The effects of chemically synthesized saposin C onglucosylceramide-β-glucosidase.
The effects of chemically synthesized saposin C onglucosylceramide-β-glucosidase.
复制标题
化学合成的皂苷 C 对葡萄糖神经酰胺-β-葡萄糖苷酶的影响。
DOI：
10.1016/j.clinbiochem.2015.06.004
复制
发表时间：
2015
影响因子：
2.8
通讯作者：
Matsuda J.
中科院分区：
文献类型：
--
作者：
Yoneshige A;Muto M;Watanabe T;Hojo H;Matsuda J.
目的血清载脂蛋白C （saposin C, SAP-C）是高谢氏病中缺乏的葡萄糖神经酰胺-β-葡萄糖苷酶（GCase）的重要激活因子。在本研究中，我们研究了化学合成的SAP-Cs（合成SAP-Cs）对GCase的影响。方法采用酶联免疫印迹（western blot）分析方法，评价两种合成SAP-Cs（非糖基化形式和an -糖基化形式）对GCase的激活、稳定和保护作用。以吡格酶（Cerezyme）作为GCase。为了模拟生理条件，我们在含有双（单酰基甘油）磷酸、1 -α-磷脂酰胆碱和胆固醇的4-硝基苯-2-氧-1,3-二唑标记的含葡萄糖脂质体中检测GCase的活性。结果gcase活性随合成SAP-Cs浓度的增加而增加。1 μM浓度的SAP-Cs可显著提高GCase活性，提高幅度为14 ~ 22倍（未糖基化SAP-C: 22.9±0.16；未糖基化SAP-C: 14.9±0.19；未糖基化SAP-C: 1.05±0.035 pmol/h/ng GCase）。这些值等于或超过了先前发表的使用重组非糖基化SAP-C获得的值。两种合成的SAP-Cs在37℃下稳定GCase的效果与牛血清白蛋白相同。Western blot分析显示合成的SAP-Cs特异性地保护GCase免受组织蛋白酶D的消化。结论这些新型化学合成的SAP-Cs具有激活剂、稳定剂和GCase保护剂的功能，可用于戈谢病患者的酶替代治疗。
ObjectiveSaposin C (SAP-C) is an essential activator of glucosylceramide (GlcCer)-β-glucosidase (GCase), the enzyme deficient in Gaucher's disease. In this study, we investigated the effects of chemically synthesized SAP-Cs (synthetic SAP-Cs) on GCase.MethodsEnzymatic assays and western blot analyses were carried out to evaluate the effects of two kinds of synthetic SAP-Cs, a non-glycosylated form and aN-glycosylated form bearing a complex type nonasaccharide, on GCase with respect to its activation, stabilization, and protection. Imiglucerase (Cerezyme) was used as the GCase. To mimic physiological conditions, GCase activity was assayed in the presence of 4-nitrobenzo-2-oxa-1,3-diazole-labeled GlcCer-containing liposomes composed of bis(monoacylglycero)phosphate,l-α-phosphatidylcholine, and cholesterol.ResultsGCase activities increased depending on the concentration of synthetic SAP-Cs. SAP-Cs at a concentration of 1 μM increased GCase activities significantly, by 14- to 22-fold (non-glycosylated SAP-C: 22.9 ± 0.16; nona-glycosylated SAP-C: 14.9 ± 0.19; without SAP-C: 1.05 ± 0.035 pmol/h/ng GCase). These values equaled or surpassed previously published values obtained using recombinant non-glycosylated SAP-C. Both synthetic SAP-Cs were as effective as bovine serum albumin in stabilizing GCase at 37 °C. Western blot analysis revealed that synthetic SAP-Cs specifically protected GCase from cathepsin D digestion.ConclusionsThe results demonstrate that these novel, chemically synthesized SAP-Cs function as activators, stabilizers, and protectors of GCase, suggesting their utility in enzyme replacement therapy in patients with Gaucher's disease.