Michael A. Davitz

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.

Neoplastic angioendotheliomatosis: a variant of malignant lymphoma immunohistochemical and ultrastructural observations of three cases

Neoplastic angioendotheliomatosis (NAE) is a rare, fatal disease characterized by widespread intravascular proliferations of malignant cells of putative endothelial origin. Clinically, dermatologic and bizarre neurological manifestations predominate, but review of the reported cases of NAE reveals ophthalmic involvement to be frequent. To our knowledge, no reports of NAE have appeared in the ophthalmic literature. We describe three cases of NAE with the ocular manifestations of visual loss, cells in the vitreous, retinal artery occlusion, retinal vascular and pigment epithelial alterations, nystagmus, and cortical blindness. Autopsies (including eyes and central nervous system) revealed pancorporal involvement by intravascular anaplastic cells in each patient. In two patients massive extravascular involvement was also present. The tumor cells lacked ultrastructural features of endothelial cells and failed to stain for factor-VIII-related antigen. Common leukocyte antigen, a maker for hematopoietic cells, particularly lymphocytes, was detected on tumor cells in all cases, indicating that NAE is probably an extranodal lymphoma. The dramatic response of the central nervous system lesions to radiotherapy in one case supports this contention. It is suggested that this disorder be treated as a malignant lymphoma.

Mouse glycosylphosphatidylinositol-specific phospholipase D (Gpld1) characterization

Abstract. 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 μg/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.

Characterization of the plasma glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD)

Proteins anchored to the cell membrane by glycosylphosphatidylinosital (GPI) are functionally diverse; they include: complement regulatory proteins, cell-adhesion molecules, ectoenzymes, lymphocyte differentiation antigens , tumor markers, both the normal and scrapie forms of the prion protein, as well as parasitic protozoan cell surface antigens (Cross, 1990). One proposed function for the GPI-anchor is that it facilitates the release of the protein from the membrane by serving as a target substrate for anchor-specific phospholipases (Low, 1990). Recently, we and others have discovered (Davitz et al., 1987; Cardoso de Almeida et al., 1988; and Low and Prasad, 1988), purified (Davitz et al., 1989; Huang et al., 1990), and cloned (Scallon et al., 1991) a GPI-specific phospholipase D (GPI-PLD) isolated from mammalian plasma. Based on a cleavage analysis of the GPI-biosynthetic precursors for the membrane form variant surface glycoprotein (mfVSG) of Trypanosorna brucei, the minimal carbohydrate structure recognized by the GPI-PLD consists of Mannose-Glucosamine-phosphatidylinositol (Man,GlcN-PI) (Masterson et al., 1989) (Figure 1). This carbohydrate unit is part of a conserved glycan core backbone that is present on both trypanosomal as well as mammalian anchors (Cross, 1990). Thus it is likely that all GPI-anchors could serve as potential substrates for the GPI-PLD.

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.