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Deletion of N‐terminus of human tyrosine hydroxylase type 1 enhances stability of the enzyme in AtT‐20 cells
Author(s) -
Nakashima Akira,
Hayashi Nobuhiro,
Kaneko Yoko S.,
Mori Keiji,
Egusa Hiromi,
Nagatsu Toshiharu,
Ota Akira
Publication year - 2005
Publication title -
journal of neuroscience research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.72
H-Index - 160
eISSN - 1097-4547
pISSN - 0360-4012
DOI - 10.1002/jnr.20540
Subject(s) - mutant , wild type , tyrosine , c terminus , n terminus , enzyme , biology , tyrosine hydroxylase , microbiology and biotechnology , amino acid , peptide sequence , biochemistry , gene
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 = 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 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. © 2005 Wiley‐Liss, Inc.