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Published on: July 11, 2020
Glial cell line-derived neurotrophic factor: characterization of mammalian posttranslational modifications
Elisa Piccinini1, Nisse Kalkkinen, Mart Saarma
1Institute of Biotechnology, University of Helsinki, PB 56 Viikinkaari 9, SF-00014, University of Helsinki, Finland.
Introduction:
Although glial cell line-derived neurotrophic factor (GDNF) has a strong clinical potential, little is known of how the posttranslational modifications of GDNF affect its biological activity and therapeutic potential. In mammalian cells GDNF is synthesized as a preproprotein. During secretion GDNF dimerizes, folds with -S-S- bonds, is modified by N-linked glycosylation, and undergoes proteolytic processing. After production in E. coli, unglycosylated GDNF is renaturated in vitro. Nevertheless, GDNF from E. coli was used in Parkinson's disease-related clinical trials.
Material And Methods:
Constructs encoding variants of human GDNF were generated and expressed in mammalian cells. The proteins were analysed by SDS-PAGE, Western blotting, RET-phosphorylation assays, and N-terminal sequencing. The stability of mammalian GDNF was compared to commercial GDNF produced in E. coli.
Results:
Posttranslational processing of mammalian GDNF depends on the expression conditions. Two forms of GDNF with different N-termini are formed. GDNF without a prosequence is secreted and biologically active. GDNF is modified by N-linked glycosylation at one (Asn(49)) out of two consensus sites. N-linked glycosylation aids proteolytic processing of GDNF. Both glycosylated and unglycosylated GDNF from mammalian cells are more stable than GDNF from E. coli.
Discussion:
Posttranslational modifications of GDNF influence its stability, which may be critical for its clinical use.
