Matsushita-Oikawa Hiroko, Komatsu Mayumi, Iida-Tanaka Naoko, Sakagami Hiromi, Kanamori Tetsuko, Matsumoto Isamu, Seno Nobuko, Ogawa Haruko
Course of Advanced Biosciences, Graduate School of Humanities and Sciences, Tokyo, Japan.
Glycobiology. 2006 Oct;16(10):891-901. doi: 10.1093/glycob/cwl016. Epub 2006 Jun 13.
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.
β-葡萄糖醛酸酶是一种溶酶体酶，在生理和炎症状态下，它在糖胺聚糖的正常周转以及细胞外基质成分的重塑中起着至关重要的作用。β-葡萄糖醛酸酶的酶活性调节机制和蛋白质靶向作用对多种治疗方法的开发具有重要意义。在本研究中，测试了各种固定化碳水化合物吸附剂从其他糖苷酶中分离牛肝β-葡萄糖醛酸酶（BLG）的有效性。商业BLG制剂中的β-葡萄糖醛酸酶和污染性糖苷酶与固定有β-葡萄糖醛酸酶底物或抑制剂的吸附剂结合并共同洗脱，而在所测试的吸附剂中，发现β-葡萄糖醛酸酶仅与内酰胺基琼脂糖结合，并能有效地与其他酶分离。结合和洗脱研究表明，β-葡萄糖醛酸酶与内酰胺基琼脂糖的相互作用是pH依赖性的且具有碳水化合物特异性。通过内酰胺基亲和色谱和随后的阴离子交换高效液相色谱（HPLC）将BLG纯化至同质。发现乳糖以非竞争性方式激活β-葡萄糖醛酸酶，这表明乳糖结合位点与底物结合位点不同。与生物素化糖蛋白、脂质和合成糖探针的结合研究表明，β-葡萄糖醛酸酶在中性pH下与含N-乙酰乳糖胺/乳糖的糖缀合物结合。结果表明BLG中存在N-乙酰乳糖胺/乳糖结合活性，并提供了一种利用新型碳水化合物结合活性的有效纯化方法。讨论了β-葡萄糖醛酸酶与底物识别不同的碳水化合物特异性相互作用的生物学意义。
