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Evaluation of artificial signal peptides for secretion of two lysosomal enzymes in CHO cells

Cheng, Kai-Wen and Wang, Feng and Lopez, George A. and Singamsetty, Srikanth and Wood, Jill and Dickson, Patricia I. and Chou, Tsui-Fen (2021) Evaluation of artificial signal peptides for secretion of two lysosomal enzymes in CHO cells. Biochemical Journal, 478 (12). pp. 2309-2319. ISSN 0264-6021. PMCID PMC9074758. doi:10.1042/bcj20210015.

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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 nine 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.

Item Type:Article
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URLURL TypeDescription CentralArticle
Cheng, Kai-Wen0000-0001-9888-9773
Chou, Tsui-Fen0000-0003-2410-2186
Additional Information:© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society. Received: January 12 2021; Revision Received: May 24 2021; Accepted: May 24 2021; Accepted Manuscript online: May 25 2021. This project was supported in part by the National Institute of Neurological Disorders and Stroke U44NS089061 to Phoenix Nest Inc. We thank William Rosencrans and Nadia Houerbi for proofreading our final manuscript. Author contributions statement: T.-F. Chou conceived the project. K.-W. Cheng designed and performed experiments. F. Wang constructed the pOptiVEC-TOPO-rhGNS-TEV-myc plasmid. G. A. Lopez constructed the pOptiVECTOPO-rhNAGLU-myc plasmid. F. Wang and G. A. Lopez assisted in eliminating problems during experiments and provided suggestions. K.-W. Cheng, and T.-F. Chou wrote the manuscript. S. Singamsetty, J. Wood, and P.I. Dickson, and T.-F. Chou provided suggestions, edited the manuscript and obtained funding for this work. The authors declare no conflicts of interest. Disclosure: SS, and JW are Phoenix Nest Inc. employees. Data availability statement: Most data generated during this study are included in the article and its Supplementary Information. Uncropped images of all gels and blots can be found in Supplementary Figures 1 to 3. Additional measurements such as protein concentrations and enzymatic activities generated during the current study and relevant information are available from the corresponding authors upon request.
Funding AgencyGrant Number
Subject Keywords:N-acetyl-α-glucosaminidase (NAGLU), enzyme replacement therapy, mucopolysaccharidosis, N-acetylglucosamine-6-sulfatase (GNS), signal peptide
Issue or Number:12
PubMed Central ID:PMC9074758
Record Number:CaltechAUTHORS:20210723-172036186
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Official Citation:Kai-Wen Cheng, Feng Wang, George A. Lopez, Srikanth Singamsetty, Jill Wood, Patricia I. Dickson, Tsui-Fen Chou; Evaluation of artificial signal peptides for secretion of two lysosomal enzymes in CHO cells. Biochem J 25 June 2021; 478 (12): 2309–2319. doi:
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:109997
Deposited By: Tony Diaz
Deposited On:26 Jul 2021 19:05
Last Modified:12 Jul 2022 18:15

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