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Genetic disruption of multiple ?1,2-mannosidases generates mammalian cells producing recombinant proteins with high-mannose-type N-glycans.

ABSTRACT: Recombinant therapeutic proteins are becoming very important pharmaceutical agents for treating intractable diseases. Most biopharmaceutical proteins are produced in mammalian cells because this ensures correct folding and glycosylation for protein stability and function. However, protein production in mammalian cells has several drawbacks, including heterogeneity of glycans attached to the produced protein. In this study, we established cell lines with high-mannose-type N-linked, low-complexity glycans. We first knocked out two genes encoding Golgi mannosidases (MAN1A1 and MAN1A2) in HEK293 cells. Single knockout (KO) cells did not exhibit changes in N-glycan structures, whereas double KO cells displayed increased high-mannose-type and decreased complex-type glycans. In our effort to eliminate the remaining complex-type glycans, we found that knocking out a gene encoding the endoplasmic reticulum mannosidase I (MAN1B1) in the double KO cells reduced most of the complex-type glycans. In triple KO (MAN1A1, MAN1A2, and MAN1B1) cells, Man9GlcNAc2 and Man8GlcNAc2 were the major N-glycan structures. Therefore, we expressed two lysosomal enzymes, ?-galactosidase-A and lysosomal acid lipase, in the triple KO cells and found that the glycans on these enzymes were sensitive to endoglycosidase H treatment. The N-glycan structures on recombinant proteins expressed in triple KO cells were simplified and changed from complex types to high-mannose types at the protein level. Our results indicate that the triple KO HEK293 cells are suitable for producing recombinant proteins, including lysosomal enzymes with high-mannose-type N-glycans.

SUBMITTER: Jin ZC

PROVIDER: S-EPMC5900765 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Genetic disruption of multiple α1,2-mannosidases generates mammalian cells producing recombinant proteins with high-mannose-type <i>N</i>-glycans.

Jin Ze-Cheng ZC Kitajima Toshihiko T Dong Weijie W Huang Yi-Fan YF Ren Wei-Wei WW Guan Feng F Chiba Yasunori Y Gao Xiao-Dong XD Fujita Morihisa M

The Journal of biological chemistry 20180223 15

Recombinant therapeutic proteins are becoming very important pharmaceutical agents for treating intractable diseases. Most biopharmaceutical proteins are produced in mammalian cells because this ensures correct folding and glycosylation for protein stability and function. However, protein production in mammalian cells has several drawbacks, including heterogeneity of glycans attached to the produced protein. In this study, we established cell lines with high-mannose-type <i>N</i>-linked, low-com ...[more]

PMID: 29475941

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Dataset Information

Genetic disruption of multiple ?1,2-mannosidases generates mammalian cells producing recombinant proteins with high-mannose-type N-glycans.

Publications

Genetic disruption of multiple α1,2-mannosidases generates mammalian cells producing recombinant proteins with high-mannose-type <i>N</i>-glycans.

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