title: Novel carbohydrate-binding activity of bovine liver beta-glucuronidase toward lactose/N-acetyllactosamine sequences.
abstract: Beta-glucuronidase is a lysosomal enzyme that plays an essential role in normal turnover of glycosaminoglycans and remodeling of the extracellular matrix components in both physiological and inflammatory states. The regulation mechanisms of enzyme activity and protein targeting of beta-glucuronidase have implications for the development of a variety of therapeutics. In this study, the effectiveness of various carbohydrate-immobilized adsorbents for the isolation of bovine liver beta-glucuronidase (BLG) from other glycosidases was tested. Beta-glucuronidase and contaminating glycosidases in commercial BLG preparations bound to and were coeluted from adsorbents immobilized with the substrate or an inhibitor of beta-glucuronidase, whereas beta-glucuronidase was found to bind exclusively with lactamyl-Sepharose among the adsorbents tested and to be effectively separated from other enzymes. Binding and elution studies demonstrated that the interaction of beta-glucuronidase with lactamyl-Sepharose is pH dependent and carbohydrate specific. BLG was purified to homogeneity by lactamyl affinity chromatography and subsequent anion-exchange high-performance liquid chromatography (HPLC). Lactose was found to activate beta-glucuronidase noncompetitively, indicating that the lactose-binding site is different from the substrate-binding site. Binding studies with biotinyl glycoproteins, lipids, and synthetic sugar probes revealed that beta-glucuronidase binds to N-acetyllactosamine/lactose-containing glycoconjugates at neutral pH. The results indicated the presence of N-acetyllactosamine/lactose-binding activity in BLG and provided an effective purification method utilizing the novel carbohydrate binding activity. The biological significance of the carbohydrate-specific interaction of beta-glucuronidase, which is different from the substrate recognition, is discussed.
authors.affiliation: Course of Advanced Biosciences, Graduate School of Humanities and Sciences, Tokyo, Japan.
authors.forename: Hiroko
authors.initials: H
authors.lastname: Matsushita-Oikawa
authors.forename: Mayumi
authors.initials: M
authors.lastname: Komatsu
authors.forename: Naoko
authors.initials: N
authors.lastname: Iida-Tanaka
authors.forename: Hiromi
authors.initials: H
authors.lastname: Sakagami
authors.forename: Tetsuko
authors.initials: T
authors.lastname: Kanamori
authors.forename: Isamu
authors.initials: I
authors.lastname: Matsumoto
authors.forename: Nobuko
authors.initials: N
authors.lastname: Seno
authors.forename: Haruko
authors.initials: H
authors.lastname: Ogawa
mesh_terms: D000606:Amino Sugars / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D050260:Carbohydrate Metabolism; D002241:Carbohydrates / Q000494:pharmacology; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods; D002847:Chromatography, Agarose / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003636:DEAE-Cellulose / Q000493:pharmacokinetics; D005966:Glucuronidase / Q000302:isolation & purification / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism; D007785:Lactose / Q000737:chemistry / Q000378:metabolism*; D050356:Lipid Metabolism; D008099:Liver / Q000201:enzymology*; D008954:Models, Biological; D011485:Protein Binding; D012685:Sepharose / Q000493:pharmacokinetics; D013379:Substrate Specificity
chemical_list: D000606:Amino Sugars; D002241:Carbohydrates; D006023:Glycoproteins; C000458:N-acetyllactosamine; D012685:Sepharose; D003636:DEAE-Cellulose; D005966:Glucuronidase; D007785:Lactose