TY - JOUR
T1 - Deletion of N-terminus of human tyrosine hydroxylase type 1 enhances stability of the enzyme in AtT-20 cells
AU - Nakashima, Akira
AU - Hayashi, Nobuhiro
AU - Kaneko, Yoko S.
AU - Mori, Keiji
AU - Egusa, Hiromi
AU - Nagatsu, Toshiharu
AU - Ota, Akira
PY - 2005/7/1
Y1 - 2005/7/1
N2 - 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.
AB - 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.
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U2 - 10.1002/jnr.20540
DO - 10.1002/jnr.20540
M3 - Article
C2 - 15898085
AN - SCOPUS:23744476295
SN - 0360-4012
VL - 81
SP - 110
EP - 120
JO - Journal of Neuroscience Research
JF - Journal of Neuroscience Research
IS - 1
ER -