Endocytosis provides a major alternative pathway for lysosomal biogenesis in kidney proximal tubular cells
Endocytosis provides a major alternative pathway for lysosomal biogenesis in kidney proximal tubular cells
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DOI：
10.1073/pnas.0700330104
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发表时间：
2007-03-27
影响因子：
11.1
通讯作者：
Christensen, Erik I.
中科院分区：
文献类型：
--
作者：
Nielsen, Rikke;Courtoy, Pierre J.;Christensen, Erik I.
酸性水解酶向溶酶体的募集通常通过基于transGolgi网络中常见甘露糖6-磷酸信号的识别的细胞内分选和选择性转运至晚期内体/溶酶体而发生。在这里，我们提供了一个替代的，有效的分泌-再捕获途径介导的巨蛋白和组织蛋白酶B在肾脏近曲小管（PCT）的例子的证据。我们发现，在megalin表达缺陷[megalin敲除（KO）]或顶端PCT运输（CIC-5 KO）的小鼠肾脏中，（原）组织蛋白酶B mRNA水平基本保持不变，但蛋白质含量大大降低，并且酶以缺乏甘露糖6-磷酸的形式经尿液排泄。在极化PCT衍生的细胞中，纯化的组织蛋白酶B被贪婪地和选择性地在顶膜上摄取，并且通过巨蛋白竞争者受体相关蛋白来消除摄取。组织蛋白酶B与megalin的直接相互作用通过表面等离子体共振证实。在正常小鼠血清中检测到组织蛋白酶原B。注射到小鼠体内的纯化组织蛋白酶B被肾脏有效吸收（约占注射量的10%），并靶向溶酶体，在溶酶体中保持活性，如放射自显影和亚细胞分级分离所示。向组织蛋白酶B KO小鼠单次注射组织蛋白酶B可重建肾脏中的全部溶酶体酶活性。这些发现证明了循环溶酶体酶在肾小球中连续过滤、通过巨蛋白介导的内吞作用重吸收并转移到溶酶体中发挥其功能的途径，为PCT提供了酶的主要来源。这些结果也扩展了巨蛋白在PCT中的意义，并具有几个生理病理学和临床意义。
Recruitment of acid hydrolases to lysosomes generally occurs by intracellular sorting based on recognition of a common mannose 6-phosphate signal in the transGolgi network and selective transport to late endosomes/lysosomes. Here we provide evidence for an alternative, efficient secretion-recapture pathway mediated by megalin and exemplified by cathepsin B in kidney proximal convoluted tubules (PCT). We found that in mouse kidneys with defective megalin expression [megalin knockout (KO)] or apical PCT trafficking (CIC-5 KO), the (pro)cathepsin B mRNA level was essentially preserved, but the protein content was greatly decreased and the enzyme was excreted in the urine as mannose 6-phosphate-devoid species. In polarized PCT-derived cells, purified cathepsin B was avidly and selectively taken up at the apical membrane, and uptake was abolished by the megalin competitor, receptor-associated protein. Direct interaction of cathepsin B with megalin was demonstrated by surface plasmon resonance. Procathepsin B was detected in normal mouse serum. Purified cathepsin B injected into mice was efficiently taken up by kidneys (approximate to 10% of injection) and targeted to lysosomes where it remained active, as shown by autoradiography and subcellular fractionation. A single cathepsin B injection into cathepsin B KO mice could reconstitute full lysosomal enzyme activity in the kidneys. These findings demonstrate a pathway whereby circulating lysosomal enzymes are continuously filtered in glomeruli, reabsorbed by megalin-mediated endocytosis, and transferred into lysosomes to exert their function, providing a major source of enzymes to PCT. These results also extend the significance of megalin in PCT and have several physiopathological and clinical implications.