Identification of functional domains of rat intestinal phospholipase B/lipase. Its cDNA cloning, expression, and tissue distribution.
A cDNA encoding a rat intestinal Ca(2+)-independent phospholipase B/lipase (PLB/LIP) was cloned from an ileac mucosa cDNA library using a probe amplified by polymerase chain reaction based on the purified enzyme's sequence. PLB/LIP consists of an NH2-terminal signal peptide, four tandem repeats of about 350 amino acids each, and a hydrophobic domain near the COOH terminus. The enzyme purified previously was found to be derived from the second repeat part. To examine the function of each domain, the full-length PLB/LIP, individual repeats, and a protein lacking the COOH-terminal hydrophobic stretch were expressed in COS-7 cells. The results showed that the second repeat, but not the other repeats, had all the activities (phospholipase A2, lysophospholipase, and lipase) found in the purified natural and expressed full-length enzymes, suggesting repeat 2 is a catalytic domain. The full-length enzyme was mainly present in membrane fractions and efficiently solubilized by treatment with 1% Triton X-100, but not with phosphatidylinositol-specific phospholipase C. Deletion of the COOH-terminal hydrophobic stretch caused the secretion of > 90% of synthesized PLB/LIP into culture media. These results suggest the hydrophobic domain is not replaced by a glycosylphosphatidylinositol anchor but serves as a membrane anchor directly. A message of the full-length PLB/LIP was abundantly expressed in the ileum and also, in a smaller, but significant amount, in the esophagus and testis. Immunohistochemistry showed that PLB/LIP is localized in brush border membranes of the absorptive cells, Paneth cells, and acrosomes of spermatid, suggesting its roles related and unrelated to intestinal digestion.
Takemori
H
H
Department of Molecular Physiological Chemistry, Osaka University Medical School, Japan.
Zolotaryov
F N
FN
Ting
L
L
Urbain
T
T
Komatsubara
T
T
Hatano
O
O
Okamoto
M
M
Tojo
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D002384:Catalysis; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D015870:Gene Expression; D007413:Intestinal Mucosa / Q000201:enzymology; D007422:Intestines / Q000201:enzymology*; D008245:Lysophospholipase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D008297:Male; D008871:Microvilli / Q000201:enzymology; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D016415:Sequence Alignment; D014018:Tissue Distribution
D018076:DNA, Complementary; D012333:RNA, Messenger; C110540:phospholipase B-lipase; D008245:Lysophospholipase