Human CD38 Stable Cell Line-HEK293T

The CD38 molecule (CD38) catalyzes the biosynthesis of the calcium mobilizing messenger cyclic ADP-ribose (cADPR). CD38 has dual membrane orientations, in which the catalytic domain of type III CD38 faces the cytosol and is less abundant, but can effectively cyclize cytosolic NAD to generate cADPR. The role of cell surface type II CD38 in cellular cADPR generation is still unclear. Here, the researchers developed a photoactivatable cross-linking probe based on CD38 nanobodies, and combined with mass spectrometry analysis, they found that cell surface CD38 interacts with CD71. CD71 knockdown increases CD38 levels, while CD38 knockout in turn increases CD71, both of which can be co-capped and co-immunoprecipitated. The researchers constructed a chimera containing the CD71 N-terminal fragment and the CD38 nanobody to mimic the ligand properties of CD71. Overexpression of this chimera led to a sharp decrease in CD38 levels in lysosomes. Notably, there was no corresponding decrease in cADPR levels in cells. Bafilomycin-mediated blockade of lysosomal degradation significantly increased active type II CD38 by trapping it in lysosomes, but this did not increase cADPR levels. Retention of type II CD38 in the ER by expressing an ER construct that prevents type II CD38 trafficking to the cell surface also did not alter cADPR levels. These results provide the first direct evidence that cADPR biogenesis occurs in the cytosol and is primarily catalyzed by type III CD38, whereas type II CD38 is compartmentalized in the ER, lysosomes, or on the cell surface and contributes little to cADPR biogenesis.

The study showed that in NPC-treated LP-1 cells, Strep magnetic beads detected CD38 and CD71 in the precipitate, but not in two control cells (NPC-treated CD38-KO cells or untreated LP-1 cells) (Figure 1A). Since both CD38 and CD71 are present on the cell surface, the researchers further confirmed this interaction using co-capping experiments. The results showed that CD38 capping and CD71 capping appeared in the same clusters in wild-type LP-1 cells (Figure 1B). The same treatment did not induce CD71 aggregation in CD38-KO LP-1 cells or in controls incubated on ice to inhibit capping (Figure 1B). These data indicate that CD71 is tightly bound to CD38, both naturally in LP-1 cells and when transfected into HEK293 cells.

Figure 1. CD71 interacts with CD38 and regulates its protein level and cellular cADPR production. (Deng Q W, et al., 2019)

Next, the researchers determined the functional consequences of the binding between CD38 and CD71. Compared with normal LP-1 cells, LP-1 cells with CD38 knocked out had significantly increased CD71 expression levels (Figure 1C). Because CD71 knockdown severely impaired cell viability, the researchers used shRNA or siRNA knockdown in LP-1 cells (Figure 1D) and CD38 overexpressing HEK293 cells (Figure 1E, CD38/HEK-293). Both methods showed that knocking down CD71 significantly increased the protein level of CD38. By measuring the activity of NADase in cell lysates, it was found that the increased CD38 was active, and the enzyme activity increased accordingly after CD71 knockdown (Figure 1F). Surprisingly, knocking down CD71 in LP-1 and CD38 overexpressing HEK293 cells resulted in an increase in both total protein and CD38 activity (Figure 1C-F), but did not lead to changes in cellular cADPR levels (Figure 1G). In fact, a slight decrease was observed in both HEK293 and LP-1 cells. This is the first time that the cellular level of surface type II CD38 is independent of cADPR levels.