Panoply™ Human CD38 Over-expressing Stable Cell Line

Nicotinic acid adenosine dinucleotide phosphate (NAADP) is a Ca2+-activated second messenger involved in regulating a variety of biological activities. However, its biosynthesis mechanism remains controversial. CD38 is the only enzyme known to catalyze the synthesis of NAADP from NADP and niacin. CD38-mediated catalysis requires acidic pH, suggesting that NAADP may be produced in acidic endolysosomes, but this hypothesis has not been verified. Here, using human cell lines, the researchers specifically directed CD38 to the endolysosomal system and assessed the production of intracellular NAADP. First, the study found that nanobodies targeting different epitopes on the CD38 C-terminal domain could bind to cell-surface-localized CD38 and induce its endocytosis. In addition, CD38 internalization was carried out through a clathrin-dependent pathway, delivering CD38 to endolysosomes and increasing intracellular NAADP levels. The researchers also constructed a CD38 variant for lysosome-specific expression that not only tolerates the degradation environment within lysosomes, but is also more active than wild-type CD38 in increasing cellular NAADP levels. Supplementing CD38-expressing cells with nicotinic acid substantially increased cellular NAADP levels. These results indicate that endolysosomal CD38 is capable of producing NAADP in human cells.

CD38-overexpressing HeLa cells were incubated with DMSO or different concentrations of PAO, Pitstop 2, MβCD, or filipin in Hanks balanced salt solution. CME inhibitors phenylarsine oxide (PAO) and Pitstop 2 significantly blocked Nb-induced endocytosis in HeLa cells at sufficient concentrations (Figure 1a), while CIE inhibitors methyl-β-cyclodextrin (MβCD) or filipin had no such effect, indicating that Nb-induced endocytosis mainly proceeds through the CME pathway. This was further verified by RNAi, using siRNA targeting clathrin heavy chain (CHC) or caveolin-1 (Cav1), the major component of caveolin membranes involved in CIE. Western blotting confirmed the knockdown efficiency (Figure 1b). Figure 1c shows the percentage of CD38 internalization at different time points after Nb-1053 treatment. The data show that CHC knockdown impairs endocytosis, while Cav1 knockdown has little effect. If Nb-induced endocytosis is indeed mediated by CME, then internalized Nbs should colocalize with clathrin (CHC). Figure 1d shows this situation. HeLa cells overexpressing CD38 were incubated with Nb-1053. Merged fluorescence micrographs showed a significant overlap of Nb and CHC fluorescence in HeLa cells 30 minutes after induced endocytosis. Statistical analysis of the colocalization of the two signals in a large number of cells showed a significant increase in the colocalization of the two signals after Nb-1053 treatment (Figure 1e). In contrast, the colocalization between Nb-1053 and Cav1 was significantly reduced (Figure 1f and g). The data confirm that Nb-1053-induced CD38 endocytosis is mainly mediated by a clathrin-dependent pathway.

Figure 1. Nanobody-induced endocytosis of CD38 was mainly via the clathrin-dependent pathway. (Fang C, et al., 2018)