Mutation at Glu23 eliminates the neuron growth inhibitory activity of human metallothionein-3

Zhi-Chun Ding, Xin Chen Teng, Bin Cai, Hui Wang, Qi Zheng, Yang Wang, Guo Ming Zhou, Ming Jie Zhang, Hou Ming Wu, Hong Zhe Sun, Zhong Xian Huang

Research output: Contribution to journalArticle

16 Scopus citations

Abstract

Human metallothionein-3 (hMT3), first isolated and identified as a neuronal growth inhibitory factor (GIF), is a metalloprotein expressed predominantly in brain. However, untill 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 β-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.

Original languageEnglish (US)
Pages (from-to)674-682
Number of pages9
JournalBiochemical and Biophysical Research Communications
Volume349
Issue number2
DOIs
StatePublished - Oct 20 2006

Keywords

  • Cell culture assay
  • Human metallothionein-3
  • Mutants
  • NMR
  • Neuron growth inhibitory factor
  • S-Nitrosylation

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Mutation at Glu23 eliminates the neuron growth inhibitory activity of human metallothionein-3'. Together they form a unique fingerprint.

  • Cite this

    Ding, Z-C., Teng, X. C., Cai, B., Wang, H., Zheng, Q., Wang, Y., Zhou, G. M., Zhang, M. J., Wu, H. M., Sun, H. Z., & Huang, Z. X. (2006). Mutation at Glu23 eliminates the neuron growth inhibitory activity of human metallothionein-3. Biochemical and Biophysical Research Communications, 349(2), 674-682. https://doi.org/10.1016/j.bbrc.2006.08.090