Glycobiology Advance Access originally published online on June 13, 2006
Glycobiology 2006 16(10):891-901; doi:10.1093/glycob/cwl016
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Novel carbohydrate-binding activity of bovine liver ß-glucuronidase toward lactose/N-acetyllactosamine sequences
3 Course of Advanced Biosciences, Graduate School of Humanities and Sciences, Tokyo, Japan; 4 Department of Food Science, Otsuma University, Tokyo, Japan; and 5 The Glycoscience Institute, Ochanomizu University, Tokyo, Japan
2 To whom correspondence should be addressed; e-mail: hogawa{at}cc.ocha.ac.jp
Received on August 31, 2005; revised on May 18, 2006; accepted on June 11, 2006
ß-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 ß-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 ß-glucuronidase (BLG) from other glycosidases was tested. ß-Glucuronidase and contaminating glycosidases in commercial BLG preparations bound to and were coeluted from adsorbents immobilized with the substrate or an inhibitor of ß-glucuronidase, whereas ß-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 ß-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 ß-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 ß-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 ß-glucuronidase, which is different from the substrate recognition, is discussed.
Key words: affinity purification / glycoprobe / lactose and N-acetyllactosamine-binding / lysosomal enzyme / zymography / ß-glucuronidase
1 These two authors contributed equally to this article.