Purification and characterization of recombinant human lysosomal α-mannosidase
Purification and characterization of recombinant human lysosomal α-mannosidase
复制标题
DOI：
10.1006/mgme.2001.3173
复制
发表时间：
2001-05-01
影响因子：
3.8
通讯作者：
Hopwood, JJ
中科院分区：
文献类型：
--
作者：
Berg, T;King, B;Hopwood, JJ
糖蛋白的天冬酰胺连接的碳水化合物的降解需要溶酶体α-甘露糖苷酶(EC 3.2.1.24)。该酶的缺乏会导致溶酶体贮积症α-甘露糖苷贮积症。作为α-甘露糖苷贮积症酶替代疗法的第一步，将人溶酶体α-甘露糖苷酶cDNA克隆到pcDNA 3.1载体中并在中国仓鼠卵巢细胞中表达。在细胞培养基中添加二甲亚砜 (DMSO) 可诱导生长停滞，导致酶产量增加 4 倍，平均产量为 3.2 mg L-1 day(-1)。 α-甘露糖苷酶作为 130 kDa 前体的活性同二聚体分泌，在随后的酶纯化过程中被蛋白水解成 55 和 72 kDa 的两种多肽。纯化酶的 N 端序列分析表明，蛋白水解发生在先前在溶酶体 α-甘露糖苷酶的细胞内形式中鉴定的切割位点附近。针对重组酶的单克隆抗体的产生使得开发α-甘露糖苷酶的单步免疫亲和纯化程序成为可能。主要由 130 kDa 前体组成的免疫亲和纯化酶表现出与加工形式相似的比活性和动力学。重组α-甘露糖苷酶被培养的α-甘露糖苷沉积成纤维细胞吸收，并通过甘露糖6-磷酸途径运输到溶酶体，在那里它减少了储存的含甘露糖寡糖的量。 (C) 2001 年学术出版社。
Lysosomal alpha -mannosidase (EC 3.2.1.24) is required in the degradation of the asparagine-linked carbohydrates of glycoproteins, Deficiency of this enzyme leads to the lysosomal storage disorder alpha -mannosidosis. As an initial step toward enzyme replacement therapy for alpha -mannosidosis, the human lysosomal alpha -mannosidase cDNA was cloned into the pcDNA 3.1 vector and expressed in Chinese hamster ovary cells. Dimethyl sulfoxide (DMSO) added to the cell culture media to induce growth arrest led to a 4-fold increase in the enzyme production, with an average yield of 3.2 mg L-1 day(-1). alpha -Mannosidase was secreted as an active homodimer of a 130-kDa precursor that was proteolyzed into two polypeptides of 55 and 72 kDa during the subsequent purification of the enzyme. N-terminal sequence analysis of the purified enzyme revealed that the proteolysis occurred close to a cleavage site previously identified in the intracellular form of lysosomal alpha -mannosidase. Generation of monoclonal antibodies against the recombinant enzyme made it possible to develop a single-step immunoaffinity purification procedure for alpha -mannosidase. The immunoaffinity-purified enzyme which mainly consisted of the 130-kDa precursor, displayed specific activity and kinetics similar to those of the processed form. Recombinant alpha -mannosidase was taken up by cultured alpha -mannosidosis fibroblasts and was trafficked to the lysosomes via the mannose 6-phosphate pathway where it reduced the amounts of stored mannose-containing oligosaccharides. (C) 2001 Academic Press.