Evaluation of artificial signal peptides for secretion of two lysosomal enzymes in CHO cells.
Evaluation of artificial signal peptides for secretion of two lysosomal enzymes in CHO cells.
复制标题
DOI：
10.1042/bcj20210015
复制
发表时间：
2021-06-25
影响因子：
4.1
通讯作者：
Chou, Tsui-Fen
中科院分区：
文献类型：
--
作者：
Cheng, Kai-Wen;Wang, Feng;Lopez, George A.;Singamsetty, Srikanth;Wood, Jill;Dickson, Patricia, I;Chou, Tsui-Fen
酶替代疗法（ERT）是一种科学合理的和临床证明的治疗溶酶体贮积病。用于ERT的大多数酶是从哺乳动物细胞的培养上清液中纯化的。然而，由于溶酶体酶在哺乳动物细胞系统中的低分泌水平，以足够的质量和数量纯化溶酶体酶以供临床使用是具有挑战性的。为了提高重组溶酶体酶的分泌效率，我们研究了人工信号肽对重组溶酶体酶在中国仓鼠卵巢（CHO）细胞系中分泌的影响。我们用9种不同的来源于高分泌蛋白的信号肽取代了两种重组人溶酶体酶N-乙酰-α-氨基葡萄糖苷酶（rhNAGLU）和氨基葡萄糖（N-乙酰）-6-硫酸酯酶（rhGNS）的天然信号肽，并在CHO K1细胞中表达。当比较天然信号肽时，我们发现rhGNS以高于rhNAGLU的水平分泌到培养基中。然而，rhNAGLU和rhGNS的分泌可以通过改变信号肽来小心地控制。小鼠IGκ轻链信号肽和人胰凝乳蛋白酶原B1信号肽对rhNAGLU的分泌作用相对较高，而IGκ轻链信号肽1和人胰凝乳蛋白酶原B1信号肽对rhGNS的分泌作用较强，提示人胰凝乳蛋白酶原B1信号肽最适合增加溶酶体酶的分泌。总的来说，我们的结果表明，改变信号肽可以调节重组溶酶体酶的分泌，并将使溶酶体酶的生产用于临床使用。
Enzyme replacement therapy (ERT) is a scientifically rational and clinically proven treatment for lysosomal storage diseases. Most enzymes used for ERT are purified from the culture supernatant of mammalian cells. However, it is challenging to purify lysosomal enzymes with sufficient quality and quantity for clinical use due to their low secretion levels in mammalian cell systems. To improve the secretion efficiency of recombinant lysosomal enzymes, we evaluated the impact of artificial signal peptides on the production of recombinant lysosomal enzymes in Chinese Hamster Ovary (CHO) cell lines. We engineered two recombinant human lysosomal enzymes, N-acetyl-α-glucosaminidase (rhNAGLU) and glucosamine (N-acetyl)-6-sulfatase (rhGNS), by replacing their native signal peptides with 9 different signal peptides derived from highly secretory proteins and expressed them in CHO K1 cells. When comparing the native signal peptides, we found that rhGNS was secreted into media at higher levels than rhNAGLU. The secretion of rhNAGLU and rhGNS can, however, be carefully controlled by altering signal peptides. The secretion of rhNAGLU was relatively higher with murine Igκ light chain and human chymotrypsinogen B1 signal peptides, whereas Igκ light chain signal peptide 1 and human chymotrypsinogen B1 signal peptides were more effective for rhGNS secretion, suggesting that human chymotrypsinogen B1 signal peptide is the most appropriate for increasing lysosomal enzyme secretion. Collectively, our results indicate that altering signal peptide can modulate the secretion of recombinant lysosome enzymes and will enable lysosomal enzyme production for clinical use.