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

Akira Nakashima, Nobuhiro Hayashi, Yoko S. Kaneko, Keiji Mori, Hiromi Egusa, Toshiharu Nagatsu, Akira Ota

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

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-Arg 38 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-52and 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 Met 1-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.

Original languageEnglish
Pages (from-to)110-120
Number of pages11
JournalJournal of Neuroscience Research
Volume81
Issue number1
DOIs
Publication statusPublished - 01-07-2005

All Science Journal Classification (ASJC) codes

  • Cellular and Molecular Neuroscience

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