Lysosomal proteolysis of prosaposin, the precursor of saposins (sphingolipid activator proteins): Its mechanism and inhibition by ganglioside
Lysosomal proteolysis of prosaposin, the precursor of saposins (sphingolipid activator proteins): Its mechanism and inhibition by ganglioside
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DOI：
10.1006/abbi.1997.9958
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发表时间：
1997-05-01
影响因子：
3.9
通讯作者：
OBrien, JS
中科院分区：
文献类型：
--
作者：
Hiraiwa, M;Martin, BM;OBrien, JS
皂苷A、B、C和D是特定溶酶体水解鞘脂所必需的，它们是由它们共同的前体蛋白——丙皂苷的蛋白水解过程产生的。我们之前的观察表明，溶酶体组织蛋白酶D可能参与了蛋白酶的蛋白水解。在此，我们报道了组织蛋白酶D在蛋白酶水解过程中的参与。一种针对人胎盘组织蛋白酶D的抗体阻断了人睾丸溶酶体蛋白酶混合物（糖蛋白部分）对丙皂苷的蛋白水解活性。使用抗人皂素C单克隆抗体进行免疫印迹分析，组织蛋白酶D显示出与人睾丸糖蛋白部分相似的蛋白水解模式，并将皂素水解成48和29 kDa的产物。K-m和V-max值分别为0.9 μ M和167 nmol/h/mg。n端序列分析表明，48kda为两种三皂苷的混合物，分别包含皂苷a、B、C和皂苷B、C、D的结构域。一项类似的研究也表明，29kda带包含两个去脂蛋白，分别包括皂苷A和B以及皂苷C和D的结构域。通过组织蛋白酶D的长时间处理，disapins被进一步加工成成熟的皂苷A和含有单个皂苷和部分结构域间序列的小片段（14.5-17.5 kDa）。这些小片段不再被组织蛋白酶D处理，而是通过大鼠溶酶体制备的与成熟皂苷相似的分子大小（10.5-11.5 kDa）的片段。这些发现表明，组织蛋白酶D参与了皂苷的成熟，但除了组织蛋白酶D之外，其他蛋白酶似乎也参与了溶酶体中皂苷B、C和D的成熟。神经节苷类与丙糖苷和皂苷形成复合物，抑制组织蛋白酶d对丙糖苷的蛋白水解。这一发现表明，在体内，通过与神经节苷类形成复合物，可以保护丙糖苷免受溶酶体蛋白水解。(C) 1997学术出版社。
Saposins A, B, C, and D, which are required for the enzymatic hydrolysis of sphingolipids by specific lysosomal hydrolases, are produced by proteolytic processing of their common precursor protein, prosaposin. Our previous observation suggested that lysosomal cathepsin D may be involved in the proteolysis of prosaposin. Herein we report the involvement of cathepsin D in the proteolytic processing of prosaposin. An antibody against human placental cathepsin D blocked the proteolytic activity toward prosaposin in a human testicular lysosomal protease mixture (glycoprotein fraction). On immunoblot analysis using a monoclonal antibody against human saposin C, cathepsin D showed a similar proteolytic pattern as that of a human testicular glycoprotein fraction and hydrolyzed prosaposin into products of 48 and 29 kDa. The K-m and V-max values were 0.9 mu M and 167 nmol/h/mg, respectively. N-Terminal sequence analysis indicated that the 48-kDa band was a mixture of two trisaposins, including domains for saposins A, B, and C and saposins B, C, and D, respectively. A similar study also showed that the 29-kDa band contained two disaposins, including domains for saposins A and B and saposins C and D, respectively. By longer treatment with cathepsin D, disaposins were further processed into mature saposin A and small fragments (14.5-17.5 kDa) containing individual saposins and portions of interdomain sequences. These small fragments were no longer processed by cathepsin D, but trimmed to fragments having similar molecular sizes (10.5-11.5 kDa) to those of mature saposins by a rat lysosome preparation. These findings indicated that cathepsin D is involved in the maturation of saposins but that, in addition to cathepsin D, other proteases appear to be involved in the maturation of saposin B, C, and D in lysosomes. Gangliosides, which specifically form complexes with prosaposin and saposins, inhibit proteolysis of prosaposin by cathepsin D. This finding indicates that prosaposin may be protected from lysosomal proteolysis by forming a complex with gangliosides in vivo. (C) 1997 Academic Press.