Defining the pathway for Tat-mediated delivery of beta-glucuronidase in cultured cells and MPS VII mice.
Defining the pathway for Tat-mediated delivery of beta-glucuronidase in cultured cells and MPS VII mice.
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定义培养细胞和 MPS VII 小鼠中 Tat 介导的 β-葡萄糖醛酸酶递送途径。
DOI：
10.1016/j.ymthe.2005.02.031
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Sly,WilliamS
中科院分区：
文献类型：
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作者：
Orii,KojiO;Grubb,JeffreyH;Vogler,Carole;Levy,Beth;Tan,Yun;Markova,Kamelia;Davidson,BeverlyL;Mao,Q;Orii,Tadao;Kondo,Naomi;Sly,WilliamS
我们使用从稳定转染的CHO细胞分泌物中纯化的重组形式的人β-葡萄糖醛酸苷酶（GUS），比较天然酶与含有11个氨基酸HIV达特蛋白转导结构域的GUS-达特C-末端融合蛋白：（1）对培养细胞内吞作用的敏感性，（2）静脉输注后的清除率，和（3）在MPS VII小鼠中输注后清除溶酶体蓄积的组织分布和有效性。我们发现：（1）天然GUS被培养的人成纤维细胞更有效地摄取，其内吞作用仅由M6 P受体介导。GUS-达特融合蛋白仅显示30-50%的M6 P-受体介导的摄取，但也通过带正电荷的达特肽与细胞表面蛋白聚糖的结合而被吸附性内吞作用摄取。(2)GUS-Tat从大鼠循环中清除的速度较慢（t1/2= 13 min vs 7 min）。(3)向MPS VII小鼠的大多数组织的递送是相似的，但GUS-Tat更有效地递送至肾脏。组织学显示GUS-Tat更有效地减少肾小管、视网膜和骨中的储存。这些研究表明，达特修饰可以扩大输注酶校正的组织范围。
We used recombinant forms of human β-glucuronidase (GUS) purified from secretions from stably transfected CHO cells to compare the native enzyme to a GUS–Tat C-terminal fusion protein containing the 11-amino-acid HIV Tat protein transduction domain for: (1) susceptibility to endocytosis by cultured cells, (2) rate of clearance following intravenous infusion, and (3) tissue distribution and effectiveness in clearing lysosomal storage following infusion in the MPS VII mouse. We found: (1) Native GUS was more efficiently taken up by cultured human fibroblasts and its endocytosis was exclusively mediated by the M6P receptor. The GUS–Tat fusion protein showed only 30–50% as much M6P-receptor-mediated uptake, but also was taken up by adsorptive endocytosis through binding of the positively charged Tat peptide to cell surface proteoglycans. (2) GUS–Tat was less rapidly cleared from the circulation in the rat (t1/2= 13 min vs 7 min). (3) Delivery to most tissues of the MPS VII mouse was similar, but GUS–Tat was more efficiently delivered to kidney. Histology showed that GUS–Tat more efficiently reduced storage in renal tubules, retina, and bone. These studies demonstrate that Tat modification can extend the range of tissues corrected by infused enzyme.