title: Glial cell line-derived neurotrophic factor: characterization of mammalian posttranslational modifications.
abstract: BACKGROUND
  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.
  
  
  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.
  
  
  CONCLUSIONS
  Posttranslational modifications of GDNF influence its stability, which may be critical for its clinical use.
authors.affiliation: Institute of Biotechnology, University of Helsinki, PB 56 Viikinkaari 9, SF-00014, University of Helsinki, Finland.
authors.forename: Elisa
authors.initials: E
authors.lastname: Piccinini
authors.forename: Nisse
authors.initials: N
authors.lastname: Kalkkinen
authors.forename: Mart
authors.initials: M
authors.lastname: Saarma
authors.forename: Pia
authors.initials: P
authors.lastname: Runeberg-Roos
mesh_terms: D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003470:Culture Media; D004926:Escherichia coli; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000737:chemistry / Q000378:metabolism*; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors / Q000378:metabolism; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D011134:Polysaccharides / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D055550:Protein Stability
chemical_list: D003470:Culture Media; D051100:Glial Cell Line-Derived Neurotrophic Factor; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors; D011134:Polysaccharides; D011498:Protein Precursors