Molecular cloning and functional expression of bovine spleen ecto-NAD(+) glycohydrolase: structural identity with human CD38

Bovine spleen ecto-NAD(+) glycohydrolase, an archetypal member of the mamma lian membrane-associated NAD(P)(+) glycohydrolase enzyme family (EC 3.2.2.6 ), displays catalytic features similar to those of CD38, i.e. a protein ori ginally described as a lymphocyte differentiation marker involved in the me tabolism of cyclic ADP-ribose and signal transduction. Using amino acid seq uence information obtained from NAD(+) glycohydrolase and from a truncated and hydrosoluble form of the enzyme (hNADase) purified to homogeneity, a fu ll-length cDNA clone was obtained. The deduced sequence indicates a protein of 278 residues with a molecular mass of 31.5 kDa. It predicts that bovine ecto-NAD(+) glycohydrolase is a type II transmembrane protein, with a very short intracellular tail. The bulk of the enzyme, which is extracellular a nd contains two potential N-glycosylation sites, yields the fully catalytic ally active hNADase which is truncated by 71 residues. Transfection of HeLa cells with the full-length cDNA resulted in the expression of the expected NAD(+) glycohydrolase, ADP-ribosyl cyclase and GDP-ribosyl cyclase activit ies at the surface of the cells. The bovine enzyme, which is the first 'cla ssical' NAD(P)(+) glycohydrolase whose structure has been established, pres ents a particularly high sequence identity with CD38, including the presenc e of 10 strictly conserved cysteine residues in the ectodomain and putative catalytic residues. However, it lacks two otherwise conserved cysteine res idues near its C-terminus. Thus hNADase, the truncated protein of 207 amino acids, represents the smallest functional domain endowed with all the cata lytic activities of CD38/NAD(+) glycohydrolases so far identified. Altogeth er, our data strongly suggest that the cloned bovine spleen ecto-NAD(+) gly cohydrolase is the bovine equivalent of CD38.