Molecular cloning, expression, and functional characterization of a novel member of the CD38 family of ADP-ribosyl cyclases.
We report the molecular cloning and functional characterization of a novel member of the CD38 family of cyclic ADP-ribose (cADPr)-generating cyclases. We cloned a cDNA insert that encoded a 298-amino-acid-long protein (M(w) approximately 39 kDa). The predicted protein displayed 69, 61, and 58% similarity, respectively, to mouse, rat, and human CD38. Rabbit CD38 was also 28% homologous to Aplysia ADP-ribosyl cyclase and leukocyte CD157 (another ADP-ribosyl cyclase); the three cyclases shared 10 cysteine and 2 adjacent proline residues. We then transfected CD38-negative NIH3T3 cells with cDNA encoding a CD38-EGFP fusion protein. Epifluorescence microscopy showed intense EGFP fluorescence confirming CD38 expression. We finally confirmed the ADP-ribosyl cyclase activity of the expressed CD38 by measuring its ability to catalyze the cyclization of the nicotinamide adenine dinucleotide (NAD(+)) surrogate, NGD(+), to its fluorescent nonhydrolyzable derivative, cGDPr.
Adebanjo
O A
OA
Mount Sinai Bone Program, Mount Sinai School of Medicine, New York, New York 10029, USA.
Koval
A
A
Moonga
B S
BS
Wu
X B
XB
Yao
S
S
Bevis
P J
PJ
Kumegawa
M
M
Zaidi
M
M
Sun
L
L
D016475:3T3 Cells; D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD; D000943:Antigens, Differentiation / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D008562:Membrane Glycoproteins; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009244:NAD+ Nucleosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D017386:Sequence Homology, Amino Acid
D015703:Antigens, CD; D000943:Antigens, Differentiation; D018076:DNA, Complementary; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D049452:Green Fluorescent Proteins; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; C498155:Cd38 protein, mouse; C498156:Cd38 protein, rat; D009244:NAD+ Nucleosidase; D051997:ADP-ribosyl Cyclase 1