the xylose moiety by linkers, a trend towards increased galactosylation with increased linker length is observed. The galactosylation is influenced by the identity and position of substituents in the aromatic framework, and generally, only xylosides with β-glycosidic linkages function as good substrates for β4GalT7. We also show that the galactosylation ability of a xyloside is increased by replacing the anomeric oxygen with sulfur, but decreased by replacing it with carbon. Finally, we propose that reaction kinetics of galactosylation by β4GalT7 is dependent on subtle differences in orientation of the xylose moiety.

Identification of Ellagic Acid Rhamnoside as a Bioactive Component of a Complex Botanical Extract with Anti-biofilm Activity

“…The product was isolated as a white solid via silica gel flash column chromatography (15:1 EtOAc/MeOH, 1% Et 3 N) in 59% yield (252 mg). The spectra match previously published data (Siegbahn et al, 2015). …”

Naphthyl Thio‐ and Carba‐xylopyranosides for Exploration of the Active Site of β‐1,4‐Galactosyltransferase 7 (β4GalT7)

“…Remarkably, the authors propose that the l ‐xylo‐derivative 6 shows a much higher antithrombotic activity compared to the d ‐xylo‐enantiomer 5 and that only the compound with l ‐xylo conformation functioned as substrate for β4GalT7 when assayed with enzyme extracts from chicken embryo. This is in stark contrast to other investigations where l ‐enantiomers of xylosides rarely act as substrates for β4GalT7 …”

“…For the measurement of the kinetic parameters for the galactosylation by β4GalT7, we used a slightly improved version of our previously published assay, which is reflected in that the kinetic parameters for the compounds analyzed might differ slightly from previous investigations (Table ) . For example, the K m value for the β‐ d ‐xyloside 7 was similar to previously reported values, whereas V max , k cat , and k cat / K m differed significantly.…”

“…Briefly, instead of a HisTrap HP (GE Life Sciences) column and manual purification, a HisTrap FF crude (GE Life Sciences) column and an ÄKTA start (GE Life Sciences) chromatography system were used during purification. The β4GalT7 activity was determined essentially as previously described, but in a 96‐well format . Briefly, in a 96‐well polypropylene plate β4GalT7 (50 ng) was mixed with 1 m m UDP‐Gal and various concentrations of the xyloside analogues, in a total volume of 50 μL MES buffer (20 m m , pH 6.2) supplemented with MnCl 2 (10 m m ).…”

‘Click’‐xylosides as initiators of the biosynthesis of glycosaminoglycans: Comparison of mono‐xylosides with xylobiosides

“…Indeed, we showed that three tyrosine residues represent key structural elements of the acceptor active site likely creating a hydrophobic environment for the aglycone moiety of exogenous xylosides . Altogether, our results indicate that the mono‐xyloside 3d is taken up by β4GalT7 as efficiently as 4‐MUX, though with different kinetic behavior, whereas the second xylose moiety present in the xylobioside 4d molecule does not accommodate the hydrophobic environment of the enzyme active site …”