Effect of alpha-domain substitution on the structure, property and function of human neuronal growth inhibitory factor.
Human metallothionein-3 (hMT3), also named human neuronal growth inhibitory factor (hGIF), is attractive due to its distinct neuronal growth inhibitory activity, which is not shown by other human MT isoforms. It has been reported that the neuronal growth inhibitory activity arises from the N-terminal beta-domain rather than its C-terminal alpha-domain. However, previous bioassay results have shown that the single beta-domain is less effective at inhibiting the neuron growth than that in intact hMT3 on a molar basis, which suggests that the alpha-domain is indispensable to the neuronal growth inhibitory activity of hMT3. In order to confirm this assumption, we constructed two domain-hybrid mutants, the beta(MT3)-beta(MT3) mutant and the beta(MT3)-alpha(MT1) mutant, and investigated their structural and metal binding properties by UV-vis spectroscopy, CD spectroscopy, pH titration, DTNB reaction, EDTA reaction, etc. The results showed that stability of the Cd(3)S(9) cluster of the beta(MT3)-beta(MT3) mutant decreased significantly while the Cd(3)S(9) cluster of the beta(MT3)-alpha(MT1) mutant had a similar stability and solvent accessibility to that of hMT3. Interestingly, the bioassay results showed that the neuronal growth inhibitory activity of the beta(MT3)-beta(MT3) mutant decreased significantly, while the beta(MT3)-alpha(MT1) mutant showed similar inhibitory activity to hMT3. Based on these results, we conclude that the alpha-domain is indispensable and plays an important role in modulating the stability of the metal cluster in the beta-domain by domain-domain interactions, thus influencing the bioactivity of hMT3.
Ding
Zhi-Chun
ZC
Chemical Biology Lab, Department of Chemistry, Fudan University, Shanghai, China.
Zheng
Qi
Q
Cai
Bin
B
Yu
Wen-Hao
WH
Teng
Xin-Chen
XC
Wang
Yang
Y
Zhou
Guo-Ming
GM
Wu
Hou-Ming
HM
Sun
Hong-Zhe
HZ
Zhang
Ming-Jie
MJ
Huang
Zhong-Xian
ZX
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D006801:Humans; D008668:Metallothionein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000090023:Metallothionein 3; D008969:Molecular Sequence Data; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology
D000090023:Metallothionein 3; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D008668:Metallothionein
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