Measuring CD38 Hydrolase and Cyclase Activities: 1,N⁶-Ethenonicotinamide Adenine Dinucleotide (ε-NAD) and Nicotinamide Guanine Dinucleotide (NGD) Fluorescence-based Methods

Background

Current studies on age-related development of metabolic dysfunction and frailty are each day in more evidence. It is known that, as the aging progresses, the NAD⁺ levels decrease in an expected process. Recent studies have shown that a reduction in nicotinamide adenine dinucleotide (NAD⁺) is implicated in the development of age-associated metabolic decline (Massudi et al., 2012). Increased NAD⁺ levels in vivo, results in activation of pro-longevity and health span-related factors and improves several physiological and metabolic parameters of aging (Camacho-Pereira et al., 2016), including muscle function, exercise capacity, glucose tolerance, and cardiac function in mouse models of natural and accelerated aging.

Due to its role in NAD⁺ metabolism, the study of CD38 and its functions has been of great importance. CD38 was first identified in 1980 as a structural cell surface marker for the characterization of immune cells (Malavasi et al., 2008; van de Donk et al., 2016), and its first association as a NAD hydrolase enzyme was in the following decade (Kontani et al., 1993). However, during the past years, its enzymatic activities were more clearly elucidated. Initially, CD38 has been implicated to be responsible for the synthesis of the second messengers, cyclic ADP-ribose (cADPR), ADPR and nicotinic acid–adenine dinucleotide phosphate (NAADP) (Chini et al., 2002). These products are involved in calcium signaling and control many biological processes including lymphocyte proliferation and insulin secretion (Kato et al., 1999). However, its major enzymatic activity is the NAD⁺ hydrolysis, placing CD38 as the major NADase in several mammalian tissues and as an important regulator of NAD⁺-dependent processes (Aksoy et al., 2006).

The primary catalytic reaction of CD38 involves the cleavage of the high energy β-glycosidic bond between nicotinamide and ribose. During catalysis, the removal of the nicotinamide from β-NAD is coupled with the formation of intermediates that are stabilized through H-bonds between their ribosyl groups and the catalytic residue Glu226, a residue required for the NADase and cyclase activity of the enzyme (Sauve et al., 2000; Liu et al., 2009). These intermediates are released from the catalytic site forming ADPR or cADPR (Figure 1). In general, the majority of the CD38 NADase catalytic activity will generate nicotinamide, but also ADPR and cADPR which have been shown to have second messenger signaling roles through the activation of ryanodine receptor (RYR2). The full description of these pathways and Ca²⁺ signaling can be found in the remarkable works of Galione (1994) and Chini and Dousa (1996). The roles of CD38 as a cyclase and of NAD-derived calcium messengers in physiology and pathology have been extensively reviewed (Sauve et al., 2000; Chini, 2009).

Physiologically, CD38 has been implicated in the regulation of metabolism and the pathogenesis of the aging process, and of multiple conditions, such as obesity, diabetes, heart disease, asthma and inflammation. Therefore, the study of CD38, its activities, and possible modulators are of great interest. Our protocol presents a method of measuring its hydrolase and cyclase activities, utilizing ε-NAD and NGD techniques (Graeff et al., 1994) with a fluorescence-based enzymatic assay performed in a plate reader, in a consistent and reproducible manner (Figure 2).


Figure 1. Schematic illustrating the reactions catalyzed by CD38 under physiological conditions


Figure 2. CD38 and substrate schematics. A. CD38 hydrolase activity (ε-NAD as substrate); B. Cyclase activity (NGD as substrate); Strong arrow indicates which product is preferentially formed in each reaction. C. Molecular structure of ε-NAD and NGD. *Nic = Nicotinamide, ε-I = enzyme-intermediate complex, (c)ADPR = (cyclic) ADP-ribose, (c)GDPR = (cyclic) GDP-ribose, Ex = excitation wavelength, Em = emission wavelength.