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

TitleMutation at Glu23 eliminates the neuron growth inhibitory activity of human metallothionein-3
Authors
KeywordsCell culture assay
Human metallothionein-3
Mutants
Neuron growth inhibitory factor
NMR
S-Nitrosylation
Issue Date2006
PublisherAcademic Press. The Journal's web site is located at http://www.elsevier.com/wps/find/journaldescription.cws_home/622790/description
Citation
Biochemical And Biophysical Research Communications, 2006, v. 349 n. 2, p. 674-682 How to Cite?
AbstractHuman 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. © 2006 Elsevier Inc. All rights reserved.
Persistent Identifierhttp://hdl.handle.net/10722/70069
ISSN
2015 Impact Factor: 2.371
2015 SCImago Journal Rankings: 1.152
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorDing, ZCen_HK
dc.contributor.authorTeng, XCen_HK
dc.contributor.authorCai, Ben_HK
dc.contributor.authorWang, Hen_HK
dc.contributor.authorZheng, Qen_HK
dc.contributor.authorWang, Yen_HK
dc.contributor.authorZhou, GMen_HK
dc.contributor.authorZhang, MJen_HK
dc.contributor.authorWu, HMen_HK
dc.contributor.authorSun, HZen_HK
dc.contributor.authorHuang, ZXen_HK
dc.date.accessioned2010-09-06T06:19:25Z-
dc.date.available2010-09-06T06:19:25Z-
dc.date.issued2006en_HK
dc.identifier.citationBiochemical And Biophysical Research Communications, 2006, v. 349 n. 2, p. 674-682en_HK
dc.identifier.issn0006-291Xen_HK
dc.identifier.urihttp://hdl.handle.net/10722/70069-
dc.description.abstractHuman 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. © 2006 Elsevier Inc. All rights reserved.en_HK
dc.languageengen_HK
dc.publisherAcademic Press. The Journal's web site is located at http://www.elsevier.com/wps/find/journaldescription.cws_home/622790/descriptionen_HK
dc.relation.ispartofBiochemical and Biophysical Research Communicationsen_HK
dc.subjectCell culture assayen_HK
dc.subjectHuman metallothionein-3en_HK
dc.subjectMutantsen_HK
dc.subjectNeuron growth inhibitory factoren_HK
dc.subjectNMRen_HK
dc.subjectS-Nitrosylationen_HK
dc.titleMutation at Glu23 eliminates the neuron growth inhibitory activity of human metallothionein-3en_HK
dc.typeArticleen_HK
dc.identifier.openurlhttp://library.hku.hk:4550/resserv?sid=HKU:IR&issn=0006-291X&volume=349&spage=674–682&epage=&date=2006&atitle=Mutation+at+Glu23+eliminates+the+neuron+growth+inhibitory+activity of+human+metallothionein-3en_HK
dc.identifier.emailSun, HZ:hsun@hkucc.hku.hken_HK
dc.identifier.authoritySun, HZ=rp00777en_HK
dc.description.naturelink_to_subscribed_fulltext-
dc.identifier.doi10.1016/j.bbrc.2006.08.090en_HK
dc.identifier.pmid16945328-
dc.identifier.scopuseid_2-s2.0-33748417843en_HK
dc.identifier.hkuros124845en_HK
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-33748417843&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume349en_HK
dc.identifier.issue2en_HK
dc.identifier.spage674en_HK
dc.identifier.epage682en_HK
dc.identifier.isiWOS:000240791800030-
dc.publisher.placeUnited Statesen_HK
dc.identifier.scopusauthoridDing, ZC=14424225400en_HK
dc.identifier.scopusauthoridTeng, XC=9842935500en_HK
dc.identifier.scopusauthoridCai, B=36484162900en_HK
dc.identifier.scopusauthoridWang, H=15052706700en_HK
dc.identifier.scopusauthoridZheng, Q=36887872800en_HK
dc.identifier.scopusauthoridWang, Y=36078812500en_HK
dc.identifier.scopusauthoridZhou, GM=8449295200en_HK
dc.identifier.scopusauthoridZhang, MJ=7601555100en_HK
dc.identifier.scopusauthoridWu, HM=13808047800en_HK
dc.identifier.scopusauthoridSun, HZ=7404827446en_HK
dc.identifier.scopusauthoridHuang, ZX=7406221847en_HK

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