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Article: Cytosolic CD38 protein forms intact disulfides and is active in elevating intracellular cyclic ADP-ribose

TitleCytosolic CD38 protein forms intact disulfides and is active in elevating intracellular cyclic ADP-ribose
Authors
KeywordsADP-ribose
Catalytic residue
Cyclic ADP-ribose (cADPR)
Cytosolic
Cytosols
Issue Date2011
PublisherAmerican Society for Biochemistry and Molecular Biology, Inc. The Journal's web site is located at http://www.jbc.org/
Citation
Journal Of Biological Chemistry, 2011, v. 286 n. 25, p. 22170-22177 How to Cite?
AbstractCD38 catalyzes the synthesis of cyclic ADP-ribose (cADPR), a Ca 2+ messenger responsible for regulating a wide range of physiological functions. It is generally regarded as an ectoenzyme, but its intracellular localization has also been well documented. It is not known if internal CD38 is enzymatically active and contributes to the Ca 2+ signaling function. In this study, we engineered a novel soluble form of CD38 that can be efficiently expressed in the cytosol and use cytosolic NAD as a substrate to produce cADPR intracellularly. The activity of the engineered CD38 could be decreased by mutating the catalytic residue Glu-226 and increased by the double mutation E146A/T221F, which increased its cADPR synthesis activity by >11-fold. Remarkably, the engineered CD38 exhibited the ability to form the critical disulfide linkages required for its enzymatic activity. This was verified by using a monoclonal antibody generated against a critical disulfide, Cys-254-Cys-275. The specificity of the antibody was established by x-ray crystallography and site-directed mutagenesis. The engineered CD38 is thus a novel example challenging the general belief that cytosolic proteins do not possess disulfides. As a further refinement of this approach, the engineered CD38 was placed under the control of tetracycline using an autoregulated construct. This study has set the stage for in vivo manipulation of cADPR metabolism. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.
Persistent Identifierhttp://hdl.handle.net/10722/137152
ISSN
2015 Impact Factor: 4.258
2015 SCImago Journal Rankings: 3.151
PubMed Central ID
ISI Accession Number ID
Funding AgencyGrant Number
Hong Kong General Research Fund769107
768408
769309
770610
765909
766510
785110
National Natural Science Foundation of China/Research Grants CouncilN_HKU 722/08
Funding Information:

This work was supported by Hong Kong General Research Fund Grants 769107, 768408, 769309, and 770610 (to H. C. L.), 765909 and 766510 (to Q. H.), and 785110 (to H. M. Z.) and National Natural Science Foundation of China/Research Grants Council Joint Research Scheme Grant N_HKU 722/08 (to H. C. L.).

References
Grants

 

DC FieldValueLanguage
dc.contributor.authorZhao, YJen_HK
dc.contributor.authorZhang, HMen_HK
dc.contributor.authorLam, CMCen_HK
dc.contributor.authorHao, Qen_HK
dc.contributor.authorLee, HCen_HK
dc.date.accessioned2011-08-26T07:49:13Z-
dc.date.available2011-08-26T07:49:13Z-
dc.date.issued2011en_HK
dc.identifier.citationJournal Of Biological Chemistry, 2011, v. 286 n. 25, p. 22170-22177en_HK
dc.identifier.issn0021-9258en_HK
dc.identifier.urihttp://hdl.handle.net/10722/137152-
dc.description.abstractCD38 catalyzes the synthesis of cyclic ADP-ribose (cADPR), a Ca 2+ messenger responsible for regulating a wide range of physiological functions. It is generally regarded as an ectoenzyme, but its intracellular localization has also been well documented. It is not known if internal CD38 is enzymatically active and contributes to the Ca 2+ signaling function. In this study, we engineered a novel soluble form of CD38 that can be efficiently expressed in the cytosol and use cytosolic NAD as a substrate to produce cADPR intracellularly. The activity of the engineered CD38 could be decreased by mutating the catalytic residue Glu-226 and increased by the double mutation E146A/T221F, which increased its cADPR synthesis activity by >11-fold. Remarkably, the engineered CD38 exhibited the ability to form the critical disulfide linkages required for its enzymatic activity. This was verified by using a monoclonal antibody generated against a critical disulfide, Cys-254-Cys-275. The specificity of the antibody was established by x-ray crystallography and site-directed mutagenesis. The engineered CD38 is thus a novel example challenging the general belief that cytosolic proteins do not possess disulfides. As a further refinement of this approach, the engineered CD38 was placed under the control of tetracycline using an autoregulated construct. This study has set the stage for in vivo manipulation of cADPR metabolism. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.en_HK
dc.languageeng-
dc.publisherAmerican Society for Biochemistry and Molecular Biology, Inc. The Journal's web site is located at http://www.jbc.org/en_HK
dc.relation.ispartofJournal of Biological Chemistryen_HK
dc.rightsJournal of Biological Chemistry. Copyright © American Society for Biochemistry and Molecular Biology, Inc.-
dc.subjectADP-ribose-
dc.subjectCatalytic residue-
dc.subjectCyclic ADP-ribose (cADPR)-
dc.subjectCytosolic-
dc.subjectCytosols-
dc.titleCytosolic CD38 protein forms intact disulfides and is active in elevating intracellular cyclic ADP-riboseen_HK
dc.typeArticleen_HK
dc.identifier.openurlhttp://library.hku.hk:4550/resserv?sid=HKU:IR&issn=0021-9258&volume=286&issue=25&spage=22170&epage=22177&date=2011&atitle=Cytosolic+CD38+protein+forms+intact+disulfides+and+is+active+in+elevating+intracellular+cyclic+ADP-ribose-
dc.identifier.emailHao, Q: qhao@hku.hken_HK
dc.identifier.authorityHao, Q=rp01332en_HK
dc.description.naturelink_to_OA_fulltext-
dc.identifier.doi10.1074/jbc.M111.228379en_HK
dc.identifier.pmid21524995-
dc.identifier.pmcidPMC3121361-
dc.identifier.scopuseid_2-s2.0-79959365485en_HK
dc.identifier.hkuros185565-
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-79959365485&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume286en_HK
dc.identifier.issue25en_HK
dc.identifier.spage22170en_HK
dc.identifier.epage22177en_HK
dc.identifier.eissn1083-351X-
dc.identifier.isiWOS:000291719900023-
dc.publisher.placeUnited Statesen_HK
dc.relation.projectChemical synthesis and biological characterizations of antagonists of a novel calcium signaling enzyme - CD38-
dc.identifier.scopusauthoridZhao, YJ=35219477000en_HK
dc.identifier.scopusauthoridZhang, HM=54409692500en_HK
dc.identifier.scopusauthoridLam, CMC=26026006700en_HK
dc.identifier.scopusauthoridHao, Q=7102508868en_HK
dc.identifier.scopusauthoridLee, HC=40761849900en_HK

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