Perkins K J, Byers S, Yogalingam G, Weber B, Hopwood J J
Lysosomal Disorders Research Unit, Department of Chemical Pathology, Women's and Children's Hospital, 72 King William Rd., North Adelaide, South Australia 5006, Australia.
J Biol Chem. 1999 Dec 24;274(52):37193-9. doi: 10.1074/jbc.274.52.37193.
Mucopolysaccharidosis IIIA (MPS-IIIA) is an autosomal recessive lysosomal storage disorder caused by the deficiency of sulfamidase (NS; EC 3.10.1.1), resulting in defective degradation and storage of heparan sulfate. This paper reports the production and characterization of monoclonal and polyclonal antibodies against recombinant human sulfamidase (rhNS) to quantitate and characterize normal and mutant sulfamidase produced from the wild type NS expression vector. Glycosylation and phosphorylation studies of immunoprecipitated rhNS show that all five potential glycosylation sites are utilized, with three high mannose/hybrid oligosaccharides and two simpler chains, with at least one functional mannose 6-phosphate group. An NS quantification system was developed to determine the effect of the three most common and severe patient mutations: S66W (Italy), R74C (Poland), and R245H (The Netherlands). The quantity and specific activity of expressed mutant rhNS was significantly lower than expressed normal rhNS, with 0.3, 0.2, and 0.05% of normal rhNS produced and 15, 17, and 83% of normal specific activity for S66W, R74C, and R245H observed, respectively. The recent structural elucidation of N-acetylgalactosamine-4-sulfatase was utilized to postulate the effect on the structure-function relationship of NS. The characterization of normal and mutated rhNS has relevance for efficient diagnosis and therapeutic developments for MPS-IIIA patients.
黏多糖贮积症IIIA型（MPS-IIIA）是一种常染色体隐性溶酶体贮积症，由硫酸酰胺酶（NS；EC 3.10.1.1）缺乏引起，导致硫酸乙酰肝素降解和贮积缺陷。本文报道了抗重组人硫酸酰胺酶（rhNS）单克隆抗体和多克隆抗体的制备及特性，以定量和表征由野生型NS表达载体产生的正常和突变硫酸酰胺酶。对免疫沉淀的rhNS进行的糖基化和磷酸化研究表明，所有五个潜在糖基化位点均被利用，有三个高甘露糖/杂合寡糖和两个较简单的链，且至少有一个功能性甘露糖6-磷酸基团。开发了一种NS定量系统，以确定三种最常见且严重的患者突变的影响：S66W（意大利）、R74C（波兰）和R245H（荷兰）。表达的突变rhNS的量和比活性显著低于表达的正常rhNS，对于S66W、R74C和R245H，观察到产生的量分别为正常rhNS的0.3%、0.2%和0.05%，比活性分别为正常的15%、17%和83%。利用最近对N-乙酰半乳糖胺-4-硫酸酯酶的结构解析来推测对NS结构-功能关系的影响。正常和突变rhNS的特性对于MPS-IIIA患者的有效诊断和治疗发展具有重要意义。
