Roberta Misasi

Prion protein is a component of the multimolecular signaling complex involved in T cell activation.

In this study we analyzed the interaction of prion protein PrPC with components of glycosphingolipid‐enriched microdomains in lymphoblastoid T cells. PrPC was distributed in small clusters on the plasma membrane, as revealed by immunoelectron microscopy. PrPC is present in microdomains, since it coimmunoprecipitates with GM3 and the raft marker GM1. A strict association between PrPC and Fyn was revealed by scanning confocal microscopy and coimmunoprecipitation experiments. The phosphorylation protein ZAP‐70 was immunoprecipitated by anti‐PrP after T cell activation. These results demonstrate that PrPC interacts with ZAP‐70, suggesting that PrPC is a component of the multimolecular signaling complex within microdomains involved in T cell activation.

Cardiolipin on the surface of apoptotic cells as a possible trigger for antiphospholipid antibodies

This study provides evidence that cardiolipin (CL) molecules are expressed on the surface of apoptotic cells and are recognized by antiphospholipid antibodies, purified from patients with the antiphospholipid antibody syndrome (APS). CL expression on cell surface was demonstrated by high performance thin layer chromatography analysis of phospholipids from plasma membrane purified fractions and by the positive staining with the CL‐specific dye nonyl‐acridine orange. This finding was complemented with the observation that aCL IgG purified from patients with APS bind to the surface of apoptotic cells. This staining shows a clustered distribution mostly localized on surface blebs. Interestingly, CL exposure on the cell surface preceded the DNA fragmentation, as shown by cytofluorimetric analysis. These findings demonstrate that exposure of CL molecules on the cell plasma membrane is an early event of the apoptotic cellular program that may represent an in vivo trigger for the generation of aCL.

Cardiolipin‐enriched raft‐like microdomains are essential activating platforms for apoptotic signals on mitochondria

Cardiolipin (CL) has recently been shown to provide an anchor and an essential activating platform for caspase‐8 on mitochondria. We hypothesize that these platforms may correspond to “raft‐like” microdomains, which have demonstrated to be detectable on mitochondrial membrane of cells undergoing apoptosis. The role for CL in “raft‐like” microdomains could be to anchor caspase‐8 at contact sites between inner and outer membranes, facilitating its self‐activation, Bid cleavage and apoptosis execution. The role played by “raft‐like” microdomains in the apoptotic program could introduce a new task in the pathogenetic studies on human diseases associated with cardiolipin dismetabolism.

Specificity of anti-phospholipid antibodies in infectious mononucleosis: a role for anti-cofactor protein antibodies

The antigen specificity of anti‐phospholipid antibodies in infectious mononucleosis (IM) was studied using ELISA for the detection of anti‐β2‐glycoprotein I (β2‐GPI), anti‐annexin V, anti‐protein S and anti‐prothrombin antibodies and TLC immunostaining for the detection of anti‐phospholipid antibodies. This technique enabled us to look at antibodies reacting to ‘pure’ phospholipid antigens in the absence of protein contamination. Sera from 46 patients with IM, 18 with systemic lupus erythematosus (SLE), 21 with primary anti‐phospholipid antibody syndrome (PAPS), 50 with Helicobacter pylori infection and 30 healthy blood donors were tested. This study highlights anti‐phospholipid antibodies in patients with IM as specific ‘pure’ anti‐cardiolipin antibodies, while in PAPS and SLE patients anti‐phosphatidylserine and anti‐phosphatidylethanolamine antibodies were also found. This investigation also shows that the anti‐cardiolipin antibodies found in IM can be present with anti‐cofactor protein antibodies. The higher prevalence of anti‐cofactor antibodies found in IM sera than in Helicobacter pylori sera may be due to the immunostimulatory effect and/or the polyclonal activation often observed in course of Epstein–Barr virus infection. However, anti‐β2‐GPI and, to a lesser extent, anti‐prothrombin antibodies occur with a significantly lower prevalence in IM than in PAPS patients. This finding suggests that these antibodies should be regarded as the expression of the broad autoimmune syndrome involving the phospholipid‐binding plasma proteins.

GD3 glycosphingolipid contributes to Fas-mediated apoptosis via association with ezrin cytoskeletal protein.

Efficiency of Fas‐mediated apoptosis of lymphoid cells is regulated, among other means, by a mechanism involving its association with ezrin, a cytoskeletal protein belonging to the 4.1 family of proteins. In the present work, we provide evidence for a further molecule that associates to ezrin in Fas‐triggered apoptosis, the disialoganglioside GD3. In fact, as an early event, GD3 redistributed in membrane‐associated domains in uropods and co‐localized with ezrin. Co‐immunoprecipitation analyses confirmed this result, indicating a GD3–ezrin association. Altogether, these results are suggestive for a role of GD3 in Fas/ezrin‐mediated apoptosis, supporting the view that uropods contain a multimolecular signaling complex involved in Fas‐mediated apoptosis.

Beta-2-glycoprotein I expression on monocytes is increased in anti-phospholipid antibody syndrome and correlates with tissue factor expression

It is well known that monocytes may play an active role in thrombogenesis, since they may express on their surface tissue factor, the major initiator of the clotting cascade. The results of this investigation demonstrate beta‐2‐glycoprotein I (β2‐GPI) mRNA expression by human peripheral blood monocytes, indicating that these cells synthesize β2‐GPI. In addition, we show β2‐GPI expression on cell surface of these cells by flow cytometric analysis, and the presence of this protein in cell lysate by Western blot. Interestingly, β2‐GPI expression on monocytes is significantly increased in patients with anti‐phospholipid syndrome (APS) or systemic lupus erythematosus (SLE) as against healthy blood donors and correlates with tissue factor expression on monocytes. These findings support the view that monocytes are able to synthesize β2‐GPI and suggest that patients with APS may have increased β2‐GPI exposure on cell surface, which leads to persistently high monocyte tissue factor expression and consequently to a prothrombotic diathesis.

Colocalization and Complex Formation Between Prosaposin and Monosialoganglioside GM3 in Neural Cells

Abstract: Prosaposin, the precursor of saposins A, B, C, and D, was recently identified as a neurotrophic factor in vitro as well as in vivo. Its neurotrophic activity has been localized to a linear 12‐amino acid sequence located in the NH2‐terminal portion of the saposin C domain. In this study, we show the colocalization of prosaposin and ganglioside GM3 on NS20Y cell plasma membrane by scanning confocal microscopy. Also, TLC and western blot analyses showed that GM3 was specifically associated with prosaposin in immunoprecipitates; this binding was Ca2+‐independent and not disassociated during sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The association of prosaposin‐GM3 complexes on the cell surface appeared to be functionally important, as determined by differentiation assays. Neurite sprouting, induced by GM3, was inhibited by antibodies raised against a 22‐mer peptide, prosaptide 769, containing the neurotrophic sequence of prosaposin. In addition, pertussis toxin inhibited prosaptide‐induced neurite outgrowth, as well as prosaptide‐enhanced ganglioside concentrations in NS20Y cells, suggesting that prosaposin acted via a G protein‐mediated pathway, affecting both ganglioside content and neuronal differentiation. Our findings revealed a direct and right GM3‐prosaposin association on NS20Y plasma membranes. We suggest that ganglioside‐protein complexes are structural components of the prosaposin receptor involved in cell differentiation.

Paracrine Diffusion of PrPC and Propagation of Prion Infectivity by Plasma Membrane-Derived Microvesicles

Cellular prion protein (PrPc) is a physiological constituent of eukaryotic cells. The cellular pathways underlying prions spread from the sites of prions infection/peripheral replication to the central nervous system are still not elucidated. Membrane-derived microvesicles (MVs) are submicron (0.1–1 µm) particles, that are released by cells during plasma membrane shedding processes. They are usually liberated from different cell types, mainly upon activation as well as apoptosis, in this case, one of their hallmarks is the exposure of phosphatidylserine in the outer leaflet of the membrane. MVs are also characterized by the presence of adhesion molecules, MHC I molecules, as well as of membrane antigens typical of their cell of origin. Evidence exists that MVs shedding provide vehicles to transfer molecules among cells, and that MVs are important modulators of cell-to-cell communication. In this study we therefore analyzed the potential role of membrane-derived MVs in the mechanism(s) of PrPC diffusion and prion infectivity transmission. We first identified PrPC in association with the lipid raft components Fyn, flotillin-2, GM1 and GM3 in MVs from plasma of healthy human donors. Similar findings were found in MVs from cell culture supernatants of murine neuronal cells. Furthermore we demonstrated that PrPSc is released from infected murine neuronal cells in association with plasma membrane-derived MVs and that PrPSc-bearing MVs are infectious both in vitro and in vivo. The data suggest that MVs may contribute both to the intercellular mechanism(s) of PrPC diffusion and signaling as well as to the process of prion spread and neuroinvasion.

Evidence for cell surface association between CXCR4 and ganglioside GM3 after gp120 binding in SupT1 lymphoblastoid cells

CXCR4 (fusin) is a chemokine receptor which is involved as a coreceptor in gp120 binding to the cell surface. In this study we provide evidence that binding of gp120 triggers CXCR4 recruitment to glycosphingolipid‐enriched microdomains. Scanning confocal microscopy showed a nearly complete localization of CXCR4 within GM3‐enriched plasma membrane domains of SupT1 cells and coimmunoprecipitation experiments revealed that CXCR4 was immunoprecipitated by IgG anti‐GM3 after gp120 pretreatment. These findings reveal that gp120 binding induces a strict association between CXCR4 and ganglioside GM3, supporting the view that GM3 and CXCR4 are components of a functional multimolecular complex critical for HIV‐1 entry.

Role of GM3-enriched microdomains in signal transduction regulation in T lymphocytes

Gangliosides, sialic acid containing glycosphigolipids, are ubiquitous constituents of cell plasma membranes. Each cell type shows a peculiar ganglioside expression pattern. In human T lymphocytes monosialoganglioside GM3 represents the main ganglioside constituent of cell plasma membrane where it is concentrated in glycosphingolipid-enriched microdomains (GEM). The presence of tyrosine kinase receptors, mono- (Ras, Rap) and heterotrimeric G proteins, Src-like tyrosine kinases (lck, lyn, fyn), PKC isozymes, glycosylphosphatidylinositol (GPI)-anchored proteins and, after T cell activation, the Syk-family kinase Zap-70, prompts these portions of the plasma membrane to be considered as “glycosignaling domains.” In particular, during T cell activation and/or other dynamic functions of the cell, such as apoptosis, key signaling molecules are recruited to these microdomains, where they strictly interact with GM3. The association of transducer proteins with GM3 in microdomains suggests that this ganglioside is the main marker of GEM in human lymphocytes and is a component of a cell plasma membrane multimolecular signaling complex involved in cell-cell interaction, signal transduction, and cell activation. Published in 2004.