POSTTRANSLATIONAL PROCESSING OF MEMBRANE-ASSOCIATED NEU DIFFERENTIATION FACTOR PROISOFORMS EXPRESSED IN MAMMALIAN-CELLS
POSTTRANSLATIONAL PROCESSING OF MEMBRANE-ASSOCIATED NEU DIFFERENTIATION FACTOR PROISOFORMS EXPRESSED IN MAMMALIAN-CELLS
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DOI：
10.1074/jbc.270.9.4775
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发表时间：
1995-03-03
影响因子：
4.8
通讯作者：
WEN, DZ
中科院分区：
文献类型：
--
作者：
LU, HS;HARA, S;WEN, DZ
将人和大鼠前神经分化因子（pro-neu differentiation factor，NDF）cDNA构建表达载体，转染中国仓鼠卵巢细胞，表达重组NDF分子。可溶性NDF形式在膜结合的pro-NDF形式的翻译后加工后释放到培养基中。不同的人和大鼠的NDF亚型，从培养基中纯化后，进行结构和生化表征。分离的人和大鼠NDF亚型已在N末端的特定位点进行了蛋白水解加工，这与从天然来源制备的大鼠或人MIF分子的加工观察到的不同。每个重组NDF亚型在其C末端的加工是异质的，但始终发生在附近的肽键。中国仓鼠卵巢细胞的特异性N-和C-末端加工导致产生两种类型（α和β）的含有222-225个氨基酸残基的重组NDF。人和大鼠NDF分子在三个潜在的Asn-连接的糖基化位点中的两个都是高度糖基化的，并且在11个Thr/Ser位点含有O-连接的糖。糖基化发生在短的富含Ser/Thr的间隔区，该间隔区将N-末端免疫球蛋白同源单元连接至表皮生长因子结构域。细胞磷酸化试验表明，这些分泌形式在乳腺肿瘤细胞受体酪氨酸自磷酸化中具有相似的生物活性。
Expression vectors constructed from human and rat pro-neu differentiation factor (NDF) cDNAs were transfected in Chinese hamster ovary cells for expression of recombinant NDF molecules. Soluble NDF forms were released into culture medium after post-translational processing of the membrane-bound pro-NDF forms. Different human and rat NDF isoforms, after being purified from the culture medium, were subjected to structural and biochemical characterizations. The isolated human and rat NDF isoforms have been proteolytically processed at a specific site at the N terminus, which is different from that observed for the processing of rat or human MIF molecule prepared from natural origins. The processing of each recombinant NDF isoform at its C terminus was heterogeneous but consistently occurred at nearby peptide bonds. Specific N- and C-terminal processing by Chinese hamster ovary cells has resulted in the production of two types (alpha and beta) of recombinant NDFs containing 222-225 amino acid residues. Both human and rat NDF molecules are heavily glycosylated at two of the three potential Asn-linked glycosylation sites and contain O-linked sugars at 11 of the Thr/Ser sites. Glycosylation occurs at a short, Ser/Thr-rich spacer region that connects the N-terminal immunoglobulin homology unit to the epidermal growth factor domain. Cellular phosphorylation assay indicated that these secreted forms contain similar biological activity in receptor tyrosine autophosphorylation of mammary tumor cells.