A likely role for a novel PH-domain containing protein, PEPP2, in connecting membrane and cytoskeleton

PH domains (pleckstrin homology) are well known to bind membrane phosphoinositides with different specificities and direct PH domain-containing proteins to discrete subcellular apartments with assistances of alternative binding partners. PH domain-containing proteins are found to be involved in a wide range of cellular events, including signalling, cytoskeleton rearrangement and vesicular trafficking. Here we showed that a novel PH domain-containing protein, PEPP2, displayed moderate phosphoinositide binding specificity. Full length PEPP2 associated with both plasma membrane and microtubules. The membrane-associated PEPP2 nucleated at cell-cell contacts and the leading edge of migrating cells. Overexpression of PEPP2 increased membrane microviscosity, indicating a potential role of PEPP2 in regulating function of membrane and microtubules.

Effect of gamma interferon and interleukin 10 on phosphoinositol turnover by human neutrophils in vitro

We evaluated the levels of inositolmono-(IP1), di-(IP2), tri- (IP3) and tetraphosphates (IP4) in human neutrophils (N) stimulated with gamma interferon (IFN-gamma) (200 mul from a pool of cell culture supernatant obtained from 1 x 10(7) PHA-primed peripheral blood mononuclear cells (30-60 min at 37 degrees Celsius, 5 per cent CO2) in the presence or in the absence of interleukin 10 (IL-10) (10 mug/10mul). The results, reported as mean + SEM cpm, showed that IFN-gamma induced a significant increase only in the IP3 level (N + medium = 1.413 + 172 and N + IFN-gamma = 8,875 + 832). However, this activation mediated by IFN-gamma was blocked partially in the presence of IL-10 (N + IFN-gamma + IL-10 = 2,430 + 239) (P<0.05). Interleukin 10 alone did not induce significant alterations in the content of IP1 (1,203 + 123), IP2 (1,880 + 163), IP3 (938 + 102) or IP4 (2,403 + 345) when compared to the respective controls in the absence of IL-10 (IP1 = 1,625 + 132; IP2 = 1,343 + 149; IP3 = 1,413 + 172 and IP4 = 3,281 + 234). We also demonstrated the inhibitory effect of IL-10 on chemoluminescence generation by human neutrophils during phagocytosis of opsonized particles (OZ). Chemoluminescence generation was enhanced by IFN-gamma (N + OZ = 42.8 + 3.9 and N + OZ + IFN-gamma = 66.5 + 4.3) and this effect was reduced by IL-10 (N + OZ + IFN-gamma + IL-10 = 37.6 + 5.1). These data suggest that IL-10 modulates the neutrophil response and may be important for the development of new treatments of inflammatory injury.

Characterization of a phospholipase C-resistant inositol containing glycolipid from Trypanosoma cruzi

Since glycosylphosphatidylinositol is the most common form of attachment of proteins to membranes in T. cruzi, and that this parasite depends on surface-mediated interactions for survival within the vector and mammalian host, it is probable that a drug which interfers with the metabolism of glycosylphosphatidylinositol (GPI) could be successfully employed in chemotherapy. Over the last few years several groups have been characterizing this mode of attachment in T. cruzi and more recently we have been concentrating our efforts on the identification of candidate precursors for protein anchors in metacyclic trypomastigotes. Previously detected GPI heterogeneity regarding solubilization of a major stage-specific antigen (1G7-Ag) by phospholipase C led us to investigate whether biosynthetic precursors with similar properties could also be identified. Two glycolipid species whose migration properties resemble glycolipids A and C of T. brucei were amenable to biosynthetic radiolabelling with palmitic acid, inositol, ethanolamine, glucosamine and mannose. Following purification, these species were submitted to classical GPI diagnostic treatments. In both cases digestion with GPI-specific phospholipase D (GPIPLD) produced phosphatidic acid and treatment with either mild base or phospholipase A2 (PLA2) produced free fatty acid, indicating an acylation at least at position 2 of the glycerol. The glycolipid A-like species proved to be susceptible to solubilization by PIPLC of B. thuriengiensis and by GPIPLC of T. brucei and the glycolipid C-like material proved to be fully resistant to both lipases. Although the glycolipid A-like species indeed presents these and other properties compatible with a precursor for the chemically characterized 1G7-Ag anchor, the PLC-resistant species which is completely insensitive to nitrous acid deamination might be an exception to the general finding of a non-acetylated glucosamine in the GPI moieties so far described

Novel GPI structures of the membrane anchor of acetylcholinesterase from the electric organ of Torpedo californica

The structure of the glycan moiety of the glycosylphosphatidylinositol (GPI) membrane anchor from Torpedo californica electric organ acetylcholinesterase was solved using nuclear magnetic resonance (NMR), methylation analysis, and chemical and enzymic microsequencing. Two structures were found to be present: Glcalfa1-2 Manalfa1-2 Manalfa1-6 Manalfa1-4 GlcNalfa1-6myo-inositol, and Glcalfa1-2 Manalfa1-2 Manalfa1-6 (GalNAcß1-4) Manalfa1-4 GlcNalfa1-6myo-inositol. The presence of glucose in this GPI anchor structure is a novel feature. The anchor was also shown to contain 2.3 residues of ethanolamine per molecule

Signal transduction in host cells mediated by glycosylphosphatidylinositols of the parasitic protozoa, or why do the parasitic protozoa have so many GPI molecules?

Considerable circumstantial evidence indicates that glycosylphosphatidylinositol (GPI) molecules of mammalian origin are able to mediate signal transduction in lymphoid cells. For example, pertubation of GPI-anchored surface proteins, but not transmembrane forms of these molecules, can lead to the activation of T lymphocytes. GPIs appear also to be precursors of pharmacologically active phosphoinositol-glycans which mediate responses to hormones such as insulin, nerve growth factor and IL-2. Nonetheless, the biochemical mechanisms of signal transduction by GPIs remain obscure. We have shown that structurally defined GPIs of protozoal parasite origin are able to mediate signal transduction in host macrophages and lymphocytes, by substituting for the putative endogenous GPI-based signalling mechanisms of the host. Signalling by parasite GPIs appears to involve the activation of protein tyrosine kinase and protein C. Evidence from other sources indicates that structurally variant GPIs may provide anergic signals to down-regulate host cell function. These phenomena may represent mechanisms by which eukaryotic parasites regulate host cell function, and can explain a variety of pathological and immunological features of protozoal infections. Furthermore, protozoal GPIs may prove to be an informative model system for the analysis of GPI-mediated signal transduction in lymphocytes and macrophages

Structure and function of GPI-anchored surface proteins of Trypanosoma brucei

While proteins modified at their COOH-terminal end by a glycosylphosphatidylinositol (GPI) membrane anchor have been found as minor components in many eukaryotic cells, they dominate surface constituents of several parasitic protozoa. In this article, GPI-anchored proteins of Trypanosoma brucei are discussed

GPI anchor-hydrolyzing phospholipases

Glycosilphosphatidylinositol(GPI) anchor-hydrolyzing phospholipases include C- and D- type phospholipases and have been described in a number of organisms including bacteria, protozoan parasites, plants, and mammals. Although these phospholipases efficiently cleave GPI structures in vitro, the physiological role of GPI hydrolysis by anchor-specific phospholipases is still unclear. In order to permit comparison of the known GPI anchor-hydrolyzing phospholipases, we studied the kinetic parameters of these enzymes and provide an overview of the currently available information

GPI-specific phospholipase D as an apolipoprotein

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) has recently been shown to be associated with high-density lipoproteins (HDL) in bovine serum. To determine the distribution of GPI-PLD among lipoproteins and characterize the GPI-PLD-containing lipoproteins in human plasma, we used dextram sulfate and immunoaffinity chromatography to isolate apolipoprotein-specific lipoproteins. This procedure allowed fractionation of lipoprotein particles into those containing apolipoprotein B (Lp B), apolipoproteins AI and AII (Lp AI/AII), or apolipoprotein AI only (Lp AI). In five plasma samples with HDL cholesterol ranging from 40 to 129 mg/dl, 75 ñ 12 percent (mean ñ SD) of the GPI-PLD activity was associated with LpAI, 11 ñ 13 percent with Lp AI/AII, while only 13 ñ 9 percent was present in plasma devoid of these lipoproteins, suggesting that most of the GPI-PLD in human plasma is associated with apolipoprotein AI. No GPI-PLD activity was detected in Lp B. Further characterization of the GPI-PLD-containing lipoproteins by gel filtration chromatography, nondenaturing poly-acrylamide and agarose gel electrophoresis revealed that GPI-PLD was restricted to an apolipoprotein AI-containing particle or complex that was small (apparent mean Mw of 140 KDa) and distinct from the bulk of HDL. Thus, the majority of plasma GPI-PLD appears to be specifically associated with a small, minor fraction of apoloprotein AI

Further characterization of the acidic GPI-hydrolyzing phospholipase present in human sera

A phospholipase from human serum capable of hydrolyzing glycosylphosphatidylinositol membrane anchors was described and partially characterized by our group some years ago. This activity presented a pH optimum between 5.0 and 6.0 and was inhibited by EDTA, EGTA and 1,10-phenanthroline. Partial purification showed that the enzyme was a glycoprotein with an apparent molecular weight of 140 kDa as judged by gel filtration. Other investigators characterized at the same time a phospholipase D with similar properties but with a pH optimum near 7.5. We now confirm that the human serum enzyme is indeed a phospholipase D capable of hydrolyzing mfVSG and glycolipids A and C from T. brucei. Isoelectric focusing of whole sera suggests the presence of two isoforms, one with a pI of 4.7 which was the form previously purified by our group, and others with pI from 6.2 to 7.4

Structural and immunological studies of GPI-anchored brush border hydrolases

The complement of brush border hydrolases provides an excellent model system for study of the structure, topology and assembly of plasma membrane proteins. Among the peptidases of the renal brush border are a number of glycosylphosphatidylinositol (GPI)-anchored proteins, especially membrane dipeptidase and aminopeptidase P. Affinity purification protocols have led to the isolation of homogeneous forms of these proteins and membrane dipeptidase has been cloned and expressed in Xenopus oocytes and Cos-1 cells. The core glycan structures of both human and porcine dipeptidase have been determined, allowing direct comparisons of the GPI anchors on the same protein in different species. Aminopeptidase P has been compared in the brush borders of pig kidney and intestine and may well be anchored in distinct ways in the two tissues. Immunological cross-reactivity of polyclonal antibodies to these two proteins has revealed the phospholipase C-cleaved GPI anchor as the common epitope and defined those components of the anchor important for recognition. These antibodies are also proving useful in characterizing GPI-derived mediators that may be involved in cell signalling processes. These abundant ectopeptidases offer a number of advantages for studies of the biochemistry of mammalian GPI anchors

Metabolism of GPIs in mammalian cells

In Trypanosoma brucei, glycosylphosphatidylinositol (GPI) anchors of proteins and free GPIs with identical structures have been characterized. This identity provides strong presumptive evidence that the free GPIs are in fact precursors of the GPI anchors on proteins. In mammalian tissues, however, rather consistent differences in the structures of free GPIs and GPI anchors are observed. The terminal GPIs produced by the mammalian biosynthetic pathway differ from GPI anchors in being almost exclusively fatty acid acylated on the inositol residue, having a greater number of phosphoethanolamine residues, and perhaps in containing a greater percentage of diacylglycerol components. While in principle these differences could be reconciled by remodeling reactions before or after attachment of GPI anchors, it is possible that some of the mammalian free GPIs play cellular roles other than as anchor precursors. We have approached this question by studying the lifetimes of the last three GPIs on the biosynthetic pathway, denoted H6, H7 and H8, in K562 cells and in K562 mutant designated class K that is devoid of GPI-anchored proteins. Pulse-chase metabolic labeling with [3H]-mannose indicated that H6 was a precursor of H7 and H8 and that the H8 lifetime was more than one hour in the parental cells and even longer in the mutant. Preliminary data indicated that the majority of each of the three GPIs was localized in the plasma membrane fraction rather than the endoplasmic reticulum. These observations argue that mammalian GPIs are not utilized exclusively as GPI anchor precursors

Characterization of phosphoinositol oligosaccharides from parasitic protozoa by fast atom bombardment and collisional activation mass spectrometry

Fast atom bombardment mass spectrometry (FAB MS) enables the rapid, accurate and sensitive determination of the molecular masses of glycosylphosphatidylinositol(GPI)-derived oligosaccharides, from which the composition in terms of monosaccharide residues and non-carbohydrate substituents can be determined. Interpretation of fragment ions in collisional activation mass spectra further enables the determination of residue sequence, the positions of branch points, and the location of non-carbohydrate substituents. We have applied these techniques to the characterization of phosphoinositol oligosaccharides from Leptomonas samueli, Endotrypanum schaudinni and Leishmania adleri. The mass spectral data permit the postulation of candidate structures for the oligosaccharides, which provide a set of constraints that can assist the interpretation of results from other techniques such as NMR

Characterization of glycosyl phosphatidylinositol lipids of parasitic protozoans: Leishmania mexicana mexicana promastigotes, Trypanosoma cruzi Peru epimastigotes and Tritrichomonas foetus.

Glycosyl phosphatidylinositol lipids of cultured L.mex, mexicana LV732 promastigotes, T. cruzi Peru epimastigotes and Tritrichomonas foetus have been isolated and characterized using metabolic labelling and chromatographic and mass spectrometric (MS) techniques. TLC of the unsaponifiable lipid fractions of L. mex. mexicana and T. cruzi obtained from DEAE Sephadex A-25 followed by Iatrobead column chromatography showed three inositol phosphate-containing lipid components. [3H]myo-inositol, [3H]palmitic acid or H3 32PO4 lipid precursors were incorporated into these three lipid components. Fraction 2 (LM2 and TCP-2) comprises inositol phosphate ceramides. The other two fractions appear to contain mono-O-alkyl and di-O-alkyl glycerol inositol phosphates. Lyso-1-O-alkyl phosphatidylinositols could be cleaved by treatment of PI-specific phosphalipase C. The di-O-alkyl-phospho inositols of these parasites being the first dialkylglycerol lipids reported from eukaryotic membranes raises the possibility of chemotherapy for leishmaniasis and trypanosomiasis based upon functional impairment of alkyl ether lipids. Tritrichomonas foetus contains two major glycophosphosphingolipids, designated TF1 and TF2, which are metabolically labelled with [3H]myo-inositol and H3 32PO4. Both lipids contained ceramides. The major ceramide contains the 18:0 and 18:1 bases and 16:0 N-acyl group. The major glycolipid fraction (TF1) contains fucose linked to inositol diphosphate; one of the phosphates being linked to the ceramide moiety, and the other to ethanolamine. TF1 appears to be a novel class of glycophosphosphingolipid, which may be a part of a membrane anchor.