The nucleotide receptor P2Y13 is a key regulator of hepatic high-density lipoprotein (HDL) endocytosis.

Cell surface receptors for high-density lipoprotein (HDL) on hepatocytes are major partners in the regulation of cholesterol homeostasis. We recently identified a cell surface ATP synthase as a high-affinity receptor for HDL apolipoprotein A-I (apoA-I) on human hepatocytes. Stimulation of this ectopic ATP synthase by apoA-I triggered a low-affinity-receptor-dependent HDL endocytosis by a mechanism strictly related to the generation of ADP. This suggests that nucleotide G-protein-coupled receptors of the P2Y family are molecular components in this pathway. Only P2Y1 and P2Y13 are present on the membrane of hepatocytes. Using both a pharmacological approach and small interference RNA, we identified P2Y13 as the main partner in hepatic HDL endocytosis, in cultured cells as well as in situ in perfused mouse livers. We also found a new important action of the antithrombotic agent AR-C69931MX as a strong activator of P2Y13-mediated HDL endocytosis.

Enterophilins, a new family of leucine zipper proteins bearing a b30.2 domain and associated with enterocyte differentiation.

Enterocyte terminal differentiation occurs at the crypt-villus junction through the transcriptional activation of cell-specific genes, many of which code for proteins of the brush border membrane such as intestinal alkaline phosphatase, sucrase-isomaltase, or the microvillar structural protein villin. Several studies have shown that this sharp increase in specific mRNA levels is intimately associated with arrest of cell proliferation. We isolated several clones from a guinea pig intestine cDNA library. They encode new proteins characterized by an original structure associating a carboxyl-terminal B30.2/RFP-like domain and a long leucine zipper at the amino terminus. The first member of this novel gene family codes for a 65-kDa protein termed enterophilin-1, which is specifically expressed in enterocytes before their final differentiation. Enterophilin-1 is the most abundant in the small intestine but is still present in significant amounts in colonic enterocytes. In Caco-2 cells, a similar 65-kDa protein was recognized by a specific anti-enterophilin-1 antibody, and its expression was positively correlated with cell differentiation status. In addition, transfection of HT-29 cells with enterophilin-1 full-length cDNA slightly inhibited cell growth and promoted an increase in alkaline phosphatase activity. Taken together, these data identify enterophilins as a new family of proteins associated with enterocyte differentiation.

New developments in phospholipase A2.

Some of the most recent data concerning various phospholipases A2, with special emphasis on secretory, cytosolic, and calcium-independent phospholipases A2 are summarized. Besides their contribution to the production of proinflammatory lipid mediators, the involvement of these enzymes in key cell responses such as apoptosis or tumor cell metastatic potential is also discussed, taking advantage of transgenic models based on gene invalidation by homologous recombination. The possible role of secretory and cytosolic platelet-activating factor acetyl hydrolases is also briefly mentioned. Finally, the ectopic expression in epididymis of an intestinal phospholipase B opens some novel issues as to the possible function of phospholipases in reproduction.

Ectopic epididymal expression of guinea pig intestinal phospholipase B. Possible role in sperm maturation and activation by limited proteolytic digestion.

Guinea pig intestinal phospholipase B is a calcium-independent phospholipase hydrolyzing sequentially the acyl ester bonds at sn-2 and sn-1 positions of glycerophospholipids, promoting the formation of sn-glycero-3-phosphocholine from phosphatidylcholine. This 140-kDa glycoprotein from the brush border membrane of differentiated enterocytes contributes to lipid digestion as an ectoenzyme. The cDNA coding for guinea pig phospholipase B was revealed to be the homologue of AdRab-B, an mRNA appearing in rabbit upon intestine development. The sequence predicts a polypeptide of 1463 amino acids displaying four homologous repeats, two of them containing the lipase consensus sequence GXSXG. A 5-kilobase transcript was particularly abundant in mature ileal and jejunal enterocytes but was also detected in epididymis, where phospholipase B displayed a higher molecular mass (170 kDa versus 140 kDa in intestine), with no obvious evidence for enzyme activity. Trypsin treatment of phospholipase B immunoprecipitated from epididymal membranes reduced its size to 140 kDa, coinciding with the appearance of a significant phospholipase A2 activity. The same results were obtained in COS cells transfected with phospholipase B cDNA. Since sn-glycero-3-phosphocholine present at high concentrations in seminal plasma mainly stems from epididymis, this suggests a possible role of phospholipase B in male reproduction. This novel localization also unravels a mechanism of phospholipase B activation by limited proteolysis involving either trypsin in the intestinal lumen or a trypsin-like endopeptidase in the male reproductive tract.

Guinea pig intestinal phospholipase B: protein expression during enterocyte maturation and effects of N-oligosaccharide removal on enzymatic activities and protein stability.

Guinea pig phospholipase B (PLB) is an intestinal brush-border hydrolase displaying a broad substrate specificity towards various dietary lipids. PLB was detected by immunoblotting as a single 140-kDa polypeptide in all cell populations isolated from guinea pig intestinal mucosa, but increased in parallel to its activity from undifferentiated to mature cells, the specific activity of the enzyme remaining constant. Moreover, N-glycosylation, which contributed to 23% of the apparent molecular mass, was identical along the cell differentiation axis. In all cell fractions, N-linked sugar chains were of the complex type, since they were removed by N-glycosidase F, whereas PLB remained insensitive to endoglycosidase H. Moreover, lack of O-glycosylation was demonstrated by the insensitivity of PLB to O-glycosidase and by its failure to interact with Helix pomatia lectin after prior treatment with neuraminidase or alpha-fucosidase. Enzymatic removal of sugar chains reduced phospholipase A2, lysophospholipase and diacylglycerol lipase activities by 27-35%, kinetic analysis indicating a decrease in apparent Vmax values for the three enzymatic activities, whereas the Km remained unchanged. Finally, the carbohydrate-depleted form of PLB did not display gross changes in thermal stability, in contrast to PLB from microorganisms previously investigated. Our data indicate that the high level of PLB N-glycosylation is poorly related to its biological function. Whether carbohydrate chains are involved in proper targeting of the enzyme to the brush-border membrane remains to be established.