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. Mobilization of Ca2ϩ from intracellular stores is of fundamental importance in virtually all aspects of cellular activity. The major intracellular Ca 2ϩ stores are in the endoplasmic reticulum (ER) 2 and are mobilized by specific messenger molecules, inositol trisphosphate and cyclic ADP-ribose (cADPR). The latter is a novel cyclic nucleotide derived from NAD. It was first described in sea urchin eggs (1, 2) but has since been established as a second messenger molecule responsible for regulating a wide range of physiological functions as diverse as abscisic acid signaling in plants (3) and sponges (4) and social behavior in mice (Ref. 5; reviewed in Refs. 6 and 7). It targets the ryanodine receptor of the endoplasmic Ca 2ϩ stores. The synthesis and hydrolysis of cADPR in mammalian cells are catalyzed by CD38 (8), a transmembrane protein ubiquitously expressed in virtually all tissues (reviewed in Ref. 9). Gene knock-out studies have established that CD38 plays a critical role in a wide range of physiological functions, including insulin secretion (10), susceptibility to bacterial infection (11), and social behavior of mice through modulating neuronal oxytocin secretion (5).CD38 is a membrane protein with a short N-terminal tail, a single transmembrane segment, and a large C-terminal domain containing all the enzymatic activities (8, 12, 13). The crystal structure of the catalytic domain of CD38 has been solved, and the mechanism of how it catalyzes the multiple reactions responsible for metabolizing cADPR has also been elucidated to atomic resolution by x-ray crystallography (14 -16). It is established that the s...

Roles and mechanisms of the CD38/cyclic adenosine diphosphate ribose/Ca2+signaling pathway

“…Since the number of positive charges that determine the polarity of membrane protein is equal on each side of the CD38 transmembrane segment, studies from protease digestion [54] and electron microscopy [55] showed that the nuclear CD38 might be a type-Ⅲ membrane protein. Most recently, Zhao et al [56] reported that expression of a cytosolic CD38 protein with deletion of both the N-terminal tail and transmembrane domain results in intact disulfides as well as active enzyme in spite of the cytosolic reductive environment; this result appears to solve the fundamental need of the six disulfides for CD38 enzymatic activity. Based on this finding, they consequently proved the coexpression of type Ⅱ and type Ⅲ CD38 on the surface of leukemia HL-60 cells during retinoic acid-induced differentiation and on interferon Υ-activated natural human monocytes and U937 cells [57] .…”

Cyclic ADP-ribose and Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) as Messengers for Calcium Mobilization

“…It is thus not clear whether type III CD38, with its C-terminal domain inside the cytosol, can actually form the important disulfides and, if not, whether it is still enzymatically functional. The question has been directly addressed by making a soluble CD38 construct containing the C-terminal domain (99). The soluble CD38 directed to express in the cytosol is found to be biologically active in elevating the intracellular cADPR level.…”

Cyclic ADP-ribose and NAADP: fraternal twin messengers for calcium signaling

“…A novel form of CD38 was engineered that expressed efficiently in the cytosol [159]. It was shown that it not only forms intact disulfides but is also fully active in elevating the cytosolic cADPR levels.…”