Glial cell line-derived neurotrophic factor: Characterization of mammalian posttranslational modifications

Abstract

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 (Asn⁴⁹) 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.

Key words::

• Clinical trial
• glial cell line-derived neurotrophic factor
• posttranslational protein processing

Acknowledgements

We thank Mari Heikkinen and Denise Heinrich for excellent technical assistance, Maria Lume and Mikko Airavaara for critical reading of the manuscript, and Icosagen Ltd (previously Quattromed Ltd) for expression of GDNF with high-expression technology in CHO cells. This work was supported by Academy of Finland (grant numbers 11411226 and 1126735), S. Jusélius foundation, an Erasmus Placement scholarship, IUBMB (Wood-Whelan fellowship), CIMO (Finnish Government Scholarship Pool), and a University of Helsinki Research Foundation grant.

Declaration of interest: The authors report no conflicts of interest.