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• W1978217169 abstract "The cellular prion protein (PrPC) is a glycosylphosphatidylinositol (GPI)-anchored protein. We investigated whether PrPC can move from one cell to another cell in a cell model. Little PrPC transfer was detected when a PrPCexpressing human neuroblastoma cell line was cultured with the human erythroleukemia cells IA lacking PrPC. Efficient transfer of PrPC was detected with the presence of phorbol 12-myristate 13-acetate, an activator of protein kinase C. Maximum PrPC transfer was observed when both donor and recipient cells were activated. Furthermore, PrPC transfer required the GPI anchor and direct cell to cell contact. However, intercellular protein transfer is not limited to PrPC, another GPI-anchored protein, CD90, also transfers from the donor cells to acceptor cells after cellular activation. Therefore, this transfer process is GPI-anchor and cellular activation dependent. These findings suggest that the intercellular transfer of GPI-anchored proteins is a regulated process, and may have implications for the pathogenesis of prion disease. The cellular prion protein (PrPC) is a glycosylphosphatidylinositol (GPI)-anchored protein. We investigated whether PrPC can move from one cell to another cell in a cell model. Little PrPC transfer was detected when a PrPCexpressing human neuroblastoma cell line was cultured with the human erythroleukemia cells IA lacking PrPC. Efficient transfer of PrPC was detected with the presence of phorbol 12-myristate 13-acetate, an activator of protein kinase C. Maximum PrPC transfer was observed when both donor and recipient cells were activated. Furthermore, PrPC transfer required the GPI anchor and direct cell to cell contact. However, intercellular protein transfer is not limited to PrPC, another GPI-anchored protein, CD90, also transfers from the donor cells to acceptor cells after cellular activation. Therefore, this transfer process is GPI-anchor and cellular activation dependent. These findings suggest that the intercellular transfer of GPI-anchored proteins is a regulated process, and may have implications for the pathogenesis of prion disease. The normal cellular prion protein (PrPC) 1The abbreviations used are: PrPC, cellular prion protein; GPI, glycosylphosphatidylinositol; PrpSc, scrapie prion protein; PI-PLC, phosphatidylinositol-phospholipase C; mAb, monoclonal antibody; PMA, phorbol 12-myristate 13-acetate; ConA, concanavalin A; PKC, protein kinase C is a highly conserved glycoprotein bound to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor (1Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Google Scholar, 2Stahl N. Borchelt D.R. Hsiao K. Prusiner S.B. Cell. 1987; 51: 229-240Google Scholar). Although the general function is not understood, recent studies have established that PrPC has several activities. There is strong evidence that PrPC is a metal-binding protein that has antioxidation property (3Kramer M.L. Kratzin H.D. Schmidt B. Romer A. Windl O. Liemann S. Hornemann S. Kretzschmar H. J. Biol. Chem. 2001; 276: 16711-16719Google Scholar, 4Brown D.R. Wong B.S. Hafiz F. Clive C. Haswell S.J. Jones I.M. Biochem. J. 1999; 344: 1-5Google Scholar, 5Brown D.R. Qin K. Herms J.W. Madlung A. Manson J. Strome R. Fraser P.E. Kruck T. von Bohlen A. Schulz-Schaeffer W. Giese A. Westaway D. Kretzschmar H. Nature. 1997; 390: 684-687Google Scholar, 6Wong B.S. Pan T. Liu T. Li R. Gambetti P. Sy M.S. Biochem. Biophys. Res. Commun. 2000; 273: 136-139Google Scholar). Cell surface PrPC has also been reported to bind laminin and glycosaminoglycans, and to participate in signal transduction (7Mouillet-Richard S. Ermonval M. Chebassier C. Laplanche J.L. Lehmann S. Launay J.M. Kellermann O. Science. 2000; 289: 1925-1928Google Scholar, 8Gauczynski S. Peyrin J.M. Haik S. Leucht C. Hundt C. Rieger R. Krasemann S. Deslys J.P. Dormont D. Lasmezas C.I. Weiss S. EMBO J. 2001; 20: 5863-5875Google Scholar, 9Hundt C. Peyrin J.M. Haik S. Gauczynski S. Leucht C. Rieger R. Riley M.L. Deslys J.P. Dormont D. Lasmezas C.I. Weiss S. EMBO J. 2001; 20: 5876-5886Google Scholar, 10Caughey B. Raymond G.J. J. Virol. 1993; 67: 643-650Google Scholar). PrPC also plays a central role in a group of fatal neurodegenerative disorders, commonly known as transmissible spongiform encephalopathies or prion diseases (1Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Google Scholar, 11Collinge J. Annu. Rev. Neurosci. 2001; 24: 519-550Google Scholar, 12Gambetti P. Parchi P. Capellari S. Russo C. Tabaton M. Teller M. Teller J.K. Chen S.G. J. Alzheimer's Dis. 2001; 3: 87-95Google Scholar), which occur in three forms: familial, sporadic, and acquired by infection. The fundamental pathogenic mechanism shared by all the three forms involves the post-translational conversion of PrPC into a pathogenic and infectious conformer, called scrapie PrP (PrPSc) (13Prusiner S.B. Annu. Rev. Microbiol. 1989; 43: 345-374Google Scholar,14Pan K.M. Baldwin M. Nguyen J. Gasset M. Serban A. Groth D. Mehlhorn I. Huang Z. Fletterick R.J. Cohen F.E. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10962-10966Google Scholar). The mechanism and the cellular locale of PrPC to PrPSc conversion remain unclear. It has been suggested that the conversion is either through the direct binding of PrPSc to PrPC or mediated by a chaperone protein (8Gauczynski S. Peyrin J.M. Haik S. Leucht C. Hundt C. Rieger R. Krasemann S. Deslys J.P. Dormont D. Lasmezas C.I. Weiss S. EMBO J. 2001; 20: 5863-5875Google Scholar, 15Horiuchi M. Baron G.S. Xiong L.W. Caughey B. J. Biol. Chem. 2001; 276: 15489-15497Google Scholar, 16Kaneko K. Zulianello L. Scott M. Cooper C.M. Wallace A.C. James T.L. Cohen F.E. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10069-10074Google Scholar, 17DebBurman S.K. Raymond G.J. Caughey B. Lindquist S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13938-13943Google Scholar). Horiuchi and Caughey (18Horiuchi M. Caughey B. EMBO J. 1999; 18: 3193-3203Google Scholar) found that direct interaction between PrPC and PrPSc through the region around amino acid residues 219 to 232 is required for the subsequent conversion in the cell-free system (18Horiuchi M. Caughey B. EMBO J. 1999; 18: 3193-3203Google Scholar). Plasma membrane and subcellular compartments of the protein recycling pathway have been suggested to be the sites of conversion (19Shyng S.L. Huber M.T. Harris D.A. J. Biol. Chem. 1993; 268: 15922-15928Google Scholar, 20Vey M. Pilkuhn S. Wille H. Nixon R. DeArmond S.J. Smart E.J. Anderson R.G. Taraboulos A. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14945-14949Google Scholar, 21Lehmann S. Harris D.A. J. Biol. Chem. 1996; 271: 1633-1637Google Scholar, 22Borchelt D.R. Taraboulos A. Prusiner S.B. J. Biol. Chem. 1992; 267: 16188-16199Google Scholar), because in the cell model, the generation of PrPSc is inhibited by brefeldin A, an agent, which blocks the delivery of membrane proteins from the cytosol to the membrane (23Taraboulos A. Raeber A.J. Borchelt D.R. Serban D. Prusiner S.B. Mol. Biol. Cell. 1992; 3: 851-863Google Scholar). Furthermore, treatment of cells with phospholipase C (PI-PLC), an enzyme that hydrolyzes the GPI anchor, or with proteases, which degrade PrPC also inhibits PrPSc formation (24Caughey B. Raymond G.J. J. Biol. Chem. 1991; 266: 18217-18223Google Scholar). In addition, PrPC is not converted to PrPSc when it is expressed as a transmembrane protein rather than a GPI-anchored protein (25Kaneko K. Vey M. Scott M. Pilkuhn S. Cohen F.E. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2333-2338Google Scholar, 26Taraboulos A. Scott M. Semenov A. Avrahami D. Laszlo L. Prusiner S.B. Avraham D. J. Cell Biol. 1995; 129: 121-132Google Scholar). The GPI anchor may be needed because GPI-anchored proteins occupy microdomains on the cell membrane, known as detergent insoluble complex or lipid rafts, in a concentrated and multimeric form (27Muniz M. Riezman H. EMBO J. 2000; 19: 10-15Google Scholar). An additional relevant issue is that although PrPC is expressed in many non-central nervous system tissues (28Liu T. Li R. Wong B.S. Liu D. Pan T. Petersen R.B. Gambetti P. Sy M.S. J. Immunol. 2001; 166: 3733-3742Google Scholar, 29Bendheim P.E. Brown H.R. Rudelli R.D. Scala L.J. Goller N.L. Wen G.Y. Kascsak R.J. Cashman N.R. Bolton D.C. Neurology. 1992; 42: 149-156Google Scholar, 30Caughey B. Race R.E. Chesebro B. J. Gen. Virol. 1988; 69: 711-716Google Scholar), the pathology of prion diseases occurs exclusively in the central nervous system (12Gambetti P. Parchi P. Capellari S. Russo C. Tabaton M. Teller M. Teller J.K. Chen S.G. J. Alzheimer's Dis. 2001; 3: 87-95Google Scholar,31DeArmond S.J. Prusiner S.B. Am. J. Pathol. 1995; 146: 785-811Google Scholar). It is believed that in prion diseases acquired by infection such as variant Creutzfeldt-Jakob disease, the PrPC to PrPSc conversion happens through a series of intermediates in different tissues until PrPSc reaches the central nervous system. GPI-anchored proteins are diverse and mediate various functions such as: cell to cell adhesion, nutrient uptake, signal transduction, and regulation of complement activity (32McConville M.J. Ferguson M.A. Biochem. J. 1993; 294: 305-324Google Scholar, 33Stevens E.M. Lament R. J. Clin. Psychiatry. 1979; 40: 445-446Google Scholar). Purified GPI-anchored proteins can incorporate spontaneously onto the target cell membranein vitro, a phenomenon known as “cell painting” (34Medof M.E. Kinoshita T. Silber R. Nussenzweig V. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 2980-2984Google Scholar,35Tykocinski M.L. Kaplan D.R. Medof M.E. Am. J. Pathol. 1996; 148: 1-16Google Scholar). Accumulated evidence also suggests that GPI-anchored proteins may detach from the cell surface and re-attach to another cell. For example, spontaneous cell to cell transfer of CD4-GPI has been demonstrated between co-cultured HeLa cells (36Anderson S.M. Yu G. Giattina M. Miller J.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5894-5898Google Scholar). In a transgenic mouse model, human CD59 and CD55, which are expressed only on red blood cells, were observed to transfer to vascular endothelial cells (37Kooyman D.L. Byrne G.W. McClellan S. Nielsen D. Tone M. Waldmann H. Coffman T.M. McCurry K.R. Platt J.L. Logan J.S. Science. 1995; 269: 89-92Google Scholar). In both cases, the transferred molecules retained their normal functions. However, any intercellular transfer of GPI-anchored proteins in vivo must be tightly regulated, to assure that GPI-anchored proteins do not lose their cell type specificity. The in vivo significance and the mechanism by which GPI-anchored protein moves between cells remain unclear (38Ilangumaran S. Robinson P. Hoessli D. Trends Cell Biol. 1996; 6: 163-167Google Scholar). In the current study, we established a co-culture system using a PrPC expressing (PrPC+) cell line M17-PrP and the cell line IA lacking PrPC (PrPC−) to study whether PrPC can move from cell to cell. We found that PrPC could be transferred from M17-PrP cells to IA cells by a GPI-dependent process in vitro. However, this process does not efficiently occur spontaneously but requires cell activation and direct cell to cell contact. The human neuroblastoma cell line M17 and the human PrP transfectant M17-PrP have been described in detail (39Petersen R.B. Parchi P. Richardson S.L. Urig C.B. Gambetti P. J. Biol. Chem. 1996; 271: 12661-12668Google Scholar). The PrP-CD44-pCEP4 expression construct was generated by replacing the GPI-anchor signaling sequence with the transmembrane and cytoplasmic domain of human CD44. The PrP-CD44-pCEP4 plasmid was transfected into M17 cells using the DOTAP Liposomal Transfection Reagent (Roche Molecular Biochemicals, Indianapolis, IN). Hygromycin-resistant cells were selected and screened for the expression of cell surface PrPC by immunofluorescent staining with anti-PrP monoclonal antibody (mAb) and flow cytometry as described below. The human erythroleukemia cell line IA, derived from K562, was kindly provided by Dr. M. E. Medof of our institution. IA cells failed to express any GPI-anchored protein because of a defect in the assembly of the GPI-anchor core structure (40Hirose S. Prince G.M. Sevlever D. Ravi L. Rosenberry T.L. Ueda E. Medof M.E. J. Biol. Chem. 1992; 267: 16968-16974Google Scholar). The original breeding pairs of FVB PrP−/− mice were kindly provided by Dr. S. Prusiner, University of California, San Francisco, CA, and Dr. G. Carlson, McLaughlin Institute, Great Falls, MT. Co-culture was carried out in 6- or 12-well tissue culture plates (Corning, Corning, NY). M17 or M17-PrP cells were first plated and allowed to grow to 90% confluence. IA cells were then added onto the plate and incubated overnight. Except in the experiments to determine the kinetics of co-culture, a 1:1 of donor cell (1 × 106) to recipient cell was used for all the experiments. Phorbol 12-myristate 13-acetate (PMA, Sigma) was added at 20 ng/ml at the beginning of the culture. After co-culture, the IA cells, which grow in suspension, were carefully collected. In some experiments, an anti-CD44 mAb was used with a secondary reagent conjugated to magnetic beads to isolate the CD44+ IA cells according to the protocol provided by the manufacturer (Matenyi Biotec, Auburn, CA). For treatment with PI-PLC, cells were incubated with 60 ng/ml phosphatidylinositol-specific phospholipase C for 30 min in a 37 °C in a CO2 incubator as described (39Petersen R.B. Parchi P. Richardson S.L. Urig C.B. Gambetti P. J. Biol. Chem. 1996; 271: 12661-12668Google Scholar). After treatment, cells were washed extensively and stained with the anti-PrP mAb as described. In some experiments, the donor and recipient cells were cultured in transwells that were separated by a membrane with a 0.4-μm pore size (Costar, Corning). IA cells were placed in the top chamber and the M17-PrP cells in the lower chamber. Single cell suspensions from the spleen of PrP−/− mice were prepared as described (41Liu T. Zwingman T. Li R. Pan T. Wong B.S. Petersen R.B. Gambetti P. Herrup K. Sy M.S. Brain Res. 2001; 896: 118-129Google Scholar). Cells were cultured in a 12-well plate at 3 × 106 per well with complete medium: RPMI, 1% antibiotics, and 10% pre-selected fetal calf serum. Either PMA (20 ng/ml) or ConA (5 μg/ml) were added to the cells. Plates were incubated in a 37 °C incubator with 5% CO2. After 48 h, the spleen cells were collected and washed twice. Next, they were co-cultured with M17-PrP cells, which had been grown to 90% confluence and were pretreated with PMA or untreated overnight. After an additional overnight co-culture without any activation reagent, cells in suspension were collected for immunostaining. The IA cells or spleen cells were collected and washed with washing buffer (phosphate-buffered saline supplemented with 0.5% newborn calf serum, 0.1% NaN3, pH 7.4), and blocked with Fc-BlockTM (Pharmingen) on ice for 25–30 min. Cells were then incubated with purified anti-PrPC mAb 8H4 or anti-human CD90 mAb 5E10 (Pharmingen) or an isotype matched, control mAb on ice for 45 min. Cells were washed twice and then incubated with a fluorescein isothiocyanate-conjugated F(ab′)2, goat anti-mouse IgG Fc-specific antibody (Chemicon, CA), for 25 min on ice. Finally, the samples were washed and immediately analyzed using a FACScanTM (BD Biosciences). At least 5,000 viable cells were analyzed in all experiments, and all experiments were repeated at least three times for consistency. Following co-culture, IA cells were collected for two-color immunofluorescent staining. The cells were first blocked with 5% human serum for 30 min on ice, then incubated with antibodies in the following sequence, anti-human CD44 mAb A3, Alexa Fluor 488 (green) conjugated goat anti-mouse IgG, F(ab′)2 (Molecular Probes), biotinylated anti-PrP mAb 8H4 and Alexa Fluor 594 (red) streptavidin conjugates (Molecular Probes). Samples were fixed with 3.7% formaldehyde after staining. A cytospin was then used at 500 rpm for 10 min to allow cells to adhere onto glass slides and mounted immediately with Permount (Sigma). The slides were analyzed using a dual scanning confocal microscopy system (LSM 510, Zeiss, Oberkochen, Germany). IA cells were covalently labeled with the amine-reactive fluorescein dye, 5-carboxyfluorescein, succinimidyl ester (Molecular Probes). The reaction was carried out at 37 °C for 15 min according to the protocol provided by the manufacturer. Labeled cells were washed extensively and loaded into the 96-well plate with M17-PrP monolayers, then co-cultured with or without PMA for 6–8 h. The plate was gently washed and the remaining fluorescent cells were quantified using a CytoFluor multiplate reader (PerSeptive Biosystems, Series 4000, MA). M17-PrP cells were first cultured with or without PMA for 24 h, the supernatants were then collected and spun at 2,000 × g to remove the cell debris. The supernatant was further subjected to a 100,000 ×g ultraspeed centrifugation for 1 h at 4 °C (42Taylor D.D. Chou I.N. Black P.H. Biochem. Biophys. Res. Commun. 1983; 113: 470-476Google Scholar). In some experiments the pellets were re-suspended in lysis buffer to determine the PrPC content on immunoblots. In other experiments, the pellets were re-suspended in culture medium and various amounts of the microvesicles were cultured with recipient cells. Cells or microvesicle fractions were incubated with lysis buffer (100 mm NaCl, 10 mmEDTA, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, 10 mmTris, pH 7.4, 2 mm phenylmethylsulfonyl fluoride). Predetermined amounts of total protein from each lysate were loaded and separated in 12% polyacrylamide gels, and then transferred to Immobilon P (Bio-Rad) for 2 h at 90 V. Membranes were incubated overnight at 4 °C with the anti-PrP mAb 8H4. Bound mAbs were detected with an horseradish peroxidase-conjugated F(ab′)2of goat anti-mouse IgG Fc region specific antibody (Chemicon, CA). The blots were developed using an enhanced chemiluminescence system (Pierce) as described by the manufacturer. Prestained molecular weight markers (Bio-Rad) were used as standards. M17-PrP, a stably transfected human neuroblastoma cell that expresses high levels of human PrPC (Fig.1 A) and IA a human erythroleukemia cell line, which does not express PrPCbecause it has a defect in the assembly of the GPI anchor (Fig.1 A), were used as donor and recipient cells, respectively. Furthermore, the M17-PrP cells adhere to the substrate and are CD44 negative (CD44−), whereas the IA cells grow in suspension and are CD44 positive (CD44+) so that the two cell lines can be easily separated to examine PrPC transfer from one cell to the other. Following co-culture of M17-PrP cells and IA cells for 12 h, little or no PrPC was detected on the cell surface of IA cells with mAb 8H4 to PrP (Fig. 1 B) (43Zanusso G. Liu D. Ferrari S. Hegyi I. Yin X. Aguzzi A. Hornemann S. Liemann S. Glockshuber R. Manson J.C. Brown P. Petersen R.B. Gambetti P. Sy M.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8812-8816Google Scholar, 44Li R. Liu T. Wong B.-S. Pan T. Morillas M. Swietnicki W. O'Rourke K. Gambetti P. Surewicz W.K. Sy M.-S. J. Mol. Biol. 2000; 301: 567-573Google Scholar). However, we detected a significant amount of PrPC in the IA cells when we co-cultured M17-PrP cells and IA cells in the presence of PMA, an activator of protein kinase C, indicating that PMA had triggered the efficient transfer of PrPC (Fig. 1 C). We next used an anti-CD44 mAb and magnetic beads to isolate IA cells after co-culture. All the preparations enriched in CD44 positive cells (>98% CD44+) were PrPC positive (Fig.1 D). This finding rules out the possibility that the presence of PrPC in IA cell preparations results from contaminating M17-PrP cells because the latter are CD44−. Incubation of IA cells or M17-PrP cells with PMA, respectively, did not induce the expression of either PrPC in IA cells or CD44 in M17-PrP cells (data not shown). Two-color confocal microscopy confirmed the presence of PrPC in the plasma membrane of IA cells only after PMA activation (Fig. 2,A–H). Furthermore, the transferred PrPC and CD44 partially co-distributed on IA cells (Fig. 2,G and H). We further investigated the role of PMA activation in PrPCtransfer and the kinetics of the transfer. Activation of either the donor or acceptor cells separately prior to co-culture was sufficient for PrPC transfer. However, the activation of both cell types consistently resulted in the highest level of transfer (Fig.3, A and B). PrPC transfer could be detected as early as 3 h after co-culture, increased subsequently, and became stable at about 12 h (Fig. 3 C). Approximately 25 ng/ml PMA was the optimal concentration for triggering intercellular transfer (Fig.3 D). The transfer was cell-dose dependent because more PrPC was detected on IA cells when IA cells were co-cultured with increased numbers of M17-PrP cells (Fig.3 E). Significant levels of free PrPC were detected in the culture supernatant of PMA-treated M17-PrP cells as determined by capture enzyme-linked immunosorbent assay (not shown). We investigated whether PrPC is transferred as soluble and free PrPC present in the culture supernatant. No PrPC was detected on the surface of IA cells after culture with supernatant from PMA-treated or nontreated M17-PrP cells, even when the IA cells were also treated with PMA (not shown). We next investigated whether purified microvesicles released by activated M17-PrP cells were the source of the transferred PrPC. The microvesicle PrPC content determined by immunoblotting showed that microvesicles from PMA-treated M17-PrP cells contained more PrPC than those from the nontreated M17-PrP cells (Fig. 4 A). Therefore, microvesicles purified from activated M17-PrP cells were added directly into IA cell cultures, which had been preactivated with PMA. Only a very low level of PrPC was detected on IA cells exposed to microvesicles isolated from a number of M17-PrP cell 10 times higher than that used in common co-culture experiments (Fig.4 B). Therefore, neither the soluble PrPC in the supernatant nor the PrPC in the isolated microvesicles is the major source of the transferred PrPC. A transwell co-culture system was then used to investigate whether cell to cell contact is required for PrPC transfer. After culture for 12 h in two chambers separated by a membrane, little or no PrPC was detected on IA cells (Fig.4 C). Finally, an adhesion assay showed that after co-culture with nonlabeled M17-PrP cells for 6 h, about 30–40% more IA cells adhered to M17-PrP cell monolayers following PMA than without PMA (Fig.4 D). Therefore, direct contact between the M17-PrP donor and IA acceptor cells appears to be required for efficient PrPC transfer and is enhanced by activation with PMA. Treatment with PI-PLC that specifically cleaves GPI anchors carrying one or two acyl substituents, drastically reduced transferred PrPC on the surface of IA recipient cells (Fig.5 A) indicating that transferred PrPC is linked to the surface of IA cell by the GPI anchor, and is sensitive to PI-PLC cleavage. We further investigated the importance of the GPI anchor in PrPCtransfer by replacing the GPI anchor of PrPC with the transmembrane and cytoplasmic domains of human CD44 to generate a transmembrane form of PrPC. In immunoblots, PrP-CD44 chimeric protein migrated as a broad band at ∼55 kDa and was expressed at a level similar to that of GPI-anchored PrPC in the M17-PrP cells (Fig. 5, B andC). Co-culture of the M17-PrP-CD44 cells with IA cells under conditions identical to those used with the M17-PrP cells did not result in PrPC transfer (Fig. 5 D) suggesting that the GPI anchor is required for PrPC transfer. We investigated whether CD90 (homologue of rodent Thy-1), another GPI-anchored protein, is transferred from M17-PrP cells to IA cells. We chose CD90 because CD90 is a small cell surface molecule, is highly expressed on neurons, and is likely to be present of M17-PrP cells. We found that a moderate level of CD90 is present on the surface of M17-PrP cells (Fig. 6 A). No CD90 immunoreactivity is detected on the surface of IA cells with or without PMA (shaded peak). We then co-cultured M17-PrP cells with IA cells either with or without PMA as described earlier. After co-culture, we found that CD90 immunoreactivity was detected on activated IA cells (Fig. 6 B, dark line), but not on nonactivated IA cells (gray line). These results provide direct evidence that the phenomenon of intercellular protein transfer is applicable at least to another GPI-anchored protein. We also examined whether PrPC could be transferred from M17-PrP cells to normal cells using spleen cells from PrPC−/− mice. Co-culture of PrPC−/− spleen cells with M17-PrP cells for 16 h without PMA did not result in PrPC transfer (Fig.7 A). Following activation by PMA or ConA, a T lymphocyte mitogen, PrPC was detected on the surface of spleen cells (Fig. 7, B and C). These results indicate that PrPC transfer also occurs in normal cells and is not species specific. The present study shows that after cellular activation PrPC, a GPI-anchored protein, can be transferred from neuronal cells to other cell types. Based on the levels of immunofluorescent intensity on M17-PrP cells and IA cells, we estimate that ∼1 to 5% of the PrPC present at the surface of the donor cells is transferred to the acceptor cell (n > 10). This transfer requires cellular activation by PMA, membrane docking by the GPI anchor, and direct cell to cell contact. These requirements imply that the GPI-anchored protein transfer is tightly regulated and depends on the physiologic state of cells as well as the microenvironment. Whereas these conclusions are based mainly on studies carried out with PrPC, it is likely that they are applicable to other GPI-anchored proteins, at least for CD90 as demonstrated here. PMA increases PrPC expression in donor M17-PrP cells, but up-regulation of PrPC expression is not required for PrPC transfer, because activation of recipient IA cells or PrP−/− spleen cells, which lack PrPC also resulted in PrPC transfer, albeit at a lower level. PMA activates protein kinase C (PKC), which is known to induce changes in plasma membrane properties, such as fluidity (45Chakraborty P. Ghosh D. Basu M.K. J. Biochem. (Tokyo). 2000; 127: 185-190Google Scholar), ruffling (46Vuori K. Ruoslahti E. J. Biol. Chem. 1993; 268: 21459-21462Google Scholar, 47Myat M.M. Anderson S. Allen L.A. Aderem A. Curr. Biol. 1997; 7: 611-614Google Scholar), and lipid domain reorganization (48Parolini I. Topa S. Sorice M. Pace A. Ceddia P. Montesoro E. Pavan A. Lisanti M.P. Peschle C. Sargiacomo M. J. Biol. Chem. 1999; 274: 14176-14187Google Scholar, 49Orito A. Kumanogoh H. Yasaka K. Sokawa J. Hidaka H. Sokawa Y. Maekawa S. J. Neurosci. Res. 2001; 64: 235-241Google Scholar, 50Pitto M. Palestini P. Ferraretto A. Flati S. Pavan A. Ravasi D. Masserini M. Bottiroli G. Biochem. J. 1999; 344: 177-184Google Scholar, 51Villalba M. Bi K. Rodriguez F. Tanaka Y. Schoenberger S. Altman A. J. Cell Biol. 2001; 155: 331-338Google Scholar). In addition to PMA, other PKC activators such as ingenol and thymeleatoxin also induce PrPC transfer. Accordingly, inhibitors of PKC such as bisindolylmaleimide I and Gö6976, which is specific for PKC α and βI isoforms, abolish PrPC transfer (not shown). Moreover, inhibitors of intracellular calcium release such as 8-(N,N′-diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride also completely inhibited PrPC transfer (not shown). Collectively, these results suggest that it is the calcium-dependent PKC isozymes (α or βI or both) that are important in PrPC transfer. We also demonstrated that activation of PrPC−/− spleen cells with ConA, a T cell mitogen also resulted in PrPC transfer. Recently, it has been shown that PrPC has signaling activity that plays an important role in neuronal differentiation (7Mouillet-Richard S. Ermonval M. Chebassier C. Laplanche J.L. Lehmann S. Launay J.M. Kellermann O. Science. 2000; 289: 1925-1928Google Scholar). This activity depends on PrPC coupling to the tyrosine kinase Fyn. It will be important to determine whether transferred PrPC contributes to, or generates, this activity. In agreement with our finding that direct cell to cell contact is essential for PrPC transfer, we consistently observed a small increase in cellular adhesion between activated donor cells and acceptor cells. Increase in cell adhesion may be because of activation of adhesion molecules, or a general effect on the membrane, such as the distribution and organization of membrane microdomains (52Galbiati F. Razani B. Lisanti M.P. Cell. 2001; 106: 403-411Google Scholar). GPI-anchored proteins associate with lipid rafts, which are membrane microdomains rich in cholesterol and glycosphingolipids (53Hoessli D.C. Rungger-Brandle E. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 439-443Google Scholar, 54Simons K. Ikonen E. Nature. 1997; 387: 569-572Google Scholar). Lipid rafts play an important role in intracellular trafficking and sorting (27Muniz M. Riezman H. EMBO J. 2000; 19: 10-15Google Scholar, 55Pomorski T. Hrafnsdottir S. Devaux P.F. van Meer G. Semin. Cell Dev. Biol. 2001; 12: 139-148Google Scholar). Although we have established that PrPC is associated with lipid rafts in M17-PrP cells, 2T. Liu, and M. S. Sy, unpublished results. it remains to be determined whether the transferred PrPC is also associated with lipid rafts on IA cells, and whether lipid rafts are important in intercellular transfer of GPI-anchored proteins. On confocal microscopy both CD44 and transferred PrPC formed a punctuate pattern, and partially co-distributed on the IA cell surface. This suggests that PrPC may transfer to IA cells as individual molecules, which are subsequently re-organized into membrane domains. Alternatively, membrane fragments or microdomains that contain multiple PrPC molecules might selectively be transferred. It has been reported that intercellular transfer of CD4-GPI occurred spontaneously from transfected HeLa cell to recipient cells, and direct cell-cell contact was not required. Transfer of CD4-GPI was mediated by microvesicles released from donor cells (36Anderson S.M. Yu G. Giattina M. Miller J.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5894-5898Google Scholar, 56Miller J.L. Giattina M. Mackie E.J. Dwyer N.K. J. Lab. Clin. Med. 1998; 131: 215-221Google Scholar). This discrepancy may be because of the nature of donor cells; M17 is a neuronal tumor cell line, HeLa is an epithelial tumor cell line. The cellular activation and cell-cell contact-dependent mechanism described here may be cell type-dependent. This interpretation is in good accordance with the observation that the efficiency of CD4-GPI transfer is cell type-dependent. Alternatively, the expression levels of the protein may determine the efficiency of transfer. GPI-anchored proteins express at very high levels, such as in the CD4-GPI-transfected HeLa cell may permit the protein to relocate to the recipient cells without activation and adhesion. Another important issue we also addressed is whether increase in adhesion alone is sufficient for the transfer to occur. We first “force” the IA cells to adhere to the M17 cell monolayer by centrifuging the co-culture plate. We found that simply physically forcing the two cells to adhere without cellular activation does not result in PrPC transfer (not shown). This result provide additional evidence that cellular activation is critical in triggering efficient PrPC transfer. We also show that the GPI anchor is required for the transfer, because the trans-membrane protein PrP-CD44 is not transferred from M17-PrP-CD44 cells to IA cells. This finding also indicates that transfer is unlikely to result from the fusion of M17-PrP cells and IA cells as a consequence of cellular activation. The importance of the GPI anchor is also supported by our findings, which showed that free PrPC in the culture supernatant of activated M17-PrP cells was not the source of transferred PrPC on IA cells. Recent study revealed that free PrPC in the supernatants of neuronal cells lacked GPI anchors (57Parizek P. Roeckl C. Weber J. Flechsig E. Aguzzi A. Raeber A.J. J. Biol. Chem. 2001; 276: 44627-44632Google Scholar). Intercellular protein transfer is not limited to PrPC because CD90, another GPI-anchored protein, is transferred from M17-PrP cells to IA cells. Recent studies suggested that PrPC and Thy-1 are organized in different domains on the surface of rat neuronal cell lines (58Madore N. Smith K.L. Graham C.H. Jen A. Brady K. Hall S. Morris R. EMBO J. 1999; 18: 6917-6926Google Scholar). Whether PrPC and CD90 (Thy-1) are also present on different membrane domains on M17-PrP cells and whether cellular activation alters the distribution of theses domains are not known. In contrast to intracellular trafficking, the mechanisms of activation-induced intercellular transfer of GPI-anchored proteins are not known. Lipid transfer has been reported to occur between parasites and human epithelial cells as well as neutrophils (59Rogers R.A. Jack R.M. Furlong S.T. J. Cell Sci. 1993; 106: 485-491Google Scholar, 60Furlong S.T. Koziel H. Bartlett M.S. McLaughlin G.L. Shaw M.M. Jack R.M. J. Infect. Dis. 1997; 175: 661-668Google Scholar). There is also evidence indicating that hemifusion of the outer leaflet of the plasma membrane can occur between two cells under in vitroconditions (61Lee J. Lentz B.R. Biochemistry. 1997; 36: 6251-6259Google Scholar, 62Melikyan G.B. Brener S.A. Ok D.C. Cohen F.S. J. Cell Biol. 1997; 136: 995-1005Google Scholar). Based on these observations, we hypothesize that after cellular activation, there is a transient and focal fusion of the outer leaflets of the donor cell and the acceptor cell membrane. This focal fusion allows the exchange of lipids, which include GPI-anchored proteins. Experiments are now in progress to determine whether cellular activation results in the exchange of lipids between M17-PrP cells and IA cells. In addition to providing new insights into the biology of GPI-anchored protein transfer our findings may also have implications for the pathogenesis of prion diseases. In prion diseases acquired by infection, such as Kuru and variant Creutzfeldt-Jakob disease in humans, scrapie in sheep, and bovine spongiform encephalopathy in cattle, the infectious prion most likely enters the host through the gastrointestinal tract, subsequently migrates to the spleen, and causes pathology in the central nervous system. Therefore, from the portal of entry to the target organ, the infectious prion must be transferred through different cell types. Infectious PrPSc has first been detected in the spleen even following PrPScintracerebral injection indicating that PrPSc can also travel from central nervous system to the peripheral tissues (63Fraser H. Dickinson A.G. Nature. 1970; 226: 462-463Google Scholar, 64Eklund C.M. Kennedy R.C. Hadlow W.J. J. Infect. Dis. 1967; 117: 15-22Google Scholar). Furthermore, it has been known since the 1970s that activation of the immune system enhances susceptibility as well as shortens the incubation time in experimentally infected mice (65Dickinson A.G. Fraser H. McConnell I. Outram G.W. Nature. 1978; 272: 54-55Google Scholar, 66Marsh R.F. Adv. Exp. Med. Biol. 1981; 134: 359-363Google Scholar). Both PrPC and PrPSc are GPI anchored on the cell surface (20Vey M. Pilkuhn S. Wille H. Nixon R. DeArmond S.J. Smart E.J. Anderson R.G. Taraboulos A. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14945-14949Google Scholar, 26Taraboulos A. Scott M. Semenov A. Avrahami D. Laszlo L. Prusiner S.B. Avraham D. J. Cell Biol. 1995; 129: 121-132Google Scholar, 67Naslavsky N. Stein R. Yanai A. Friedlander G. Taraboulos A. J. Biol. Chem. 1997; 272: 6324-6331Google Scholar). Whether PrPSc also transfers between cells in a similar manner to PrPC is not known. It has been reported that PrPC to PrPSc conversion required PrPSc and PrPC to be present in the same continuous membrane (68Baron G.S. Wehrly K. Dorward D.W. Chesebro B. Caughey B. EMBO J. 2002; 21: 1031-1040Google Scholar). This result suggests that PrPSchas to be transferred from infected cell to the uninfected cell surface in order for the conversion to occur. The GPI anchor-mediated intercellular transfer of PrPSc might provide a mechanism for this transfer. Our results indicate that activation-induced PrPC transfer may promote or enhance the conversion by providing PrPC negative cells or cells with low levels of PrPC with more substrate. On the other hand, the transfer of PrPSc would implant the seed of the infectious agent. Experiments are now in progress to determine whether transfer of PrPSc also occur under similar culture conditions. We thank Dr. Alan Tartakoff, Dr. Karl Herrup, and Dr. Claudio Fiocchi for discussion and suggestion. We also thank Dr. E. Medof for providing the IA cell line. We thank Dr. Minh Lam of the Confocal Microscopy Core Facility at the CWRU Cancer Center for help with the confocal microscope." @default.
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• W1978217169 title "Intercellular Transfer of the Cellular Prion Protein" @default.
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