Deletion of N-terminus of human tyrosine hydroxylase type 1 enhances stability of the enzyme in AtT-20 cells
✍ Scribed by Akira Nakashima; Nobuhiro Hayashi; Yoko S. Kaneko; Keiji Mori; Hiromi Egusa; Toshiharu Nagatsu; Akira Ota
- Publisher
- John Wiley and Sons
- Year
- 2005
- Tongue
- English
- Weight
- 558 KB
- Volume
- 81
- Category
- Article
- ISSN
- 0360-4012
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✦ Synopsis
Wildtype human tyrosine hydroxylase (TH) type 1 and 4 mutants (del-52, a form with the first 52 amino acid residues deleted; del-157, one with the first 157 amino acid residues deleted; RR-EE, one in which Arg 37 -Arg 38 was replaced by Glu 37 -Glu 38 ; and S40D, one in which Ser 40 was replaced by Asp 40 ) were expressed in AtT-20 mouse neuroendocrine cells in order to clarify how deeply the N-terminus of TH is involved in the efficient production of dopamine (DA) in mammalian cells. The amounts of DA that accumulated in AtT-20 cells expressing these human TH type 1 (hTH1) phenotypes were in the following order: del-52 ¼ del-157 ¼ RR-EE > S40D > wildtype, although the enzyme activities of del-52 and del-157 were lower than those of wildtype, RR-EE, and S40D. The observation on immunoblot analyses that the N-terminus-deleted hTH1 mutants were much more stable than wildtype can reconcile the discrepant results. Computer-assisted analysis of the spatial configuration of hTH1 identified five newly recognized PEST motifs, one of which was located in the Nterminus sequence of Met 1 -Lys 12 and predicted that deletion of the N-terminus region would alter the secondary structure within the catalytic domain. Collectively, the high stability of the N-terminus-deleted hTH1 mutants can be generated by the loss of a PEST motif in their N-termini and the structural change in the catalytic domain, which would promise an efficient production of DA in mammalian cells expressing N-terminus deleted hTH1. V