Mutation at Glu23 eliminates the neuron growth inhibitory activity of human metallothionein-3.
Human metallothionein-3 (hMT3), first isolated and identified as a neuronal growth inhibitory factor (GIF), is a metalloprotein expressed predominantly in brain. However, until now, the exact mechanism of the bioactivity of hMT3 is still unknown. In order to study the influence of acid-base catalysis on S-nitrosylation of hMT3, we constructed the E23K mutant of hMT3. During the course of bioassay, we found out unexpectedly that mutation at E23 of hMT3 eliminates the neuronal growth inhibitory activity completely. To the best of our knowledge, it is the first report that other residues, besides the TCPCP motif, in the beta-domain can alter the bioactivity of hMT3. In order to figure out the causes for the loss of bioactivity of the E23K mutant, the biochemical properties were characterized by UV-vis spectroscopy, CD spectroscopy, pH titration, DTNB reaction, EDTA reaction, and SNOC reaction. All data demonstrated that stability of the metal-thiolate cluster and overall structure of the E23K mutant were not altered too much. However, the reaction of the E23K mutant with SNOC exhibited biphasic kinetics and the mutant protein released zinc ions much faster than hMT3 in the initial step, while hMT3 exhibited single kinetic process. The 2D [1H-15N] HSQC was also employed to characterize structural changes during the reaction of hMT3 with varying mounts of nitric oxide. It was shown that the resonance of Glu23 disappeared at a molar ratio of NO to protein of 4. Based on these results, we suggest that mutation at Glu23 may alter the NO metabolism and/or affect zinc homeostasis in brain, thus altering the neuronal growth inhibitory activity.
Ding
Zhi-Chun
ZC
Chemical Biology Lab, Department of Chemistry, Fudan University, Shanghai 200433, China.
Teng
Xin-Chen
XC
Cai
Bin
B
Wang
Hui
H
Zheng
Qi
Q
Wang
Yang
Y
Zhou
Guo-Ming
GM
Zhang
Ming-Jie
MJ
Wu
Hou-Ming
HM
Sun
Hong-Zhe
HZ
Huang
Zhong-Xian
ZX
D000818:Animals; D001921:Brain / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D018698:Glutamic Acid / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D000090023:Metallothionein 3; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D051381:Rats; D014466:Ultraviolet Rays
D000090023:Metallothionein 3; D009419:Nerve Tissue Proteins; D009569:Nitric Oxide; D018698:Glutamic Acid