Deletion of N-terminus of human tyrosine hydroxylase type 1 enhances stability of the enzyme in AtT-20 cells.
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.
Nakashima
Akira
A
Department of Physiology, School of Medicine, Fujita Health University, Toyoake, Aichi, Japan.
Hayashi
Nobuhiro
N
Kaneko
Yoko S
YS
Mori
Keiji
K
Egusa
Hiromi
H
Nagatsu
Toshiharu
T
Ota
Akira
A
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D020134:Catalytic Domain / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D004298:Dopamine / Q000378:metabolism*; D004795:Enzyme Stability / Q000235:genetics / Q000502:physiology; D025461:Feedback, Physiological / Q000502:physiology; D017353:Gene Deletion; D000882:Haplorhini; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009490:Neurosecretory Systems / Q000166:cytology / Q000201:enzymology*; D010446:Peptide Fragments / Q000378:metabolism; D017354:Point Mutation / Q000502:physiology; D013329:Structure-Activity Relationship; D014162:Transfection; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics / Q000378:metabolism*
D010446:Peptide Fragments; D014446:Tyrosine 3-Monooxygenase; D004298:Dopamine