GPI-specific Phospholipase D Research Articles

Age-dependent membrane release and degradation of full-length glycosylphosphatidylinositol-anchored proteins in rats

Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are associated with the surface of eucaryotic cells only through a covalently coupled carboxy-terminal GPI glycolipid structure which is anchored at the outer leaflet of plasma membranes. This mode of membrane association may be responsible for the recent observations that full-length GPI-APs harbouring the complete GPI anchor are (i) released from isolated rat adipocytes in vitro and (ii) expressed in rat and human serum. The upregulation of the adipocyte release in response to increased cell size and blood glucose/insulin levels of the donor rats and downregulation of the expression in serum of insulin resistant and diabetic rats have been reconciled with enhanced degradation of the full-length GPI-APs released into micelle-like complexes together with (lyso) phospholipids and cholesterol by serum GPI-specific phospholipase D (GPI-PLD).Here by using a sensitive and reliable sensing method for full-length GPI-APs, which relies on surface acoustic waves propagating over microfluidic chips, the upregulation of (i) the release of the full-length GPI-APs CD73, alkaline phosphatase and CD55 from isolated adipocyte plasma membranes monitored in a “lab-on-the-chip” configuration, (ii) their release from isolated rat adipocytes into the incubation medium and (iii) the lipolytic cleavage of their GPI anchors in serum was demonstrated to increase with age (3–16 weeks) and body weight (87−477 g) of (healthy) donor rats. In contrast, the amount of full-length GPI-APs in rat serum, as determined by chip-based sensing, turned out to decline with age/body weight. These correlations suggest that age-/weight-induced alterations (in certain biophysical/biochemical characteristics) of plasma membranes are responsible for the release of full-length GPI-APs which becomes counteracted by elevated GPI-PLD activity in serum. Thus, sensitive and specific measurement of these GPI-AP-relevant parameters may be useful for monitoring of age-related cell surface changes, in general, and diseases, in particular.

Regulation of glycosylphosphatidylinositol-anchored proteins and GPI-phospholipase D in a c-Myc transgenic mouse model of hepatocellular carcinoma and human HCC.

Recent research implicated glycosylphosphatidylinositol-anchored proteins (GPI-AP) and GPI-specific phospholipase D (GPI-PLD) in the pathogenesis of fatty liver disease and hepatocellular carcinoma (HCC). Given that c-Myc is frequently amplified in HCC, we investigated their regulation in a c-Myc transgenic disease model of liver cancer and HCC patient samples. Whole genome scans defined 54 significantly regulated genes coding for GPI-AP of which 29 and 14 were repressed in expression in transgenic tumors and steatotic human hepatocyte cultures, respectively, to influence lipid-mediated signal transduction, extracellular matrix and immunity pathways. Analysis of gene specific promoter revealed >95% to carry c-Myc binding sites thus establishing a link between c-Myc activity and transcriptional response. Alike, serum GPI-PLD activity was increased 4-fold in transgenic mice; however its tissue activity was reduced by 70%. The associated repression of the serine/threonine phosphatase 2A (PP2A), i.e. a key player of c-Myc proteolysis, indicates co-ordinate responses aimed at impairing tissue GPI-PLD anti-proliferative activities. Translational research identified >4-fold increased GPI-PLD serum protein expression though enzyme activities were repressed by 60% in NASH and HCC patients. Taken collectively, c-Myc influences GPI-AP signaling transcriptionally and posttranslational and represses GPI-AP anti-proliferative signaling in tumors. The findings broaden the perspective of molecular targeted therapies and disease monitoring.

Wnt Signaling Is Regulated by Endoplasmic Reticulum Retention

Precise regulation of Wnt signaling is important in many contexts, as in development of the vertebrate forebrain, where excessive or ectopic Wnt signaling leads to severe brain defects. Mutation of the widely expressed oto gene causes loss of the anterior forebrain during mouse embryogenesis. Here we report that oto is the mouse ortholog of the gpi deacylase gene pgap1, and that the endoplasmic reticulum (ER)-resident Oto protein has a novel and deacylase-independent function during Wnt maturation. Oto increases the hydrophobicities of Wnt3a and Wnt1 by promoting the addition of glycophosphatidylinositol (gpi)-like anchors to these Wnts, which results in their retention in the ER. We also report that oto-deficient embryos exhibit prematurely robust Wnt activity in the Wnt1 domain of the early neural plate. We examine the effect of low oto expression on Wnt1 in vitro by knocking down endogenous oto expression in 293 and M14 melanoma cells using shRNA. Knockdown of oto results in increased Wnt1 secretion which is correlated with greatly enhanced canonical Wnt activity. These data indicate that oto deficiency increases Wnt signaling in vivo and in vitro. Finally, we address the mechanism of Oto-mediated Wnt retention under oto-abundant conditions, by cotransfecting Wnt1 with gpi-specific phospholipase D (GPI-PLD). The presence of GPI-PLD in the secretory pathway results in increased secretion of soluble Wnt1, suggesting that the gpi-like anchor lipids on Wnt1 mediate its retention in the ER. These data now provide a mechanistic framework for understanding the forebrain defects in oto mice, and support a role for Oto-mediated Wnt regulation during early brain development. Our work highlights a critical role for ER retention in regulating Wnt signaling in the mouse embryo, and gives insight into the notoriously inefficient secretion of Wnts.

Activated human neutrophils rapidly release the chemotactically active D2D3 form of the urokinase-type plasminogen activator receptor (uPAR/CD87)

The urokinase-type plasminogen activator receptor (uPAR/CD87) exists both in cell-bound and soluble forms. Neutrophils contain extensive intracellular pools of uPAR that are translocated to the plasma membrane upon activation. In the present study, we investigated the ability of human neutrophils to shed uPAR from cell surface following activation and addressed the possible involvement of the released receptor in the inflammatory response. We first observed that the spontaneous release of suPAR by resting neutrophils was strongly and rapidly (within minutes) enhanced by calcium ionophore ionomycin and to a lesser extent when cells were primed with TNF-alpha and then stimulated with fMLP or IL-8. We demonstrated that suPAR is produced by resting and activated neutrophils predominantly as a truncated form devoid of N-terminal D1 domain (D2D3 form) that lacks GPI anchor. Migration of formyl peptide receptor-like 1 (FPRL1)-transfected human embryonic kidney (HEK) 293 cells toward the supernatants harvested from activated neutrophils was significantly diminished when D2D3 form of suPAR was immunodepleted from the supernatants. We conclude that activated neutrophils release the chemotactically active D2D3 form of suPAR that acts as a ligand of FPRL1. Interestingly, we present evidence that GPI-specific phospholipase D (GPI-PLD) that has previously been shown to shed uPAR in cancer cells is not involved in suPAR release from human neutrophils. We suggest that production of the chemotactically active D2D3 form of suPAR by activated human neutrophils in vivo could contribute to the recruitment of monocytes and other formyl peptide receptors-expressing cells to the sites of acute inflammation where neutrophil accumulation and activation occur.

Ethanolamine Phosphate Linked to the First Mannose Residue of Glycosylphosphatidylinositol (GPI) Lipids Is a Major Feature of the GPI Structure That Is Recognized by Human GPI Transamidase

Glycosylphosphatidylinositol (GPI) anchoring of proteins is catalyzed by GPI transamidase (GPIT), a multisubunit, endoplasmic reticulum (ER)-localized enzyme. GPIT recognizes ER-translocated proteins that have a GPI-directing C-terminal signal sequence and replaces this sequence with a preassembled GPI anchor. Although the GPI signal sequence has been extensively characterized, little is known about the structural features of the GPI lipid substrate that enable its recognition by GPIT. In a previous study we showed that mature GPIs could be co-immunoprecipitated with GPIT complexes containing functional subunits (Vainauskas, S., and Menon, A. K. (2004) J. Biol. Chem. 279, 6540-6545). We now use this approach, as well as a method that reconstitutes the interaction between GPIs and GPIT, to define the basis of the interaction between GPI and human GPIT. We report that (i) human GPIT can interact with GPI biosynthetic intermediates, not just mature GPIs competent for transfer to protein, (ii) the ethanolamine phosphate group on the third mannose residue of the GPI glycan is not critical for GPI recognition by GPIT, (iii) the ethanolamine phosphate residue linked to the first mannose of the GPI structure is a major feature of GPIs that is recognized by human GPIT, and (iv) the simplest GPI recognized by human GPIT is EtN-P-2Manalpha1-4GlcN-(acyl)-phosphatidyl-inositol. These studies define the molecular characteristics of GPI that are recognized by GPIT and open the way to identifying GPIT subunits that are involved in this process.

Removal or Maintenance of Inositol-linked Acyl Chain in Glycosylphosphatidylinositol Is Critical in Trypanosome Life Cycle

The protozoan parasite Trypanosoma brucei is coated by glycosylphosphatidylinositol (GPI)-anchored proteins. During GPI biosynthesis, inositol in phosphatidylinositol becomes acylated. Inositol is deacylated prior to attachment to variant surface glycoproteins in the bloodstream form, whereas it remains acylated in procyclins in the procyclic form. We have cloned a T. brucei GPI inositol deacylase (GPIdeAc2). In accordance with the acylation/deacylation profile, the level of GPIdeAc2 mRNA was 6-fold higher in the bloodstream form than in the procyclic form. Knockdown of GPIdeAc2 in the bloodstream form caused accumulation of an inositol-acylated GPI, a decreased VSG expression on the cell surface and slower growth, indicating that inositol-deacylation is essential for the growth of the bloodstream form. Overexpression of GPIdeAc2 in the procyclic form caused an accumulation of GPI biosynthetic intermediates lacking inositol-linked acyl chain and decreased cell surface procyclins because of release into the culture medium, indicating that overexpression of GPIdeAc2 is deleterious to the surface coat of the procyclic form. Therefore, the GPI inositol deacylase activity must be tightly regulated in trypanosome life cycle.

Cleavage with phospholipase of the lipid anchor in the cell adhesion molecule, csA, from Dictyostelium discoideum

A cell adhesion molecule, 80-kDa csA, is involved in EDTA-resistant cell contact at the aggregation stage of Dictyostelium discoideum. A 31-kDa csA was isolated from the 80-kDa csA by treatment with Achromobacter protease I. Results from thin-layer chromatography and MALDI-TOF MS analysis indicated that the 31-kDa csA contains ceramide as a component of glycosylphosphatidyl-inositol (GPI). Comparison between the 80-kDa csA and the 31-kDa csA treated with phosphatidylinositol-specific phospholipase C (PI-PLC) or GPI-specific phospholipase D (GPI-PLD) was carried out. Our results indicated that the GPI-anchor of the 31-kDa csA was more sensitive to PI-PLC treatment than that of the 80-kDa csA, and that the anchor in both was easily cleaved by GPI-PLD treatment. They suggested that the resistance of 80-kDa csA to PI-PLC treatment was due to steric hindrance and myo-inositol modification. The results of the 80-kDa csA and the 31-kDa csA treated with sphingomyelinase were similar to those with PI-PLC treatment. In the presence of 1,10-phenanthroline, a GPI-PLD inhibitor, development of Dictyostelium was markedly inhibited, suggesting that GPI-PLD is functional in developmental regulation through cell adhesion.

GPI-specific phospholipase D (GPI-PLD) is expressed during mouse development and is localized to the extracellular matrix of the developing mouse skeleton

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is abundant in serum and has a well-characterized biochemistry; however, its physiological role is completely unknown. Previous investigations into GPI-PLD have focused on the adult animal or on in vitro systems and a putative role in development has been neither proposed nor investigated. We describe the first evidence of GPI-PLD expression during mouse embryonic ossification. GPI-PLD expression was detected predominantly at sites of skeletal development, increasing during the course of gestation. GPI-PLD was observed during both intramembraneous and endochondral ossification and localized predominantly to the extracellular matrix of chondrocytes and to primary trabeculae of the skeleton. In addition, the mouse chondrocyte cell line ATDC5 expressed GPI-PLD after experimental induction of differentiation. These results implicate GPI-PLD in the process of bone formation during mouse embryogenesis.

A distant evolutionary relationship between GPI-specific phospholipase D and bacterial phosphatidylcholine-preferring phospholipase C

In eukaryotes some surface proteins are attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. A GPI-specific phospholipase D (GPI-PLD) activity has been characterized and implicated in the regulation of anchoring, thereby influencing the dispersal of anchored proteins or their maintenance on the cell surface, and possibly in cell signalling. Despite its biological and medical importance, little is known of the structure of GPI-PLD. Here, a distant relationship between the catalytic domains of GPI-PLD and some bacterial phospholipases C is demonstrated. A model of the GPI-PLD catalytic site sheds light on catalysis and highlights possibilities for design of improved and more specific GPI-PLD inhibitors. The databases contain hitherto unnoticed close homologues of GPI-PLD from yeast and Dictyostelium discoideum.

Different distributions of human bone alkaline phosphatase isoforms in serum and bone tissue extracts

Background: In vitro, bone alkaline phosphatase (BALP) is released from the osteoblast membrane with its glycosylphosphatidylinositol (GPI) anchor still attached (i.e., in an anchor-intact form); however, in vivo, BALP circulates as a variable mixture of anchorless isoforms, which can be identified by high-performance liquid chromatography (HPLC). Previous studies have shown that the relative abundance of these BALP isoforms in serum may be clinically useful for the diagnosis and management of metabolic bone disease. Methods: In the current studies, we describe a method for the determination of anchorless BALP isoforms in extracts of bone and we present novel data on the conversion of anchor-intact to anchorless BALP by incubation with endogenous circulating GPI-specific phospholipase D (GPI-PLD). Results: A 72-h extraction with 0.1% Triton X-100 released approximately 90% of the BALP activity from powdered bone. An average of 19% of this activity was anchorless, but essentially all of the activity could be converted to the anchorless form by incubation with partially purified GPI-PLD from human serum. Using HPLC, we detected four BALP isoforms (B/I, B1x, B1, and B2) in these GPI-PLD-treated extracts of bone. An additional BALP fraction was also detected in the samples during the initial phase of GPI-PLD treatment. Conclusions: The abundance of the BALP isoforms differed between bone and serum, particularly for the B/I isoform, which comprised, on average, 18% of the BALP in GPI-PLD-treated extracts of healthy bone tissue, but only 6% of the total BALP activity in serum from healthy individuals. Based on our recent finding of differences in the number of sialic acid residues between the BALP isoforms, we hypothesize that this difference between BALP isoforms in serum and extracts of bone is due to the different patterns of glycosylation, which results in different biological half-lives in the circulation. A preliminary application of our method to the extraction of BALP isoforms from a small number of human bone samples suggests that the method should be useful for studies of human skeletal site-specific and metabolic bone disease-specific differences in the amounts and distributions of the BALP isoforms in bone.

Purification and Characterization of Insulin-Mimetic Inositol Phosphoglycan-Like Molecules From Grass Pea (Lathyrus sativus) Seeds

Signal transduction through the hydrolysis of glycosyl-phosphatidylinositol (GPI) leading to the release of the water-soluble inositol phosphoglycan (IPG) molecules has been demonstrated to be important for mediating some of the actions of insulin and insulin-like growth factor-I (IGF-I). In the present study, GPI from grass pea (Lathyrus sativus) seeds has been purified and partially characterized on the basis of its chromatographic properties and its compositional analysis. The results indicate that it shows similarities to GPI previously isolated from other sources such as rat liver. IPG was generated from L. sativus seed GPI by hydrolysis with a GPI-specific phospholipase D (GPI-PLD). This IPG inhibited protein kinase A (PKA) in an in vitro assay, caused cell proliferation in explanted cochleovestibular ganglia (CVG), and decreased 8-Br-cAMP-induced phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression in cultured hepatoma cells. Our data indicate that L. sativus seed IPG possess insulin-mimetic activities. This may explain why L. sativus seeds have been used in some traditional medicines to ameliorate diabetic symptoms.

Pig-n, a Mammalian Homologue of Yeast Mcd4p, Is Involved in Transferring Phosphoethanolamine to the First Mannose of the Glycosylphosphatidylinositol

Many cell surface proteins are anchored to the membrane via a glycosylphosphatidylinositol (GPI) moiety, which is attached to the C terminus of the proteins. The core of the GPI anchor is conserved in all eukaryotes but is modified by various side chains. We cloned a mouse phosphatidylinositol glycan-class N (Pig-n) gene that encodes a 931amino acid protein expressed in the endoplasmic reticulum, which is homologous to yeast Mcd4p. We disrupted the gene in F9 embryonal carcinoma cells. In the Pig-n knockout cells, the first mannose in the GPI precursors was not modified by phosphoethanolamine. Nevertheless, further biosynthetic steps continued with the addition of the third mannose and the terminal phosphoethanolamine. The surface expression of Thy-1 was only partially affected, indicating that modification of the first mannose by phosphoethanolamine is not essential for attachment of GPI anchors in mammalian cells. An inhibitor of GPI biosynthesis, YW3548/BE49385A, inhibited transfer of phosphoethanolamine to the first mannose in mammalian cells but only slightly affected the surface expression of GPI-anchored proteins. Biosynthesis of GPI in the Pig-n knockout cells was not affected by YW3548/BE49385A, and yeast overexpressing MCD4 was highly resistant to YW3548/BE49385A, suggesting that Pig-n and Mcd4p are targets of this drug.

Intracellular cleavage of glycosylphosphatidylinositol by phospholipase D induces activation of protein kinase Cα

Many proteins are anchored to the cell membrane by glycosylphosphatidylinositol (GPI). One of the functions proposed for the GPI anchor is as a possible mediator in signal transduction through its hydrolysis. GPI-specific phospholipase D (GPI-PLD) is a secretory protein that is suggested to be involved in the release of GPI-anchored protein from the membrane. In the present study we examined how GPI-PLD is involved in signal transduction. When introduced exogenously and overexpressed in cells, GPI-PLD cleaved the GPI anchors in the early secretory pathway, possibly in the endoplasmic reticulum, resulting in an increased production of diacylglycerol. Experiments in vitro and in vivo showed that the association of protein kinase Calpha (PKCalpha) with membranes was increased markedly by expression of GPI-PLD in cells. Furthermore, sucrose-density-gradient centrifugation and immunofluorescence microscopy demonstrated that PKCalpha was translocated to the endoplasmic reticulum membrane in cells expressing GPI-PLD, in contrast with its association with the plasma membrane in cells treated with PMA. We also confirmed that the phosphorylation of c-Fos as well as PKCalpha itself was greatly enhanced by the expression of GPI-PLD. Taken together, these results suggest that GPI-PLD is involved in intracellular cleavage of the GPI anchor, which is a new potential source of diacylglycerol production to activate PKCalpha.

Cellular glycosylphosphatidylinositol-specific phospholipase D regulates urokinase receptor shedding and cell surface expression.

The glycosylphosphatidylinositol (GPI)-anchored, multifunctional receptor for the serine proteinase, urokinase plasminogen activator (uPAR, CD87), regulates plasminogen activation and cell migration, adhesion, and proliferation. uPAR occurs in functionally distinct, membrane-anchored and soluble isoforms (s-uPAR) in vitro and in vivo. Recent evidence indicates that s-uPAR present in the circulation of cancer patients correlates with tumor malignancy and represents a valuable prognostic marker in certain types of cancer. We have therefore analyzed the mechanism of uPAR shedding in vitro. We present evidence that uPAR is actively released from ovarian cancer cells since the rate of receptor shedding did not correlate with uPAR expression. While s-uPAR was derived from the cell surface, it lacked the hydrophobic portion of the GPI moiety indicating anchor cleavage. We show that uPAR release is catalyzed by cellular GPI-specific phospholipase D (GPI-PLD), an enzyme cleaving the GPI anchor of the receptor. Thus, recombinant GPI-PLD expression increased receptor release up to fourfold. Conversely, a 40% reduction in GPI-PLD activity by GPI-PLD antisense mRNA expression inhibited uPAR release by more than 60%. We found that GPI-PLD also regulated uPAR expression, possibly by releasing a GPI-anchored growth factor. Our data suggest that cellular GPI-PLD might be involved in the generation of circulating prognostic markers in cancer and possibly regulate the function of GPI-anchored proteins by generating functionally distinct, soluble counterparts.

Subcellular distribution of glycosylphosphatidylinositol-specific phospholipase D in rat liver.

Glycosylphosphatidylinositol (GPI)-hydrolysing enzymes have been described in many mammalian tissues and body fluids; however, their site(s) of action and in vivo functions have remained unclear. In order to identify a possible intracellular site of GPI hydrolysis, we studied the subcellular distribution of GPI-hydrolysing activity in rat liver. We found that purified fractions from rat liver hydrolysed the GPI moieties of two GPI-anchored proteins with the specificity of a phospholipase D. This GPI-specific phospholipase D (GPI-PLD) activity was found to be highly enriched in a lysosomal fraction and showed a similar intracellular distribution to that of typical lysosomal enzymes. Our results indicate that lysosomes may represent a possible intracellular site of GPI-PLD action.

Hydrolysis of membrane-bound liver alkaline phosphatase by GPI-PLD requires bile salts.

Circulating liver plasma membrane fragments (LPMF) were purified from human serum by means of a monoclonal antileucine aminopeptidase antibody, AD-1. This was done by immunoaffinity chromatography or by incubating the sera with AD-1-coated nitrocellulose disks. Alkaline phosphatase (ALP, EC 3.1.3.1) is bound to these LPMF through a glycosylphosphatidylinositol (GPI) anchor and is referred to as membrane-bound liver ALP (Mem-LiALP). Low concentrations of Triton X-100 or high bile salt concentrations released GPI anchor-bearing LiALP (Anch-LiALP) from purified LPMF; once released, Anch-LiALP was slowly and progressively converted to hydrophilic dimeric LiALP [soluble LiALP (Sol-LiALP)], free from its GPI anchor. Low levels of GPI-specific phospholipase D (GPI-PLD) activity were measured in the pure LPMF. Apparently, this membrane-associated GPI-PLD was released by the action of detergents and contributed to the spontaneous conversion of Anch-LiALP to Sol-LiALP. In the absence of detergents, GPI-PLD had little effect on Mem-LiALP, both in purified form as well as in serum. In vitro, isolated Anch-LiALP was converted to Sol-LiALP by both GPI-specific phospholipase C and GPI-PLD. Sol-LiALP in serum, however, appeared to be the product of GPI-PLD activity only. Five- to tenfold higher concentrations of Triton X-100 were needed to release Anch-LiALP from LPMF in serum, compared with those required in a solution of purified LPMF. In serum, as well as in purified conditions, only a small range of detergent of bile salt concentrations permitted the conversion of Mem-LiALP to Sol-LiALP. A model is proposed for the release in the circulation of Mem-LiALP, Anch-LiALP, and Sol-LiALP, involving both LPMF-associated GPI-PLD and liver sinusoid bile salts.

Cell surface expression of a functional rubella virus E1 glycoprotein by addition of a GPI anchor.

Rubella virus (RV) envelope glycoproteins E1 and E2 are targeted to the Golgi as heterodimers. While E2 contains a transmembrane Golgi retention signal, E1 is arrested in a pre-Golgi compartment in the absence of E2, and appears to require heterodimerization in order to reach the Golgi. Various forms of E1 with deletions in the ectodomain or lacking the cytoplasmic (CT) and transmembrane (TM) domains, as well as the 29 C-terminal amino acid residues of the ectodomain were also retained intracellularly. We therefore investigated the possibility of targetting E1 to the plasma membrane by addition of a glycosylphosphatidylinositol (GPI) anchor. We found that E1GPI was transported to the cell surface where it retained the hemadsorption activity characteristic of the wild-type E1/E2 heterodimer. Furthermore, coexpression of a mammalian GPI-specific phospholipase D (GPI-PLD) resulted in the release of E1GPI and in constitutive expression of a soluble form of E1. This study thus demonstrates that the GPI anchor has a dominant effect over the E1 pre-Golgi retention signal and that E1 is sufficient for hemadsorption.

Streptolysin-O induces release of glycosylphosphatidylinositol-anchored alkaline phosphatase from ROS cells by vesiculation independently of phospholipase action.

Streptolysin-O (SLO), a cholesterol-binding agent, was used for studies on the release of glycosylphosphatidylinositol (GPI)-anchored alkaline phosphatase (AP) from ROS cells. Treatment of cells with SLO resulted in a time- and concentration-dependent release of AP into the extracellular medium. This release was potentiated by Ca2+ and bovine serum, but not by GPI-specific phospholipase D (GPI-PLD) purified from bovine serum. The released AP distributed to the detergent phase after Triton X-114 phase separation. This result suggested that the released AP contained an intact GPI anchor, and thus both proteolysis and anchor degradation by anchor-specific hydrolases, including GPI-PLD, as the potential mechanisms for SLO-mediated AP release were ruled out. The released AP sedimented at 100,000 g. A substantial amount of lipids was detected in the 100,000 g pellet. Cholesterol and sphingomyelin were enriched in SLO-released material, compared with intact cells. These results were consistent with vesiculation as the mechanism for SLO induction of AP release. Two other cholesterol-binding agents, saponin and digitonin, were also able to release AP, possibly by a similar vesiculation mechanism, whereas others, including nystatin, filipin and beta-escin, failed to elicit any AP release. Eight GPI-anchored proteins were identified in ROS cells, and all were substantially enriched in the vesicles released by SLO. Taken together, these results do not provide any support for the hypothesis that the clustering of GPI-anchored proteins in the plasma membrane is responsible for their resistance to GPI-PLD cleavage.

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.

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.