title: Cytosolic CD38 protein forms intact disulfides and is active in elevating intracellular cyclic ADP-ribose.
abstract: CD38 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.
authors.affiliation: Department of Physiology, The University of Hong Kong, Hong Kong, China.
authors.forename: Yong Juan
authors.initials: YJ
authors.lastname: Zhao
authors.forename: Hong Min
authors.initials: HM
authors.lastname: Zhang
authors.forename: Connie Mo Ching
authors.initials: CM
authors.lastname: Lam
authors.forename: Quan
authors.initials: Q
authors.lastname: Hao
authors.forename: Hon Cheung
authors.initials: HC
authors.lastname: Lee
mesh_terms: D051997:ADP-ribosyl Cyclase 1 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D036563:Cyclic ADP-Ribose / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry*; D057809:HEK293 Cells; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism*; D019169:Jurkat Cells; D051379:Mice; D008958:Models, Molecular; D041681:NIH 3T3 Cells; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D012995:Solubility
chemical_list: D004220:Disulfides; D036563:Cyclic ADP-Ribose; D051997:ADP-ribosyl Cyclase 1