title: Revealing CD38 cellular localization using a cell permeable, mechanism-based fluorescent small-molecule probe.
abstract: Nicotinamide adenine dinucleotide (NAD) is increasingly recognized as an important signaling molecule that affects numerous biological pathways. Thus, enzymes that metabolize NAD can have important biological functions. One NAD-metabolizing enzyme in mammals is CD38, a type II transmembrane protein that converts NAD primarily to adenosine diphosphate ribose (ADPR) and a small amount of cyclic adenosine diphosphate ribose (cADPR). Localization of CD38 was originally thought to be only on the plasma membrane, but later reports showed either significant or solely, intracellular CD38. With the efficient NAD-hydrolysis activity, the intracellular CD38 may lead to depletion of cellular NAD, thus producing harmful effects. Therefore, the intracellular localization of CD38 needs to be carefully validated. Here, we report the synthesis and application of a cell permeable, fluorescent small molecule (SR101-F-araNMN) that can covalently label enzymatically active CD38 with minimal perturbation of live cells. Using this fluorescent probe, we revealed that CD38 is predominately on the plasma membrane of Raji and retinoic acid (RA)-treated HL-60 cells. Additionally, the probe revealed no CD38 expression in K562 cells, which was previously reported to have solely intracellular CD38. The finding that very little intracellular CD38 exists in these cell lines suggests that the major enzymatic function of CD38 is to hydrolyze extracellular rather than intracellular NAD. The fluorescent activity-based probes that we developed allow the localization of CD38 in different cells to be determined, thus enabling a better understanding of the physiological function.
authors.affiliation: Department of Chemistry and Chemical Biology, Cornell University , Ithaca, New York 14853, United States.
authors.forename: Jonathan H
authors.initials: JH
authors.lastname: Shrimp
authors.forename: Jing
authors.initials: J
authors.lastname: Hu
authors.forename: Min
authors.initials: M
authors.lastname: Dong
authors.forename: Brian S
authors.initials: BS
authors.lastname: Wang
authors.forename: Robert
authors.initials: R
authors.lastname: MacDonald
authors.forename: Hong
authors.initials: H
authors.lastname: Jiang
authors.forename: Quan
authors.initials: Q
authors.lastname: Hao
authors.forename: Andrew
authors.initials: A
authors.lastname: Yen
authors.forename: Hening
authors.initials: H
authors.lastname: Lin
mesh_terms: D051997:ADP-ribosyl Cyclase 1 / Q000378:metabolism*; D045744:Cell Line, Tumor; D002463:Cell Membrane Permeability; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000493:pharmacokinetics; D006801:Humans; D018613:Microscopy, Confocal
chemical_list: D005456:Fluorescent Dyes; D051997:ADP-ribosyl Cyclase 1