EXPRESSION OF HUMAN CATHEPSIN D IN XENOPUS OOCYTES - PHOSPHORYLATION AND INTRACELLULAR TARGETING
EXPRESSION OF HUMAN CATHEPSIN D IN XENOPUS OOCYTES - PHOSPHORYLATION AND INTRACELLULAR TARGETING
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DOI：
10.1083/jcb.105.5.1937
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发表时间：
1987-11-01
影响因子：
7.8
通讯作者：
KORNFELD, S
中科院分区：
文献类型：
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作者：
FAUST, PL;WALL, DA;KORNFELD, S
我们获得了人组织蛋白酶D cDNA克隆在非洲爪蟾卵母细胞中的表达。用[35S]甲硫氨酸标记的生物合成研究表明，大部分组织蛋白酶D留在细胞内并进行蛋白水解裂解，将Mr 47,000 D的前体与Mr 43,000-41,000 D的加工中间体从Mr 39,000 D转化为成熟的组织蛋白酶D，其中90%的组织蛋白酶D是由卵母细胞结合到甘糖6-磷酸（Man-6-P）受体亲和柱合成的，表明存在磷酸甘酰基残基。对[2-3H]甘露糖标记的寡糖的分析直接证明了组织蛋白酶D上的磷甘露糖基残基。蔗糖梯度分离用于确定组织蛋白酶D在其生物合成过程中穿过的膜室，表明组织蛋白酶D靶向卵黄血小板亚群，卵黄血小板相当于卵母细胞的溶酶体。爪蟾卵母细胞能够从培养基中内吞糖溶酶体酶，这种摄取被Man-6-P抑制，从而表明这些细胞中存在Man-6-P受体。因此，整个Man-6-P靶向溶酶体酶的途径存在于卵母细胞中。非洲爪蟾卵母细胞应该是一种有用的系统，用于检测负责溶酶体酶特异性靶向溶酶体的信号。
We have obtained expression of a cDNA clone for human cathepsin D in Xenopus laevis oocytes. Biosynthetic studies with [35S]methionine labeling demonstrated that most of the cathepsin D remained intracellular and underwent proteolytic cleavage, converting a precursor of Mr 47,000 D to mature from of Mr 39,000 D with processing intermediates of Mr 43,000-41,000 D. > 90% of the cathepsin D synthesized by oocytes bound to a mannose 6-phosphate (Man-6-P) receptor affinity column, indicating the presence of phosphomannosyl residues. An analysis of [2-3H]mannose-labeled oligosaccharides directly demonstrated phosphomannosyl residues on cathepsin D. Sucrose-gradient fractionation, performed to define the membranous compartments that cathepsin D traversed during its biosynthesis, demonstrated that cathepsin D is targeted to a subpopulation of yolk platelets, the oocyte equivalent of a lysosome. Xenopus oocytes were able to endocytose lysosomal enzymes from the medium and this uptake was inhibited by Man-6-P, thus demonstrating the presence of Man-6-P receptors in these cells. Therefore, the entire Man-6-P dependent pathway for targeting of lysosomal enzymes is present in the oocytes. Xenopus oocytes should be a useful system for examining signals responsible for the specific targeting of lysosomal enzymes to lysosomes.