Deletion of N-terminus of human tyrosine hydroxylase type 1 enhances stability of the enzyme in AtT-20 cells

• Published in: Journal of neuroscience research Vol. 81; no. 1; pp. 110 - 120
• Main Authors: Nakashima, Akira, Hayashi, Nobuhiro, Kaneko, Yoko S., Mori, Keiji, Egusa, Hiromi, Nagatsu, Toshiharu, Ota, Akira
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 01.07.2005
• Subjects: Amino Acid Sequence; Animals; Catalytic Domain - genetics; Catalytic Domain - physiology; Cell Line; COS Cells; dopamine; Dopamine - metabolism; Enzyme Stability - genetics; Enzyme Stability - physiology; Feedback, Physiological - physiology; Gene Deletion; Haplorhini; Humans; Mice; Molecular Sequence Data; Neurosecretory Systems - cytology; Neurosecretory Systems - enzymology; Peptide Fragments - metabolism; PEST motif; Point Mutation - physiology; stability; Structure-Activity Relationship; Transfection; Tyrosine 3-Monooxygenase - genetics; Tyrosine 3-Monooxygenase - metabolism
• ISSN: 0360-4012; 1097-4547
• DOI: 10.1002/jnr.20540

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 Arg37‐Arg38 was replaced by Glu37‐Glu38; and S40D, one in which Ser40 was replaced by Asp40) 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 = del‐157 = 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 N‐terminus sequence of Met1‐Lys12 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. © 2005 Wiley‐Liss, Inc.