Cloning and expression of rat lung acidic Ca(2+)-independent PLA2 and its organ distribution.
A clone for a rat acidic Ca(2+)-independent phospholipase A2 (aiPLA2) was isolated from a cDNA library prepared from rat granular pneumocytes with a probe based on the human aiPLA2 sequence (T.S. Kim, C.S. Sundaresh, S. I. Feinstein, C. Dodia, W. R. Skach, M. K. Jain, T. Nagase, N. Seki, K. Ishikawa, N. Nomura, and A. B. Fisher. J. Biol. Chem. 272: 2542-2550, 1997). In addition, a consensus sequence for mouse aiPLA2 was constructed from several mouse cDNA clones in the GenBank and dbEST databases. Each sequence codes for a 224-amino acid protein with 88% identity of the amino acids among the three species and conservation of a putative lipase motif (GDSWG). Translation of mRNA produced from the rat clone in a wheat germ system resulted in expression of PLA2 activity with properties similar to those of the human enzyme, i.e., acidic pH optimum and Ca2+ independence. The localization of aiPLA2 in rat tissues was studied with the human cDNA probe, polyclonal and monoclonal antibodies, and aiPLA2 activity. aiPLA2 is present in the lung as evidenced by high levels of mRNA and protein expression and by enzymatic activity that is inhibited by anti-PLA2 antibody and by the transition state analog 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33). Immunocytochemistry showed the presence of aiPLA2 in alveolar type II cells, alveolar macrophages, and bronchiolar epithelium. In the brain, heart, liver, kidney, spleen, and intestine, aiPLA2 mRNA content was < 50% of that in the lung, immunoreactive protein was not detectable, and enzymatic activity was not inhibited by MJ33 or aiPLA2 antibody. These results show marked enrichment of aiPLA2 in the lung compared with the other organs and suggest translational control of aiPLA2 expression.
Kim
T S
TS
Institute for Environmental Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
Dodia
C
C
Chen
X
X
Hennigan
B B
BB
Jain
M
M
Feinstein
S I
SI
Fisher
A B
AB
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D054522:Group VI Phospholipases A2; D006801:Humans; D007158:Immunologic Techniques; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000235:genetics* / Q000378:metabolism*; D054467:Phospholipases A2; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D014018:Tissue Distribution; D014908:Triticum / Q000235:genetics
D018076:DNA, Complementary; D012333:RNA, Messenger; D010741:Phospholipases A; D054522:Group VI Phospholipases A2; C516475:PLA2G6 protein, human; D054467:Phospholipases A2; C516476:Pla2g6 protein, mouse