Requirement of Cys399 for processing of the human ecto-ATPase (NTPDase2) and its implications for determination of the activities of splice variants of the enzyme.
Ecto-ATPase (CD39L1) corresponds to the type 2 enzyme of the ecto-nucleoside triphosphate diphosphohydrolase family (E-NTPDase). We have isolated from human ECV304 cells three cDNAs with high homology with members of the E-NTPDase family that encode predicted proteins of 495, 472, and 450 amino acids. Sequencing of a genomic DNA clone confirmed that these three sequences correspond to splice variants of the human ecto-ATPase (NTPDase2 alpha,-2 beta, and -2 gamma). Although all three enzyme forms were expressed heterologously to similar levels in Chinese hamster ovary cells clone K-1 (CHO-K1) cells, only the 495-amino acid protein (NTPDase2 alpha exhibited ecto-ATPase activity. Immunolocalization studies demonstrated that NTPDase2 alpha is fully processed and trafficked to the plasma membrane, whereas the NTPDase2 beta and -2 gamma splice variants were retained in not fully glycosylated forms in the endoplasmic reticulum. The potential roles of two highly conserved residues, Cys399 and Asn443, in the activity and cellular trafficking of the ecto-ATPase were examined. Mutation of Cys399, which is absent in NTPDase2 beta and -2 gamma, produced a protein completely devoid of nucleotidase activity, while mutation of Asn443 to Asp resulted in substantial loss of activity. Neither the Cys399 nor Asn443 mutants were fully glycosylated, and both were retained in the endoplasmic reticulum. These results indicate that the lack of ecto-nucleotidase activity exhibited by NTPDase2 beta and -2 gamma and the C399S mutant, as well as the large reduction of activity in the N443D mutant are due to alterations in the folding/maturation of these proteins.
Mateo
Jesús
J
Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599-7365, USA.
Kreda
Silvia
S
Henry
Christopher E
CE
Harden
T Kendall
TK
Boyer
José L
JL
D016475:3T3 Cells; D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000737:chemistry; D001483:Base Sequence; D016466:CHO Cells; D002460:Cell Line; D017124:Conserved Sequence; D006224:Cricetinae; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012995:Solubility
D018076:DNA, Complementary; D011994:Recombinant Proteins; D001216:Asparagine; D000251:Adenosine Triphosphatases; C027201:ectoATPase; D003545:Cysteine