Mouse glycosylphosphatidylinositol-specific phospholipase D (Gpld1) characterization.

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is an 110-kDa monomeric protein found in the circulation that is capable of degrading the GPI anchor utilized by dozens of cell-surface proteins in the presence of detergent. This protein is relatively abundant (5-10 microgram/ml in human serum), yet its sites of synthesis, gene structure, and overall function are unclear. It is our purpose to use the mouse system to determine its putative roles in lipid transport, pathogen control, and diabetes. We have isolated murine full-length cDNA for GPI-PLD from a pancreatic alpha cell library. The deduced amino acid sequence shows 74% homology to bovine and human GPI-PLD. There is a single structural gene (Gpld1) mapping to mouse Chromosome (Chr) 13, and among nine tissues, liver showed the greatest abundance of GPI-PLD mRNA. Genetic differences in serum GPI-PLD activity were seen among four mouse strains, and no correlation was seen between GPI-PLD activity and circulating levels of high density lipoproteins in these mice. This is the first report of map position and genetic regulation for Gpld1. This information will enable us to further study the expression and function of GPI-PLD in normal and pathological conditions.

Glycosylphosphatidylinositol-phospholipase D: a tool for glycosylphosphatidylinositol structural analysis.

Cleavage by the GPI-PLD provides definitive evidence of a minimal GPI structure: glucosamine-phosphatidylinositol. Unlike the case for PI-PLC, cleavage by the GPI-PLD is unaffected by acylation of the inositol ring. Thus the GPI-PLD provides an excellent simple enzymatic tool for analyzing the basic core structure of GPI anchors.

How GPI-anchored proteins turnover: or where do they go after arrival at the plasma membrane.

Glycosylphosphatidylinositol (GPI)-anchored proteins comprise a diverse class of membrane molecules. They protect cells from complement-mediated lysis, control cell to cell adhesion, activate T cells, and play a role in the etiology of slow viral diseases. Despite their functional diversity, GPI-anchored proteins are all attached to the plasma membrane by a common glycolipid anchor. In this review we will examine how the GPI anchor is metabolized after arrival at the cell membrane. The conclusion is that break-down of the GPI anchor is potentially as diverse as the proteins themselves. The GPI-anchored protein can be endocytosed and degraded, cleaved and released, vesiculated, or exchanged onto another cell. The chimeric nature--lipid and protein--of the GPI-anchored molecules affords a unique window into the dynamic processes of lipid biosynthesis, movement, transport and maintenance.

Release of GPI-anchored membrane proteins by a cell-associated GPI-specific phospholipase D.

Although many glycosylphosphatidylinositol (GPI)-anchored proteins have been observed as soluble forms, the mechanisms by which they are released from the cell surface have not been demonstrated. We show here that a cell-associated GPI-specific phospholipase D (GPI-PLD) releases the GPI-anchored, complement regulatory protein decay-accelerating factor (DAF) from HeLa cells, as well as the basic fibroblast growth factor-binding heparan sulfate proteoglycan from bone marrow stromal cells. DAF found in the HeLa cell culture supernatants contained both [3H]ethanolamine and [3H]inositol, but not [3H]palmitic acid, whereas the soluble heparan sulfate proteoglycan present in bone marrow stromal cell culture supernatants contained [3H]ethanolamine. 125I-labeled GPI-DAF incorporated into the plasma membranes of these two cell types was released in a soluble form lacking the fatty acid GPI-anchor component. GPI-PLD activity was detected in lysates of both HeLa and bone marrow stromal cells. Treatment of HeLa cells with 1,10-phenanthroline, an inhibitor of GPI-PLD, reduced the release of [3H]ethanolamine-DAF by 70%. The hydrolysis of these GPI-anchored molecules is likely to be mediated by an endogenous GPI-PLD because [3H]ethanolamine DAF is constitutively released from HeLa cells maintained in serum-free medium. Furthermore, using PCR, a GPI-PLD mRNA has been identified in cDNA libraries prepared from both cell types. These studies are the first demonstration of the physiologically relevant release of GPI-anchored proteins from cells by a GPI-PLD.

An endogenous glycosylphosphatidylinositol-specific phospholipase D releases basic fibroblast growth factor-heparan sulfate proteoglycan complexes from human bone marrow cultures.

Basic fibroblast growth factor (bFGF) is a hematopoietic cytokine that stimulates stromal and stem cell growth. It binds to a glycosylphosphatidylinositol (GPI)-anchored heparan sulfate proteoglycan on human bone marrow (BM) stromal cells. The bFGF-proteoglycan complex is biologically active and is released by addition of exogenous phosphatidylinositol-specific phospholipase C. In this study, we show the presence of an endogenous GPI-specific phospholipase D (GPI-PLD) that releases the bFGF-binding heparan sulfate proteoglycan and the variant surface glycoprotein (a model GPI-anchored protein) from BM cultures. An involvement of proteases in this process is unlikely, because released proteoglycan contained the GPI anchor component, ethanol-amine, and protease inhibitors did not diminish the release. The mechanism of release is likely to involve a GPI-PLD and not a GPI-specific phospholipase C, because the release of variant surface glycoprotein did not reveal an epitope called the cross-reacting determinant that is exposed by phospholipase C-catalyzed GPI anchor cleavage. In addition, phosphatidic acid (which is specifically a product of GPI-PLD-catalyzed anchor cleavage) was generated during the spontaneous release of the GPI-anchored variant surface glycoprotein. We also detected GPI-PLD-specific enzyme activity and mRNA in BM cells. Therefore, we conclude that an endogenous GPI-PLD releases bFGF-heparan sulfate proteoglycan complexes from human BM cultures. This mechanism of GPI anchor cleavage could be relevant for mobilizing biologically active bFGF in BM. An endogenous GPI-PLD could also release other GPI-anchored proteins important for hematopoiesis and other physiologic processes.

The fate of GPI-anchored molecules.

Glycosylphosphatidylinositol (GPI)-anchored proteins comprise a diverse class of membrane molecules. They protect cells from complement-mediated lysis, control cell to cell adhesion, activate T cells, and play a role in the etiology of slow viral diseases. Despite their functional diversity, GPI-anchored proteins are all attached to the plasma membrane by a common glycolipid anchor. We will examine one aspect of GPI-anchor metabolism, namely, the processing of the molecule after it arrives at the plasma membrane. After biosynthesis and transport to the plasma membrane, the GPI-anchored protein can be endocytosed and degraded or cleaved and released. The enzymatic machinery controlling the catabolism of GPI-anchored molecules at the plasma membrane is likely to play a central role in regulating the cell surface expression of these molecules.

Binding of GPI-PLD-treated DAF to the surface of Schistosoma mansoni schistosomula.

Decay accelerating factor (DAF,CD55) is a 70-kDa glycosylphosphatidylinositol (GPI)-anchored protein that protects human erythrocytes (HuE) from complement-mediated damage by regulation of the C3-convertase. Purified human DAF can be incorporated into sheep red blood cell (SRBC) membrane and confer complement resistance on these DAF-deficient cells. Here, we demonstrate that normal HuE or their stroma (HuES) incubated at 37 degrees C for 24 h release soluble DAF in a biologically active form into the culture medium. This soluble DAF neither inserts into SRBC plasma membranes nor presents the cross-reacting determinant (CRD) characteristic of the hydrolysis by phosphatidylinositol-specific phospholipases C (PI-PLC) but binds to schistosomula of S. mansoni protecting them from antibody-mediated complement-dependent damage. To study the binding of DAF to schistosomula in vitro, we have used purified human DAF labeled with 125I(125I-DAF), intact or treated with either PI-PLC or GPI-PLD (glycosylphosphatidylinositol-specific phospholipase D). We have found that GPI-PLD-treated DAF binds to the surface of parasites more readily than intact or PI-PLC-treated DAF. Immunoprecipitation of the samples with a monoclonal anti-human DAF antibody (IA10) revealed that schistosomula incubated with GPI-PLD-treated 125I-DAF emit a stronger signal than their counterparts. This result indicates that the surface of schistosomula is capable of acquiring GPI-PLD-treated DAF more effectively than intact or PI-PLC-treated molecules.

The fate of GPI-anchored molecules

Glycosylphosphatidylinositol (GPI)-anchored proteins comprise a diverse class of membrane molecules. They protect cells from complement-mediated lysis, control cell to cell adhesion, activate T cells, and play a role in the etiology of slow viral diseases. Despite their functional diversity, GPI-anchored proteins are all attached to the plasma membrane by a common glycolipid anchor. We will examine one aspect of GPI-anchor metabolism, namely, the processing of the molecule after it arrives at the plasma membrane. After biosynthesis and transport to the plasma membrane, the GPI-anchored protein can be endocytosed and degraded or cleaved and released. The enzymatic machinery controlling the catabolism of GPI-anchored molecules at the plasma membrane is likely to play a central role in regulating the cell surface expression of these molecules

Immunolocalization of a glycosylphosphatidylinositol-specific phospholipase D in mast cells found in normal tissue and neurofibromatosis lesions.

A large number of eukaryotic proteins have been shown to be anchored to the cell membrane by glycosylphosphatidylinositol (GPI). This glycolipid anchor can serve as a substrate for anchor-specific phospholipases that convert the GPI-anchored membrane proteins into soluble forms. Soluble forms of many GPI anchored proteins have been identified in vivo in connective tissue, plasma, and urine. The authors have discovered that mammalian plasma contains a GPI-specific phospholipase D (GPI-PLD). Because it recognizes a portion of the conserved glycan core structure, all GPI-anchored proteins are potential substrates. The authors report the development of a murine monoclonal antibody specific for one form of the human GPI-PLD and the immunohistochemical localization of this enzyme to mast cells.

Production of the glycosylphosphatidylinositol-specific phospholipase D by the islets of Langerhans.

A large number of diverse cell surface proteins are anchored to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. One proposed function for the GPI anchor is that it facilitates the release of the protein from the cell by acting as a target for anchor-specific phospholipases. We and others have discovered that mammalian plasma contains a GPI-specific phospholipase D (GPI-PLD) (Cardoso de Almeida, M. L., Turner, M. J., Stambuk, B. V., and Schenkman, S. (1988) Biochem, Biophys. Res. Commun. 150, 476-482; Davitz, M. A., Hereld, D., Shak, S., Krakow, J., Englund, P. T., and Nussenzweig, V. (1987) Science 238, 81-84; Low, M. G., and Prasad, A. R. S. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 980-984). Because the GPI-PLD recognizes a conserved portion of the anchor, all GPI-anchored proteins are potential substrates for the enzyme. We demonstrate in this communication the production of the plasma GPI-PLD by the islets of Langerhans. GPI-PLD enzymatic activity was found in dog pancreatic microsomes, but not pancreatic juice. Both the pancreatic and plasma enzymes were divalent cation-dependent and had identical substrate specificities. Purified murine islets of Langerhans, as well as alpha and beta cells, contained and released GPI-PLD activity. A GPI-PLD DNA fragment was amplified by polymerase chain reaction from a normal human islet cDNA library; the amplified fragment hybridized with the GPI-PLD cDNA clone. These findings represent the first demonstration of the production of the plasma GPI-PLD by a specific tissue site as well as cell type.

A glycophospholipid membrane anchor acts as an apical targeting signal in polarized epithelial cells.

Glycosyl-phosphatidylinositol- (GPI) anchored proteins contain a large extracellular protein domain that is linked to the membrane via a glycosylated form of phosphatidylinositol. We recently reported the polarized apical distribution of all endogenous GPI-anchored proteins in the MDCK cell line (Lisanti, M. P., M. Sargiacomo, L. Graeve, A. R. Saltiel, and E. Rodriguez-Boulan. 1988. Proc. Natl. Acad. Sci. USA. 85:9557-9561). To study the role of this mechanism of membrane anchoring in targeting to the apical cell surface, we use here decay-accelerating factor (DAF) as a model GPI-anchored protein. Endogenous DAF was localized on the apical surface of two human intestinal cell lines (Caco-2 and SK-CO15). Recombinant DAF, expressed in MDCK cells, also assumed a polarized apical distribution. Transfer of the 37-amino acid DAF signal for GPI attachment to the ectodomain of herpes simplex glycoprotein D (a basolateral antigen) and to human growth hormone (a regulated secretory protein) by recombinant DNA methods resulted in delivery of the fusion proteins to the apical surface of transfected MDCK cells. These results are consistent with the notion that the GPI anchoring mechanism may convey apical targeting information.

Purification of a glycosyl-phosphatidylinositol-specific phospholipase D from human plasma.

Mammalian plasma contains a phospholipase D, which is specific for the glycosyl-phosphatidylinositol anchor found on many eukaryotic cell surface proteins (Davitz, M. A., Hereld, D., Shak, S., Krakow, J., Englund, P. T., and Nussenzweig, V. (1987) Science 238, 81-84; Low, M. G., and Prasad, A. R. S. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 980-984; Cardoso de Almeida, M. L., Turner, M. J., Stambuk, B. V., and Schenkman, S. (1988) Biochem. Biophys. Res. Commun. 150, 476-482). We have purified this phospholipase D to homogeneity by a four-step procedure involving a Mono Q and phenyl-5PW columns, followed by wheat germ lectin affinity chromatography and finally another Mono Q column. A 4,500-fold purification was achieved with a 5% yield. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the homogeneous enzyme has a Mr of 110,000 and appears to consist of a single polypeptide chain. It exhibits identical substrate specificity as compared with the crude preparation, is active over a broad pH range (4.0-8.5), inhibited by the thiol-blocking agent p-chloromercuriphenylsulfonic acid and by 1,10-phenanthroline, and is partially heat-labile.

Partial characterization of the cross-reacting determinant, a carbohydrate epitope shared by decay accelerating factor and the variant surface glycoprotein of the African Trypanosoma brucei.

The variant surface glycoprotein (VSG) of the African trypanosome is anchored in the cell membrane by a complex glycan attached to phosphatidylinositol. The carboxyl terminal portion of VSG contains a cryptic carbohydrate epitope, the cross-reacting determinant (CRD), that is revealed only after removal of the diacylglycerol by phosphatidylinositol-specific phospholipase C (PIPLC) or VSG lipase. Recently, we have shown that after hydrolysis by PIPLC, decay-accelerating factor (DAF)--a mammalian phosphatidylinositol-anchored protein--also contains the CRD epitope. Using a two site immunoradiometric assay in which the capturing antibody is a monoclonal antibody to DAF and the revealing antibody is anti-CRD, we now show that sugar phosphates significantly inhibited the binding of anti-CRD antibody to DAF released by PIPLC. DL-myo-inositol 1,2-cyclic phosphate was the most potent inhibitor of binding (IC50 less than 10(-8) M). Other sugar phosphates, such as alpha-D-glucose-1-phosphate, which also possess adjacent hydroxyl and phosphate moieties in cis also inhibited binding at low concentrations (IC50 = 10(-5) to 10(-4) M). In contrast, sugar phosphates which do not possess adjacent hydroxyl and phosphate moieties in cis and simple sugars weakly inhibited binding (IC50 greater than 10(-3) M). These results suggest that myo-inositol 1,2-cyclic phosphate contributes significantly to the epitope recognized by the anti-CRD antibody and is consistent with analysis of the carboxyl terminus of VSG, which also suggested the presence of the cyclic inositol phosphate. In light of the recent findings that human serum contains a glycan-phosphatidyl-inositol-specific phospholipase D, which converts DAF from a hydrophobic to a hydrophilic form lacking the CRD, the observation that the phosphate is crucial for expression of the epitope may be relevant in understanding the origin of CRD-negative DAF in urine and plasma.

Signal for attachment of a phospholipid membrane anchor in decay accelerating factor.

Decay accelerating factor (DAF) belongs to a novel group of membrane proteins anchored to the cell surface by a glycophospholipid membrane anchor that is covalently attached to the carboxyl terminus of the protein. The last 37 amino acids of membrane DAF, when fused to the carboxyl terminus of a secreted protein, are sufficient to target the fusion protein to the plasma membrane by means of a glycophospholipid anchor. This approach provides a novel means of targeting proteins to the cell-surface membrane.

A glycan-phosphatidylinositol-specific phospholipase D in human serum.

A group of proteins anchored to the cell by phosphatidylinositol (PI) has recently been identified. The significance of this new class of membrane anchor is unknown; one possibility is that it facilitates release of the molecule by phospholipases. In fact, phospholipase C enzymes specific for the complex carboxyl-terminal glycolipids of these proteins have been isolated from African trypanosomes and from hepatocyte plasma membranes. This study reports the discovery of a glycan-PI-specific phospholipase D in human serum that cleaves both the membrane form of the variant surface glycoprotein of African trypanosomes and its glycolipid precursor, but not phosphatidylethanolamine, phosphatidylcholine, or phosphatidylinositol. Decay-accelerating factor, another PI-anchored molecule, is also cleaved by the enzyme and converted from a hydrophobic to a soluble protein. The enzyme is Ca2+-dependent, heat labile, and not affected by the inhibitor of serine proteases, phenylmethylsulfonylfluoride. Its function is not known, but the present findings indicate that it participates in the metabolism of glycolipid-anchored membrane proteins.

Cloning of decay-accelerating factor suggests novel use of splicing to generate two proteins.

Decay-accelerating factor (DAF), a glycoprotein that is anchored to the cell membrane by phosphatidylinositol, binds activated complement fragments C3b and C4b, thereby inhibiting amplification of the complement cascade on host cell membranes. Here, we report the molecular cloning of human DAF from HeLa cells. Analysis of DAF complementary DNAs revealed two classes of DAF messenger RNA, one apparently derived from the other by a splicing event that causes a coding frameshift near the C terminus. The apparent 'intron' sequence contains an Alu family member and encodes contiguous protein sequence. Two DAF proteins are therefore possible, having divergent C-terminal domains which differ in their hydrophobicity. Both mRNAs are found on polysomes, suggesting that both are translated. We propose that the major (90%) spliced DAF mRNA encodes membrane-bound DAF whereas the minor (10%) unspliced DAF mRNA may encode secreted DAF and we present expression data supporting this. The deduced DAF sequence contains four repeating units homologous to a consensus repeat found in a recently described family of complement proteins.

Isolation of decay accelerating factor (DAF) by a two-step procedure and determination of its N-terminal sequence.

Decay-accelerating factor (DAF) from human red cell membranes was purified by a two-step procedure involving anion exchange and immunoaffinity chromatography. The DAF preparations were purified to homogeneity as judged by silver staining. In several experiments, the final product yields were approximately 23% of the total DAF present in the initial membrane extracts. The purified DAF retained its ability to inhibit the classical pathway C3-convertase and to reincorporate into cell membranes. An amino-terminal sequence was obtained by gas-phase sequencing. Rabbit antibodies to a synthetic peptide representing part of this sequence reacted with purified reduced membrane DAF by Western blotting and by a solid-phase immunoradiometric assay.

Decay-accelerating factor (DAF) shares a common carbohydrate determinant with the variant surface glycoprotein (VSG) of the African Trypanosoma brucei.

Decay-accelerating factor (DAF) is an integral membrane protein that inhibits amplification of the complement cascade on the cell surface. We and other investigators have shown that DAF is part of a newly characterized family of proteins that are anchored to the cell membrane by phosphatidylinositol (PI). The group includes the variant surface glycoprotein (VSG) of African trypanosomes, the p63 protein of Leishmania, acetylcholinesterase (AChE), alkaline phosphatase, Thy-1, 5'-nucleotidase, and RT6.2--an alloantigen from rat T cells. The structure of the membrane anchor has been best characterized for VSG, but chemical studies of the membrane anchors of AChE and Thy-1 suggest that similar glycolipid moieties anchor these proteins to the cell surface. In the VSG, the membrane anchor consists of an ethanolamine linked covalently to an oligosaccharide and glucosamine; the entire complex is anchored to the cell membrane by PI. Immunologically, this glycolipid defines an epitope, the cross-reacting determinant (CRD), that is only revealed after removal of the diacyl glycerol anchor by a phospholipase C. By Western blotting, we show here that DAF-S (DAF released from the membrane by PI-specific phospholipase C [PIPLC]) also contains CRD. Using a newly developed immunoradiometric assay (IRMA) in which the solid-phase capturing antibody is a monoclonal antibody to DAF and the second antibody is anti-CRD, we have been able to quantitate DAF-S. By IRMA, we show that the reaction between anti-CRD and DAF-S is specific, since the binding is competitively inhibited only by the soluble form of the VSG. These observations further support the concept that the glycolipid anchors of this new family of proteins have similar structures. DAF is also found as a soluble protein in various tissue fluids as well as in Hela cell supernatants. No evidence for the presence of the CRD epitope was found on these proteins, suggesting that these forms of DAF are not released from the surface of cells by endogenous phospholipases.