title: Effect of alpha-domain substitution on the structure, property and function of human neuronal growth inhibitory factor.
abstract: 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.
authors: Chemical Biology Lab, Department of Chemistry, Fudan University, Shanghai, China.
authors: Zhi-Chun
authors: ZC
authors: Ding
authors: Qi
authors: Q
authors: Zheng
authors: Bin
authors: B
authors: Cai
authors: Wen-Hao
authors: WH
authors: Yu
authors: Xin-Chen
authors: XC
authors: Teng
authors: Yang
authors: Y
authors: Wang
authors: Guo-Ming
authors: GM
authors: Zhou
authors: Hou-Ming
authors: HM
authors: Wu
authors: Hong-Zhe
authors: HZ
authors: Sun
authors: Ming-Jie
authors: MJ
authors: Zhang
authors: Zhong-Xian
authors: ZX
authors: Huang
mesh_terms: 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
chemical_list: D000090023:Metallothionein 3; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D008668:Metallothionein
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