Surface Expression Of GPI-anchored Proteins Research Articles

Paroxysmal nocturnal hemoglobinuria caused by CN-LOH of constitutional PIGB mutation and 70-kbp microdeletion on 15q

AbstractParoxysmal nocturnal hemoglobinuria (PNH) is a rare hematopoietic stem cell (HSC) disorder characterized by defective synthesis of the glycosylphosphatidylinositol (GPI) anchors as a result of somatic mutations in the X-linked PIGA gene. The disease is acquired. No constitutional PNH has been described. Here, we report familial PNH associated with unusual inflammatory symptoms. Genetic analysis revealed a germline heterozygous PIGB mutation on chromosome 15 without mutations in PIGA or any of the other genes involved in GPI biosynthesis. In vitro data confirmed that transfection of the mutant PIGB could not restore the surface expression of GPI-anchored proteins (APs) in PIGB-deficient Chinese hamster ovary cells. Homozygosity was caused by copy number–neutral loss of heterozygosity (CN-LOH) of the germline PIGB mutation, leading to deficient expression of GPI-APs in the affected blood cells of the index patient and her mother. The somatic event leading to homozygosity of the germline mutant PIGB gene involved a 70-kbp microdeletion of chromosome 15q containing the TM2D3 and TARSL2 genes, which was implicated in chromosome 15q mosaicism. Interestingly, we detected the deletion in both the patient and her mother. A sister of the mother, who carried the same germline PIGB mutation but without this microdeletion involving TM2D3 and TARSL2, did not have a PNH clone or CN-LOH. In conclusion, we describe PNH caused by CN-LOH of a germline heterozygous PIGB mutation in a patient and her mother and hypothesize that the 70-kbp microdeletion may have contributed to the PNH clone in both.

A PIGH mutation leading to GPI deficiency is associated with developmental delay and autism.

We identified an individual with a homozygous missense variant (p.Ser103Pro) in a conserved residue of the glycosylphosphatidylinositol (GPI) biosynthesis gene PIGH. This gene encodes an essential component of the phosphatidylinositol N-acetylglucosaminyltransferase complex, in the first step of the biosynthesis of GPI, a glycolipid anchor added to more than one hundred human proteins, several being critical for embryogenesis and neurological functions. The affected individual had hypotonia, moderate developmental delay, and autism. Unlike other reported individuals with GPI deficiency, the proband did not have epilepsy; however, he did have two episodes of febrile seizures. He had normal alkaline phosphatase and no brachytelephalangy. Upon analysis of the surface expression of GPI-anchored proteins on granulocytes, he was demonstrated to have GPI deficiency. This suggests that PIGH mutations may cause a syndrome with developmental delay and autism, but without an epileptic encephalopathy, and should increase the awareness of the potentially deleterious nature of biallelic variants in this gene.

Genome-Wide Screening of Genes Required for Glycosylphosphatidylinositol Biosynthesis.

Glycosylphosphatidylinositol (GPI) is synthesized and transferred to proteins in the endoplasmic reticulum (ER). GPI-anchored proteins are then transported from the ER to the plasma membrane through the Golgi apparatus. To date, at least 17 steps have been identified to be required for the GPI biosynthetic pathway. Here, we aimed to establish a comprehensive screening method to identify genes involved in GPI biosynthesis using mammalian haploid screens. Human haploid cells were mutagenized by the integration of gene trap vectors into the genome. Mutagenized cells were then treated with a bacterial pore-forming toxin, aerolysin, which binds to GPI-anchored proteins for targeting to the cell membrane. Cells that showed low surface expression of CD59, a GPI-anchored protein, were further enriched for. Gene trap insertion sites in the non-selected population and in the enriched population were determined by deep sequencing. This screening enriched 23 gene regions among the 26 known GPI biosynthetic genes, which when mutated are expected to decrease the surface expression of GPI-anchored proteins. Our results indicate that the forward genetic approach using haploid cells is a useful and powerful technique to identify factors involved in phenotypes of interest.

Stable cell surface expression of GPI-anchored proteins, but not intracellular transport, depends on their fatty acid structure.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are a class of lipid anchored proteins expressed on the cell surface of eukaryotes. The potential interaction of GPI-APs with ordered lipid domains enriched in cholesterol and sphingolipids has been proposed to function in the intracellular transport of these lipid anchored proteins. Here, we examined the biological importance of two saturated fatty acids present in the phosphatidylinositol moiety of GPI-APs. These fatty acids are introduced by the action of lipid remodeling enzymes and required for the GPI-AP association within ordered lipid domains. We found that the fatty acid remodeling is not required for either efficient Golgi-to-plasma membrane transport or selective endocytosis via GPI-enriched early endosomal compartment (GEEC)/ clathrin-independent carrier (CLIC) pathway, whereas cholesterol depletion significantly affects both pathways independent of their fatty acid structure. Therefore, the mechanism of cholesterol dependence does not appear to be related to the interaction with ordered lipid domains mediated by two saturated fatty acids. Furthermore, cholesterol extraction drastically releases the unremodeled GPI-APs carrying an unsaturated fatty acid from the cell surface, but not remodeled GPI-APs carrying two saturated fatty acids. This underscores the essential role of lipid remodeling to ensure a stable membrane association of GPI-APs particularly under potential membrane lipid perturbation.

Differential Gene Expression Profile in the T Cell Compartment Between Classic PNH and Aplastic Anemia/PNH Syndrome.

Abstract 3011Poster Board II-987Somatic mutation of the X- linked gene PIG-A results in partial or absolute deficiency of cell surface expression of glycosylphosphatidylinositol-anchored proteins (GPI-APs). In humans, this genetic event in a hematopoietic stem cell leads to the blood disease paroxysmal nocturnal hemoglobinuria (PNH). PNH has two major clinical presentations: predominantly hemolytic without overt marrow failure, referred to as classic PNH, and PNH clonal expansion in the setting of marrow failure, or the aplastic anemia/PNH syndrome (AA/PNH). The diagnosis of PNH is based on the presence of GPI-AP-deficient clones mainly within erythrocytes and granulocytes. Since PNH is a stem cell disorder, all hematopoietic lineages are involved and GPI-AP-deficient clones are found among platelets, monocytes and lymphocytes. Except for their different clinical presentations, classic PNH and AA-PNH have never been shown to have specific discriminating biological features. Indeed, we previously reported no difference in the expression profile of CD34 cells in hemolytic and aplastic forms of PNH (Chen G et al, Leukemia 2005; 19:862). As marrow failure in PNH, as in aplastic anemia, is immune-mediated, and T cells are affected by the PIG-A mutation, we hypothesized that the mutant subset of lymphocytes might differ in PNH. We compared by microarray the T cell compartment in both forms of the disease. We sorted peripheral blood GPI-AP-normal and -deficient pooled CD4 and CD8 T cells using CD59 and CD55 as GPI-AP markers, and subjected each subset to array hybridization and analysis. Samples were obtained from 1 patient with classic PNH, 3 patients with AA-PNH syndrome and 3 age-matched normal donors as controls. In preliminary control experiments, we determined using microarray of total RNA derived from healthy donors' T cells, with and without GPI-AP-specific antibodies, the absence of altered gene expression pattern or activation of specific genes due to selective antibody binding to normal cells. By principal component analysis (PCA), the phenotype of the PNH clone in classic PNH was divergent from the AA-PNH clone and from that of normal donors. By consistency test, only 15 probesets/genes resulted common in the comparison between GPI-AP-deficient and GPI-AP-normal cells between classic PNH and AA-PNH syndrome. Using Ingenuity Software, we found that the represented probesets/genes were involved in some major canonical pathways such as cell death, immune and lymphatic system development, immune response, and cell-to-cell signaling interaction. In order to investigate the different gene expression profile derived from specific subset of T cells, we sorted GPI-AP-deficient and GPI-AP-normal CD8 T cells using CD48 and CD52 as markers. When we compared GPI-AP-deficient and GPI-AP-normal cells between classic PNH and AA-PNH syndrome, we found that only 23 probesets/genes were up-regulated and 132 down-regulated confirming the divergent gene expression profile between the two diseases (Figure 1). We conclude that the restricted similarity in the gene expression profile in the GPI-AP-deficient T cells between classic PNH and AA-PNH syndrome leads to a specific “signature” of genes that might support the different clinical presentation and evolution of the two diseases. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

TbGPI16 is an essential component of GPI transamidase in Trypanosoma brucei

Glycosylphosphatidylinositol (GPI) is widely used by eukaryotic cell surface proteins for membrane attachment. De novo synthesized GPI precursors are attached to proteins post-translationally by the enzyme complex, GPI transamidase. TbGPI16, a component of the trypanosome transamidase, shares similarity with human PIG-T. Here, we show that TbGPI16 is the orthologue of PIG-T and an essential component of GPI transamidase by creating a TbGPI16 knockout. TbGPI16 forms a disulfide-linked complex with TbGPI8. A cysteine to serine mutant of TbGPI16 was unable to fully restore the surface expression of GPI-anchored proteins upon transfection into the knockout cells, indicating that its disulfide linkage with TbGPI8 is important for the full transamidase activity.

Trypanosoma brucei: TbRAB4 regulates membrane recycling and expression of surface proteins in procyclic forms

TbRAB4 is the Trypanosoma brucei orthologue of the small GTPase Rab4, which is implicated in the control of early endocytosis and recycling processes. TbRAB4 is expressed constitutively in the procyclic and bloodstream stages suggesting an important function throughout the trypanosome life-cycle. Previous work from our laboratory has shown TbRAB4 to be essential in the bloodstream form. Induction of double-stranded TbRAB4 RNA expression leads to a specific reduction in TbRAB4 protein levels and inhibition of growth in procyclic form T. brucei, with alterations in uptake and recycling as measured with the fluorophore FM4-64. Trypanosomes overexpressing GTP-locked TbRAB4 QL mutants exhibit significant perturbations of endocytic and recycling pathways as well as disruption of surface expression of GPI-anchored proteins. Most significantly, both the endogenous GPI-anchored procyclins and an ectopically expressed GPI-anchored protein, the variant surface glycoprotein, are relocated from the surface to internal sites in TbRAB4 mutant cells. These data indicate that TbRAB4 is important in maintenance of normal surface expression of lipid-anchored proteins, and implicate recycling pathways as factors for modulation of surface protein expression in the procyclic trypanosome. The conservation of function of Rab4 throughout eukaryotic evolution demonstrated here indicates that the Rab4-mediated trafficking pathway is an extremely ancient component of the endocytic system.

The Initial Enzyme for Glycosylphosphatidylinositol Biosynthesis Requires PIG-Y, a Seventh Component

Biosynthesis of glycosylphosphatidylinositol (GPI) is initiated by an unusually complex GPI-N-acetylglucosaminyltransferase (GPI-GnT) consisting of at least six proteins. Here, we report that human GPI-GnT requires another component, termed PIG-Y, a 71 amino acid protein with two transmembrane domains. The Burkitt lymphoma cell line Daudi, severely defective in the surface expression of GPI-anchored proteins, was a null mutant of PIG-Y. A complex of six components was formed without PIG-Y. PIG-Y appeared to be directly associated with PIG-A, implying that PIG-Y is the key molecule that regulates GPI-GnT activity by binding directly to the catalytic subunit PIG-A. PIG-Y is probably homologous to yeast Eri1p, a component of GPI-GnT. We did not obtain evidence for a functional linkage between GPI-GnT and ras GTPases in mammalian cells as has been reported for yeast cells. A single transcript encoded PIG-Y and, to its 5' side, another protein PreY that has homologues in a wide range of organisms and is characterized by a conserved domain termed DUF343. These two proteins are translated from one mRNA by leaky scanning of the PreY initiation site.

Two Subunits of Glycosylphosphatidylinositol Transamidase, GPI8 and PIG-T, Form a Functionally Important Intermolecular Disulfide Bridge

Many eukaryotic proteins are tethered to the plasma membrane via glycosylphosphatidylinositol (GPI). GPI transamidase is localized in the endoplasmic reticulum and mediates post-translational transfer of preformed GPI to proteins bearing a carboxyl-terminal GPI attachment signal. Mammalian GPI transamidase is a multimeric complex consisting of at least five subunits. Here we report that two subunits of mammalian GPI transamidase, GPI8 and PIG-T, form a functionally important disulfide bond between conserved cysteine residues. GPI8 and PIG-T mutants in which relevant cysteines were replaced with serines were unable to fully restore the surface expression of GPI-anchored proteins upon transfection into their respective mutant cells. Microsomal membranes of these transfectants had markedly decreased activities in an in vitro transamidase assay. The formation of this disulfide bond is not essential but required for full transamidase activity. Antibodies against GPI8 and PIG-T revealed that endogenous as well as exogenous proteins formed a disulfide bond. Furthermore trypanosome GPI8 forms a similar intermolecular disulfide bond via its conserved cysteine residue, suggesting that the trypanosome GPI transamidase is also a multimeric complex likely containing the orthologue of PIG-T. We also demonstrate that an inactive human GPI transamidase complex that consists of non-functional GPI8 and four other components was co-purified with the proform of substrate proteins, indicating that these five components are sufficient to hold the substrate proteins.

Cloning of Trypanosoma brucei and Leishmania major Genes Encoding the GlcNAc-Phosphatidylinositol De-N-acetylase of Glycosylphosphatidylinositol Biosynthesis That Is Essential to the African Sleeping Sickness Parasite

The second step of glycosylphosphatidylinositol anchor biosynthesis in all eukaryotes is the conversion of D-GlcNAcalpha1-6-d-myo-inositol-1-HPO(4)-sn-1,2-diacylglycerol (GlcNAc-PI) to d-GlcNalpha1-6-d-myo-inositol-1-HPO(4)-sn-1,2-diacylglycerol by GlcNAc-PI de-N-acetylase. The genes encoding this activity are PIG-L and GPI12 in mammals and yeast, respectively. Fragments of putative GlcNAc-PI de-N-acetylase genes from Trypanosoma brucei and Leishmania major were identified in the respective genome project data bases. The full-length genes TbGPI12 and LmGPI12 were subsequently cloned, sequenced, and shown to complement a PIG-L-deficient Chinese hamster ovary cell line and restore surface expression of GPI-anchored proteins. A tetracycline-inducible bloodstream form T. brucei TbGPI12 conditional null mutant cell line was created and analyzed under nonpermissive conditions. TbGPI12 mRNA levels were reduced to undetectable levels within 8 h of tetracycline removal, and the cells died after 3-4 days. This demonstrates that TbGPI12 is an essential gene for the tsetse-transmitted parasite that causes Nagana in cattle and African sleeping sickness in humans. It also validates GlcNAc-PI de-N-acetylase as a potential drug target against these diseases. Washed parasite membranes were prepared from the conditional null mutant parasites after 48 h without tetracycline. These membranes were shown to be greatly reduced in GlcNAc-PI de-N-acetylase activity, but they retained their ability to make GlcNAc-PI and to process d-GlcNalpha1-6-d-myo-inositol-1-HPO(4)-sn-1,2-diacylglycerol to later glycosylphosphatidylinositol intermediates. These results suggest that the stabilities of other glycosylphosphatidylinositol pathway enzymes are not dependent on GlcNAc-PI de-N-acetylase levels.

Requirement of PIG-F and PIG-O for Transferring Phosphoethanolamine to the Third Mannose in Glycosylphosphatidylinositol

Many eukaryotic proteins are anchored by glycosylphosphatidylinositol (GPI) to the cell surface membrane. The GPI anchor is linked to proteins by an amide bond formed between the carboxyl terminus and phosphoethanolamine attached to the third mannose. Here, we report the roles of two mammalian genes involved in transfer of phosphoethanolamine to the third mannose in GPI. We cloned a mouse gene termed Pig-o that encodes a 1101-amino acid PIG-O protein bearing regions conserved in various phosphodiesterases. Pig-o knockout F9 embryonal carcinoma cells expressed very little GPI-anchored proteins and accumulated the same major GPI intermediate as the mouse class F mutant cell, which is defective in transferring phosphoethanolamine to the third mannose due to mutant Pig-f gene. PIG-O and PIG-F proteins associate with each other, and the stability of PIG-O was dependent upon PIG-F. However, the class F cell is completely deficient in the surface expression of GPI-anchored proteins. A minor GPI intermediate seen in Pig-o knockout but not class F cells had more than three mannoses with phosphoethanolamines on the first and third mannoses, suggesting that this GPI may account for the low expression of GPI-anchored proteins. Therefore, mammalian cells have redundant activities in transferring phosphoethanolamine to the third mannose, both of which require PIG-F.

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.

Signal Transduction via Glycosyl Phosphatidylinositol-anchored Proteins in T Cells Is Inhibited by Lowering Cellular Cholesterol

Glycosylphosphatidylinositol (GPI)-anchored proteins can deliver costimulatory signals to lymphocytes, but the exact pathway of signal transduction involved is not yet characterized. GPI-anchored proteins are fixed to the cell surface solely by a phospholipid moiety and are clustered in distinct membrane domains that are formed by an unique lipid composition requiring cholesterol. To elucidate the role of membrane lipids for signal transduction via GPI-anchored proteins, we studied the influence of reduced cellular cholesterol content on calcium signaling via GPI-anchored CD59 and CD48 in Jurkat T cells. Lowering cholesterol by different inhibitors of cellular cholesterol synthesis suppressed calcium response via GPI-anchored proteins by about 50%, whereas stimulation via CD3 was only minimally affected (<10%). The decrease in overall calcium response via GPI-anchored proteins was reflected by inhibition of calcium release from intracellular stores. Cell surface expression of GPI-anchored proteins was not changed quantitatively by lowering cellular cholesterol, and neither was the pattern of immunofluorescence in microscopic examination. In addition, the distribution of GPI-anchored proteins in detergent-insoluble complexes remained unaltered. These results suggest that cellular cholesterol is an important prerequisite for signal transduction via GPI-anchored proteins beyond formation of membrane domains.

A Patient With Paroxysmal Nocturnal Hemoglobinuria Bearing Four Independent PIG-A Mutant Clones

AbstractParoxysmal nocturnal hemoglobinuria (PNH) is characterized by clonal blood cells that are deficient in the surface expression of glycosylphosphatidylinositol-anchored proteins due to somatic mutation in the X-linked gene PIG-A. In some patients, more than one abnormal clone may be present. Analysis of bulk DNA/RNA from granulocytes has been useful in identifying the predominant PIG-A mutation in each patient. However, it is often not useful in determining the presence of minor clones. Many patients have cells with partial deficiency. Here, we analyzed the PIG-A gene in two B-cell lines bearing complete or partial deficiencies, cells of hematopoietic progenitor colonies and peripheral blood granulocytes from the same patient. We found that two B-cell lines had different mutations, the granulocytes contained at least two mutants, and the hematopoietic progenitors contained four mutants. Three of the four were shared by B cells and/or granulocytes whereas the other one was found only in the hematopoietic progenitors. The partial deficiency was caused by a point mutation near an alternative splice site within exon 2 that resulted in partial decreases of activity and quantity of the full-length transcript. These results further show the oligoclonal nature of PNH and differences in extent of expansion among mutant clones.

Glycosylphosphatidylinositol-Anchor-Deficient Mice: Implications for Clonal Dominance of Mutant Cells in Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic stem cell disorder characterized by complement-mediated hemolysis. Abnormal hematopoietic cells from patients with PNH are deficient in glycosylphosphatidylinositol (GPI)-anchored proteins and clonally dominate various hematopoietic lineages in the bone marrow and the peripheral blood. Analysis of many patients with PNH has showed that somatic mutation in the X-linked gene PIG-A is responsible for the GPI-anchor deficiency in PNH. The PIG-A mutation must also be relevant to the clonal dominance of GPI-anchor deficient (GPI-) blood cells because two or more PIG-A mutant clones become dominant in many patients. However, whether the PIG-A mutation alone is sufficient for clonal dominance is not known. To address this question, we generated chimeric mice using Pig-a (the murine homologue of PIG-A) disrupted embryonic stem (ES) cells, in which the animals are chimeric with respect to the surface expression of GPI-anchored proteins. The chimerism of hematopoietic and nonhematopoietic tissues in such mice was always low, suggesting that the higher contribution of Pig-a disrupted GPI- cells had a lethal effect on the chimera. GPI- cells appeared in the peripheral blood of some of the chimeric mice. However, the percentage of GPI- erythrocytes did not increase for 10 months after birth, implying that the Pig-a mutation alone does not immediately cause the clonal dominance of GPI- blood cells; another pathologic or physiologic change(s) in the hematopoietic environments or in the clone itself may be necessary.

Defective Glycosyl Phosphatidylinositol Anchor Synthesis and Paroxysmal Nocturnal Hemoglobinuria

Publisher Summary The glycosyl phosphatidylinositol (GPI) anchor is synthesized in the endoplasmic reticulum (ER) and post-translationally linked to the nascent peptides in the ER. If the peptides are not GPI anchored, they are not expressed on the cell surface due to intracellular retention, degradation, and secretion. The human disease, paroxysmal nocturnal hemoglobinuria (PNH), is caused by this mechanism. PNH is a hematopoietic stem cell disorder characterized by the presence of abnormal cells of various hematopoietic cell lineages that are deficient in the surface expression of GPI-anchored proteins. The abnormal red blood cells of patients with PNH are sensitive to the hemolytic action of complement due to the deficient surface expression of decay accelerating factor and CD59, which are GPI-anchored complement inhibitors. The first step of GPI anchor biosynthesis is deficient in abnormal blood cells from patients with PNH. The X-linked gene PIG-A is cloned and somatically mutated in abnormal cells from patients with PNH. Due to its X-chromosomal location, a single inactivating mutation results in a loss of GPI anchor synthesis, even in a female hematopoietic stem cell, if it occurs in the active allele of the PIG-A gene. More than 60 patients with PNH from various countries were analyzed and the PIG-A gene was responsible for PNH in all of them. The X-chromosomal location of the PIG-A gene would also account for this uniformity of the responsible gene among the 10 or so genes involved in GPI anchor synthesis. Patients with PNH have one or more mutant hematopoietic stem cell clones that are deficient in GPI anchor synthesis due to somatic mutations in the X-linked gene, PIG-A. The somatic mutations of PIGA are widely distributed in the coding regions and splice sites, indicating that they occur at random sites.

Expression and secretion of glycosylphosphatidylinositol-specific phospholipase D by myeloid cell lines

Glycosylphosphatidylinositol (GPI)-specific phospholipase D (GPI-PLD) is abundant in mammalian plasma. It could potentially regulate the surface expression of GPI-anchored proteins, but it remains to be established which tissue(s) or cell type(s) are the principal sources of the circulating enzyme. Here we report that all the myeloid cell lines tested, including K562 (multipotential blast), KG-1 (human myeloblast), HL-60, NB4, PLB-985 (human promyelocyte), U937 (human promonocyte), THP-1 (human monocyte) and J774, RAW264.7 (mouse monocyte/macrophage), contained GPI-degrading activity. T.l.c. analysis of reaction products confirmed the activity as a phospholipase D. These cells also exhibited positive immunofluorescent staining with an anti-GPI-PLD monoclonal antibody. The expression of GPI-PLD activity was not substantially reduced when the cells were cultured in either serum-free medium or GPI-PLD-depleted regular medium. Both granulocytic and monocytic differentiation of myelomonoblastic lines (e.g. HL-60) induced by dimethyl sulphoxide or phorbol diester respectively was accompanied by a 2-3-fold increase in GPI-PLD activity. J774 and HL-60 cells secreted GPI-PLD into the medium constitutively. Taken together, these data suggest that myeloid cells are a potential contributor to the circulating GPI-PLD pool. As leucocytes express many important GPI-anchored surface antigens, these cells may prove to be a valuable model system for studying the physiological functions of GPI-PLD.

[56] Glycosylphosphatidylinositol-specific phospholipase D

The glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) was first observed as a result of its ability to degrade the GPI anchor of alkaline phosphatase during extraction from mammalian tissues with butanol. The GPI-PLD has recently been purified from human and bovine serum. The physiological function of this enzyme has not been determined, but it is proposed to play a role in the regulation of cell surface expression of GPI-anchored proteins. The GPI-PLD hydrolyzes the phosphodiester linkage of the phosphatidylinositol moiety in the GPI anchor of several membrane proteins. The products of this reaction are phosphatidic acid and an inositol residue, which is attached to the C terminus of the protein through the glycan. The assay methods that have been used in the purification procedure described in the chapter monitor production of either of these products. In the assay where alkaline phosphatase is used as substrate, the loss of the hydrophobic part of the anchor from the protein is determined by partitioning in Triton X-114 H and measuring the distribution of the protein between the hydrophilic, detergent-poor phase and the hydrophobic, detergent-rich phase. The substrate is human placental alkaline phosphatase, and its distribution in Triton X-114 is determined by measuring its ability to hydrolyze p -nitrophenyl phosphate in a subsequent incubation. The major disadvantage of this type of assay is that it does not distinguish between GPI-PLD-mediated cleavage and those catalyzed by other hydrolases especially GPI-PLC. It is also more time consuming as it involves a second incubation to assay for the degraded alkaline phosphatase. However, an advantage over the other assay procedure is the ready availability of the starting material for preparation of the substrate.