MAPPING OF THE CATALYTIC AND EPITOPIC SITES OF HUMAN CD38 NAD(+) GLYCOHYDROLASE TO A FUNCTIONAL DOMAIN IN THE CARBOXYL-TERMINUS/

We reported that 1) ecto-NAD(+) glycohydrolase (NADase) activity induc ed upon differentiation of HL-60 cells is localized on the extracellul ar carboxyl-terminal side of CD38 and that 2) CD38 ligation by specifi c mAbs is followed by protein tyrosine phosphorylation in the cells. T he strategy selected for identifying the relevant catalytic domains of the molecule relies upon the production in COS-7 cells of carboxyl-te rminal deletion mutants of CD38. The mutants with fewer than 15 amino acids deleted at the carboxyl terminus of the 300-amino acid wild-type molecule maintained NADase activity, whereas those with more than 27 amino acids deleted did not. The general inference is that the carboxy l-terminal 273-285 sequence bears the site of enzyme activity. introdu ction of site-directed mutation of a conserved cysteine residue (Cys(2 75)), located in the 273-285 sequence, completely abolished NADase act ivity. The second issue resolved in this work is the definition of an epitope of the agonistic anti-CD38 mAbs. To this aim, a panel of selec ted anti-CD38 mAbs was tested using these mutants and various CD38 fra gments as the target in immunoblot analyses. All of the epitopes recog nized by mAbs inducing protein tyrosine phosphorylation were mapped on an identical site containing the carboxyl-terminal sequence of 273-28 5. The conclusion is that the discrete carboxyl-terminal sequence iden tified in the present study not only plays a key role in its ecto-NADa se activity, but actually constitutes the epitopes exploited by the ag onistic anti-CD38 mAbs for transmembrane signaling.