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 Citations (Scopus)

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 [ 1 H- 15 N] 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

Fingerprint

Human Activities
Neurons
Mutation
Growth
Bioactivity
Zinc
Brain
Spectrum Analysis
growth inhibitory factor
Metalloproteins
Dithionitrobenzoic Acid
Kinetics
Bioassay
Mutant Proteins
Ultraviolet spectroscopy
Catalysis
Titration
Metabolism
Edetic Acid
Biological Assay

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

Cite this

Mutation at Glu23 eliminates the neuron growth inhibitory activity of human metallothionein-3. / Ding, Zhi-Chun; Teng, Xin Chen; Cai, Bin; Wang, Hui; Zheng, Qi; Wang, Yang; Zhou, Guo Ming; Zhang, Ming Jie; Wu, Hou Ming; Sun, Hong Zhe; Huang, Zhong Xian.

In: Biochemical and Biophysical Research Communications, Vol. 349, No. 2, 20.10.2006, p. 674-682.

Research output: Contribution to journalArticle

Ding, Z-C, Teng, XC, Cai, B, Wang, H, Zheng, Q, Wang, Y, Zhou, GM, Zhang, MJ, Wu, HM, Sun, HZ & Huang, ZX 2006, 'Mutation at Glu23 eliminates the neuron growth inhibitory activity of human metallothionein-3', Biochemical and Biophysical Research Communications, vol. 349, no. 2, pp. 674-682. https://doi.org/10.1016/j.bbrc.2006.08.090
Ding, Zhi-Chun ; Teng, Xin Chen ; Cai, Bin ; Wang, Hui ; Zheng, Qi ; Wang, Yang ; Zhou, Guo Ming ; Zhang, Ming Jie ; Wu, Hou Ming ; Sun, Hong Zhe ; Huang, Zhong Xian. / Mutation at Glu23 eliminates the neuron growth inhibitory activity of human metallothionein-3. In: Biochemical and Biophysical Research Communications. 2006 ; Vol. 349, No. 2. pp. 674-682.
@article{7236994f27e4462ab5be58e06c59c896,
title = "Mutation at Glu23 eliminates the neuron growth inhibitory activity of human metallothionein-3",
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 [ 1 H- 15 N] 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.",
keywords = "Cell culture assay, Human metallothionein-3, Mutants, NMR, Neuron growth inhibitory factor, S-Nitrosylation",
author = "Zhi-Chun Ding and Teng, {Xin Chen} and Bin Cai and Hui Wang and Qi Zheng and Yang Wang and Zhou, {Guo Ming} and Zhang, {Ming Jie} and Wu, {Hou Ming} and Sun, {Hong Zhe} and Huang, {Zhong Xian}",
year = "2006",
month = "10",
day = "20",
doi = "10.1016/j.bbrc.2006.08.090",
language = "English (US)",
volume = "349",
pages = "674--682",
journal = "Biochemical and Biophysical Research Communications",
issn = "0006-291X",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - Mutation at Glu23 eliminates the neuron growth inhibitory activity of human metallothionein-3

AU - Ding, Zhi-Chun

AU - Teng, Xin Chen

AU - Cai, Bin

AU - Wang, Hui

AU - Zheng, Qi

AU - Wang, Yang

AU - Zhou, Guo Ming

AU - Zhang, Ming Jie

AU - Wu, Hou Ming

AU - Sun, Hong Zhe

AU - Huang, Zhong Xian

PY - 2006/10/20

Y1 - 2006/10/20

N2 - 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 [ 1 H- 15 N] 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.

AB - 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 [ 1 H- 15 N] 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.

KW - Cell culture assay

KW - Human metallothionein-3

KW - Mutants

KW - NMR

KW - Neuron growth inhibitory factor

KW - S-Nitrosylation

UR - http://www.scopus.com/inward/record.url?scp=33748417843&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33748417843&partnerID=8YFLogxK

U2 - 10.1016/j.bbrc.2006.08.090

DO - 10.1016/j.bbrc.2006.08.090

M3 - Article

C2 - 16945328

AN - SCOPUS:33748417843

VL - 349

SP - 674

EP - 682

JO - Biochemical and Biophysical Research Communications

JF - Biochemical and Biophysical Research Communications

SN - 0006-291X

IS - 2

ER -