Purification and characterization of recombinant, human acid ceramidase - Catalytic reactions and interactions with acid sphingomyelinase
Purification and characterization of recombinant, human acid ceramidase - Catalytic reactions and interactions with acid sphingomyelinase
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DOI：
10.1074/jbc.m301936200
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发表时间：
2003-08-29
影响因子：
4.8
通讯作者：
Schuchman, EH
中科院分区：
文献类型：
--
作者：
He, XX;Okino, N;Schuchman, EH
通过扩增转染的全长 cDNA，在中国仓鼠卵巢细胞中过表达人酸性神经酰胺酶。大部分过表达的酶被分泌到培养基中并纯化至明显的同质性。纯化的蛋白质含有与天然来源的人类酸性神经酰胺酶相同的 13-(α) 和 40(β)-kDa 亚基，具有酸性最适 pH (4.5)，并遵循正常的 Michaelis-Menten 动力学，使用 C-14 和 BODIPY 标记的 C-12-神经酰胺作为底物。去糖基化研究表明，重组酶主要含有“高甘露糖”型寡糖，并且存在两个不同的β亚基。这些亚基多肽的氨基酸测序揭示了单个 N 末端，表明类似的 2-4-kDa 分子量差异可能是由于 C 末端加工造成的。纯化的酶还使用 C-14 标记的 C12 脂肪酸和鞘氨醇作为底物在体外催化神经酰胺合成。令人惊讶的是，我们发现来自过度表达仓鼠细胞的培养基具有增加的酸性鞘磷脂酶活性，并且该活性可以使用抗神经酰胺酶抗体与酸性神经酰胺酶共沉淀。正常人皮肤成纤维细胞中酸性神经酰胺酶的过度表达也导致酸性鞘磷脂酶分泌增强，但在尼曼-匹克病细胞中没有观察到这种情况。 RNA研究表明，这种活性的增加并不是由于内源性酸性鞘磷脂酶基因的过度表达。使用小鼠巨噬细胞进行的摄取研究表明，仓鼠细胞培养基中的酸性神经酰胺酶活性可快速内化，但酸性神经鞘磷脂酶则不然。这些研究为酸性神经酰胺酶和相关的脂质水解酶酸性鞘磷脂酶提供了新的见解。
Human acid ceramidase was overexpressed in Chinese hamster ovary cells by amplification of the transfected, full-length cDNA. The majority of the overexpressed enzyme was secreted into the culture media and purified to apparent homogeneity. The purified protein contained the same 13- (alpha) and 40 (beta)-kDa subunits as human acid ceramidase from natural sources, had an acidic pH optimum (4.5), and followed normal Michaelis-Menten kinetics using C-14- and BODIPY-labeled C-12-ceramide as substrates. Deglycosylation studies showed that the recombinant enzyme contained mostly "high mannose" type oligosaccharides and that two distinct beta-subunits were present. Amino acid sequencing of these subunit polypeptides revealed a single N terminus, suggesting that the similar to2-4-kDa molecular mass difference was likely due to C-terminal processing. The purified enzyme also catalyzed ceramide synthesis in vitro using C-14-labeled C12 fatty acid and sphingosine as substrates. Surprisingly, we found that media from the overexpressing hamster cells had increased acid sphingomyelinase activity and that this activity could be coprecipitated with acid ceramidase using anti-ceramidase antibodies. Overexpression of acid ceramidase in normal human skin fibroblasts also led to enhanced acid sphingomyelinase secretion, but this was not observed in Niemann-Pick disease cells. RNA studies showed that this increased activity was not due to overexpression of the endogenous acid sphingomyelinase gene. Uptake studies using mouse macrophages revealed rapid internalization of the acid ceramidase activity from the hamster cell media but not acid sphingomyelinase. These studies provide new insights into acid ceramidase and the related lipid hydrolase, acid sphingomyelinase.