Identification of Functional Domains of Rat Intestinal Phospholipase B/Lipase

Abstract

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 NH 2 -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 A 2 , 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.Digestion of phospholipids and triacylglycerol in gastrointestinal tract involves several hydrolysis reactions. A variety of lipases, including acid lipase in lingual gland or stomach, and pancreatic lipase (which is of primary importance in luminal digestion of fats), hydrolyze triacylglycerol to produce monoacylglycerols and free fatty acids (see Refs. 1-3 for reviews)).Pancreatic phospholipases A 2 (PLA 2 ) 1 hydrolyze ester bonds at the sn-2 position of glycerophospholipids and produce fatty acids and lysophospholipids. These steps are prerequisites for lipid absorption by intestinal epithelium cells (4). Lysophospholipid can be directly absorbed, or hydrolyzed by pancreatic lysophospholipase and converted to glycerol 3-phosphate esters and fatty acids.Until recently, all those processes were believed to proceed in the lumen of alimentary tracts by the action of secretory enzymes mentioned above. However, recent studies suggest the presence of a lipid-hydrolyzing enzyme associated with intestinal brush border membranes (5, 6), named phospholipase B/lipase (PLB/LIP) because this enzyme displayed broad lipolytic activities (PLA 2 , lysophospholipase, and lipase activities) (7). PLB/LIP might participate in terminal digestion, or membrane digestion, of dietary lipids and biliary phospholipids, like well established glycosidases and pe...

Identification of phospholipase B from Dictyostelium discoideum reveals a new lipase family present in mammals, flies and nematodes, but not yeast

“…The intestinal enzyme is also expressed in the epididymis and it may play a role in sperm maturation [9]. It is found mainly in membranes, and comprises a signal peptide, four tandem repeats of about 350 amino acids, two of them containing the lipase consensus sequence GxSxG, and a hydrophobic domain near the C-terminus [8,9,17,19]. However, the consensus sequence GxSxG was dispensable for activity and the nucleophile serine residue was located within the conserved GDSL sequence.…”

“…Essentially these enzymes display phospholipase A/lysophospholipase activity, catalysing the total deacylation of phospholipids. Phosphatidylcholine (PC)-hydrolysing PLB activities have been described in bacteria [1], fungi [2], Dictyostelium discoideum [3] and in mammalian cells [4][5][6][7][8][9][10][11], and three distinct gene families have been identified from bacteria, fungi and mammals. In the microorganism Moraxella bovis, the plb gene encodes a protein of 616 amino acids and the enzyme hydrolyses PC/LPC (lysoPC) to produce NEFAs (non-esterified fatty acids, also known as free fatty acids) and glycerophosphorylcholine (GPC) [1].…”

“…The enzyme was initially characterized from the intestine and is now known to be present in the external face of the brushborder membranes of mature enterocytes where it is involved in digestion of dietary lipids [4,[7][8][9][10][11][17][18][19]. The enzyme is an ecto-phospholipase with a short C-terminal domain, inserted in the membrane by a hydrophobic segment connected to a larger, heavily glycosylated globular domain [8,19]. The latter contains four tandem repeat domains and several lipase and phospholipase consensus sequences, i.e.…”

Adipose-specific Expression, Phosphorylation of Ser794 in Insulin Receptor Substrate-1, and Activation in Diabetic Animals of Salt-inducible Kinase-2

“…Antibodies-A specific antibody to SIK2 was raised against a peptide at the COOH-terminal side (346-931 residues) in rabbits as described (34). To produce the antigen, a cDNA fragment encoding the above peptide was amplified by PCR with primers, 5Ј-AAGGATCCGTGGAG-CAGAGACTTGATG and 5Ј-TGCGGCCGCTAGGTCTCCCGGGCTA-AG, digested by BamHI/NotI, and cloned into a BamHI/NotI site of pET28a (Novagen).…”

Physiological Significance of Lysophospholipids that Act on the Lumen Side of Mammalian Lower Digestive Tracts

“…28) Reduced digestion of phospholipids by a PLA 2 inhibitor, FPL67047XX, retarded cholesterol absorption. 29) Because the cholesterol absorption efficacies of PLA 2 (+/+) and PLA 2 (−/−) mice were similar, however, phospholipase B located in distal intestine 30) would participate in the phospholipid digestion to compensate for malfunction of pancreatic PLA 2 . 29) While mice with targeted inactivation of the group IB PLA 2 gene were reported to show no significant abnormality when raised on a chow diet, 29) the PLA 2 -deficient mice fed a high-fat diet displayed lower postprandial glycemia than wild-type mice, causing a decreased level of plasma LPC.…”