Complementary DNA cloning and sequencing of the chicken muscle ecto-ATPase. Homology with the lymphoid cell activation antigen CD39.
The ecto-ATPase from chicken gizzard (smooth muscle) was solubilized, and the 66-kDa cell membrane ecto-ATPase protein was purified. The protein was then subjected to both enzymatic and chemical cleavage, and the resultant peptides were purified by reverse phase high pressure liquid chromatography and sequenced. Several of these internal peptide sequences were used to design oligonucleotides to screen a chicken muscle library to identify the cDNA encoding the ecto-ATPase. Two overlapping partial clones were sequenced, yielding the complete coding region and a long 3'-untranslated sequence. The deduced amino acid sequence is in agreement with the N-terminal and peptide sequences obtained from the purified protein. The chicken muscle ecto-ATPase is a slightly basic (predicted pI = 7.93) 494-amino acid protein (54.4 kDa), containing a single transmembrane domain at each end of the protein. The majority of the protein is predicted to be extracellular, making it a Type Ia plasma membrane protein. There are four putative N-glycosylation sites, a single potential cAMP/cGMP-dependent protein kinase phosphorylation site, as well as a single putative tyrosine kinase phosphorylation site. Analysis of the sequence using the BLAST programs demonstrated homology with other ecto-ATPases and ecto-apyrases, including those from the parasitic protozoan Toxoplasma gondii, potato tubers, and garden pea, as well as a guanosine diphosphohydrolase from yeast. However, the most striking homology observed was to the human and mouse lymphoid cell activation antigen 39 (CD39), a molecule now known to have apyrase activity. The chicken ecto-ATPase showed considerable amino acid sequence homology with CD39 over the entire length of the sequence, excluding about 30-40 amino acids at the extreme ends of the protein (which include the two membrane-spanning helices). The sequence homology between the gizzard ecto-ATPase and CD39 was confirmed by Western blots demonstrating immunocross-reactivity between mono- and polyclonal antibodies raised against the chicken ecto-ATPase and two commercially available monoclonal antibodies against the human CD39 protein. The results suggest that the muscle ecto-ATPase may be involved in cell adhesion, since the highly homologous CD39 protein is involved in homotypic adhesion of activated B lymphocytes.
Kirley
T L
TL
Department of Pharmacology and Cell Biophysics, College of Medicine, University of Cincinnati, Ohio 45267-0575, USA. terry.kirley@uc.edu
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000737:chemistry / Q000235:genetics*; D001081:Apyrase; D001483:Base Sequence; D015153:Blotting, Western; D002645:Chickens; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry*; D005895:Gizzard, Avian / Q000201:enzymology; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000201:enzymology; D016415:Sequence Alignment
D015703:Antigens, CD; D018076:DNA, Complementary; D000251:Adenosine Triphosphatases; C027201:ectoATPase; D001081:Apyrase; C067869:CD39 antigen