Antibody M-19 distinguishes intracellular and extracellular CD38

Abstract

CD38 is an enzyme capable of synthesizing two second messengers, cyclic ADP ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). Generally, CD38 is considered as a Type II membrane protein, which means the catalytic active domain faces the extracellular space. This raised the topological paradox, in which the NAD substrate and enzyme are on opposite sides of the plasma membrane. We have previously published a “Type III CD38” hypothesis [1], in which we proposed that a small amount of biologically functional Type III CD38 co-exists with Type II CD38. In this study, we found that a polyclonal antibody against mouse CD38, M-19, only recognizes the intracellular CD38, but not the extracellular Type II CD38. We deciphered that M-19 recognizes the C-terminal 19 amino-acid sequence, FLQCVKNPEHPSCRLNT. In this region, there are two cysteines, which form a disulfide bond in Type II CD38. Considering that commercial antibodies are generally raised against peptides that do not contain disulfides, we hypothesize that M-19 recognizes CD38 with C-terminus in a reduced state. To test this hypothesis, we made C290S and C299S mutants of rat CD38, which constitutively destroyed the disulfide bridge. The mutants-transfected cells were stained on the plasma membrane by M-19, which supported the above hypothesis and indicated the intracellular CD38 might be in a Type III orientation. In addition, we found the intracellular staining pattern might be an inheritable feature in the CD38 over-expressing stable cell line. These results indicate the cells have a different genetic background, which causes the intracellular expression of CD38 and support the hypothesis that cells can express CD38 in two different orientations.