Stress-inducible protein 1 is a cell surface ligand for cellular prion that triggers neuroprotection
2002; Springer Nature; Volume: 21; Issue: 13 Linguagem: Inglês
10.1093/emboj/cdf325
ISSN1460-2075
Autores Tópico(s)Nerve injury and regeneration
ResumoArticle1 July 2002free access Stress-inducible protein 1 is a cell surface ligand for cellular prion that triggers neuroprotection Silvio M. Zanata Silvio M. Zanata Ludwig Institute for Cancer Research, São Paulo Branch, Rua Prof. Antônio Prudente 109 4A, São Paulo, 01509010 Brasil Departamento de Bioquímica, São Paulo, Brasil Search for more papers by this author Marilene H. Lopes Marilene H. Lopes Ludwig Institute for Cancer Research, São Paulo Branch, Rua Prof. Antônio Prudente 109 4A, São Paulo, 01509010 Brasil Search for more papers by this author Adriana F. Mercadante Adriana F. Mercadante Ludwig Institute for Cancer Research, São Paulo Branch, Rua Prof. Antônio Prudente 109 4A, São Paulo, 01509010 Brasil Search for more papers by this author Glaucia N. M. Hajj Glaucia N. M. Hajj Ludwig Institute for Cancer Research, São Paulo Branch, Rua Prof. Antônio Prudente 109 4A, São Paulo, 01509010 Brasil Departamento de Bioquímica, São Paulo, Brasil Search for more papers by this author Luciana B. Chiarini Luciana B. Chiarini Laboratório de Neurogênese, Instituto de Biofísica da UFRJ, Rio de Janeiro, Brasil Search for more papers by this author Regina Nomizo Regina Nomizo Centro de Tratamento e Pesquisa Hospital do Câncer, São Paulo, Brasil Search for more papers by this author Adriana R. O. Freitas Adriana R. O. Freitas Ludwig Institute for Cancer Research, São Paulo Branch, Rua Prof. Antônio Prudente 109 4A, São Paulo, 01509010 Brasil Departamento de Bioquímica, São Paulo, Brasil Search for more papers by this author Ana L. B. Cabral Ana L. B. Cabral Ludwig Institute for Cancer Research, São Paulo Branch, Rua Prof. Antônio Prudente 109 4A, São Paulo, 01509010 Brasil Departamento de Bioquímica, São Paulo, Brasil Search for more papers by this author Kil S. Lee Kil S. Lee Ludwig Institute for Cancer Research, São Paulo Branch, Rua Prof. Antônio Prudente 109 4A, São Paulo, 01509010 Brasil Departamento de Bioquímica, São Paulo, Brasil Search for more papers by this author Maria A. Juliano Maria A. Juliano INFAR, Universidade Federal de São Paulo, São Paulo, Brasil Search for more papers by this author Elizabeth de Oliveira Elizabeth de Oliveira Departamento de Química Fundamental, Instituto de Química da USP, São Paulo, Brasil Search for more papers by this author Saul G. Jachieri Saul G. Jachieri Centro de Tratamento e Pesquisa Hospital do Câncer, São Paulo, Brasil Search for more papers by this author Alma Burlingame Alma Burlingame Department of Pharmaceutical Chemistry, USCF, CA, USA Search for more papers by this author Lan Huang Lan Huang Department of Pharmaceutical Chemistry, USCF, CA, USA Search for more papers by this author Rafael Linden Rafael Linden Laboratório de Neurogênese, Instituto de Biofísica da UFRJ, Rio de Janeiro, Brasil Search for more papers by this author Ricardo R. Brentani Ricardo R. Brentani Ludwig Institute for Cancer Research, São Paulo Branch, Rua Prof. Antônio Prudente 109 4A, São Paulo, 01509010 Brasil Search for more papers by this author Vilma R. Martins Corresponding Author Vilma R. Martins Ludwig Institute for Cancer Research, São Paulo Branch, Rua Prof. Antônio Prudente 109 4A, São Paulo, 01509010 Brasil Search for more papers by this author Silvio M. Zanata Silvio M. Zanata Ludwig Institute for Cancer Research, São Paulo Branch, Rua Prof. Antônio Prudente 109 4A, São Paulo, 01509010 Brasil Departamento de Bioquímica, São Paulo, Brasil Search for more papers by this author Marilene H. Lopes Marilene H. Lopes Ludwig Institute for Cancer Research, São Paulo Branch, Rua Prof. Antônio Prudente 109 4A, São Paulo, 01509010 Brasil Search for more papers by this author Adriana F. Mercadante Adriana F. Mercadante Ludwig Institute for Cancer Research, São Paulo Branch, Rua Prof. Antônio Prudente 109 4A, São Paulo, 01509010 Brasil Search for more papers by this author Glaucia N. M. Hajj Glaucia N. M. Hajj Ludwig Institute for Cancer Research, São Paulo Branch, Rua Prof. Antônio Prudente 109 4A, São Paulo, 01509010 Brasil Departamento de Bioquímica, São Paulo, Brasil Search for more papers by this author Luciana B. Chiarini Luciana B. Chiarini Laboratório de Neurogênese, Instituto de Biofísica da UFRJ, Rio de Janeiro, Brasil Search for more papers by this author Regina Nomizo Regina Nomizo Centro de Tratamento e Pesquisa Hospital do Câncer, São Paulo, Brasil Search for more papers by this author Adriana R. O. Freitas Adriana R. O. Freitas Ludwig Institute for Cancer Research, São Paulo Branch, Rua Prof. Antônio Prudente 109 4A, São Paulo, 01509010 Brasil Departamento de Bioquímica, São Paulo, Brasil Search for more papers by this author Ana L. B. Cabral Ana L. B. Cabral Ludwig Institute for Cancer Research, São Paulo Branch, Rua Prof. Antônio Prudente 109 4A, São Paulo, 01509010 Brasil Departamento de Bioquímica, São Paulo, Brasil Search for more papers by this author Kil S. Lee Kil S. Lee Ludwig Institute for Cancer Research, São Paulo Branch, Rua Prof. Antônio Prudente 109 4A, São Paulo, 01509010 Brasil Departamento de Bioquímica, São Paulo, Brasil Search for more papers by this author Maria A. Juliano Maria A. Juliano INFAR, Universidade Federal de São Paulo, São Paulo, Brasil Search for more papers by this author Elizabeth de Oliveira Elizabeth de Oliveira Departamento de Química Fundamental, Instituto de Química da USP, São Paulo, Brasil Search for more papers by this author Saul G. Jachieri Saul G. Jachieri Centro de Tratamento e Pesquisa Hospital do Câncer, São Paulo, Brasil Search for more papers by this author Alma Burlingame Alma Burlingame Department of Pharmaceutical Chemistry, USCF, CA, USA Search for more papers by this author Lan Huang Lan Huang Department of Pharmaceutical Chemistry, USCF, CA, USA Search for more papers by this author Rafael Linden Rafael Linden Laboratório de Neurogênese, Instituto de Biofísica da UFRJ, Rio de Janeiro, Brasil Search for more papers by this author Ricardo R. Brentani Ricardo R. Brentani Ludwig Institute for Cancer Research, São Paulo Branch, Rua Prof. Antônio Prudente 109 4A, São Paulo, 01509010 Brasil Search for more papers by this author Vilma R. Martins Corresponding Author Vilma R. Martins Ludwig Institute for Cancer Research, São Paulo Branch, Rua Prof. Antônio Prudente 109 4A, São Paulo, 01509010 Brasil Search for more papers by this author Author Information Silvio M. Zanata1,2, Marilene H. Lopes1, Adriana F. Mercadante1, Glaucia N. M. Hajj1,2, Luciana B. Chiarini4, Regina Nomizo3, Adriana R. O. Freitas1,2, Ana L. B. Cabral1,2, Kil S. Lee1,2, Maria A. Juliano5, Elizabeth de Oliveira6, Saul G. Jachieri3, Alma Burlingame7, Lan Huang7, Rafael Linden4, Ricardo R. Brentani1 and Vilma R. Martins 1 1Ludwig Institute for Cancer Research, São Paulo Branch, Rua Prof. Antônio Prudente 109 4A, São Paulo, 01509010 Brasil 2Departamento de Bioquímica, São Paulo, Brasil 3Centro de Tratamento e Pesquisa Hospital do Câncer, São Paulo, Brasil 4Laboratório de Neurogênese, Instituto de Biofísica da UFRJ, Rio de Janeiro, Brasil 5INFAR, Universidade Federal de São Paulo, São Paulo, Brasil 6Departamento de Química Fundamental, Instituto de Química da USP, São Paulo, Brasil 7Department of Pharmaceutical Chemistry, USCF, CA, USA ‡S.M.Zanata, M.H.Lopes, A.F.Mercadante and G.N.M.Hajj contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:3307-3316https://doi.org/10.1093/emboj/cdf325 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Prions are composed of an isoform of a normal sialoglycoprotein called PrPc, whose physiological role has been under investigation, with focus on the screening for ligands. Our group described a membrane 66 kDa PrPc-binding protein with the aid of antibodies against a peptide deduced by complementary hydropathy. Using these antibodies in western blots from two-dimensional protein gels followed by sequencing the specific spot, we have now identified the molecule as stress-inducible protein 1 (STI1). We show that this protein is also found at the cell membrane besides the cytoplasm. Both proteins interact in a specific and high affinity manner with a Kd of 10−7 M. The interaction sites were mapped to amino acids 113–128 from PrPc and 230–245 from STI1. Cell surface binding and pull-down experiments showed that recombinant PrPc binds to cellular STI1, and co-immunoprecipitation assays strongly suggest that both proteins are associated in vivo. Moreover, PrPc interaction with either STI1 or with the peptide we found that represents the binding domain in STI1 induce neuropro tective signals that rescue cells from apoptosis. Introduction Prions, the agents of transmissible spongiform encephalopathies (reviewed by Prusiner, 1998), require the expression of a glycosylphosphatidylinositol (GPI)-anchored cell surface sialoglycoproteic homolog (PrPc) to propagate disease (Büeler et al., 1993). Mutations in the gene coding for PrPc are also the cause of hereditary neurological (Prusiner, 1998) and possibly psychiatric (Samaia et al., 1997) disorders. Since clinical manifestations may occur either before or without characteristic PrPc deposits (Collinge et al., 1990; Medori et al., 1992), it has been suggested that loss of PrPc function may concur for the etiology of such diseases (Aguzzi and Weissmann, 1997; Samaia and Brentani, 1998). Recently, certain biological functions of PrPc have been uncovered. PrPc strongly binds Cu2+ and thus may be involved in both copper metabolism (Brown et al., 1997a) and protection against oxidative stress (Brown et al., 1997b). Since PrPc is localized mainly in synaptosomal fractions, it may serve as a copper buffer in the synaptic cleft or in the re-uptake of copper into the presynaptic terminal (Kretzschmar et al., 2000). Moreover, it is known that PrPc plays a role in the modulation of neuronal survival, both in vivo (Walz et al., 1999) and in vitro (Kuwahara et al., 1999; Chiarini et al., 2002), and is also involved in signal transduction (Mouillet-Richard et al., 2000; Chiarini et al., 2002). We have also shown that PrPc is a specific receptor for the C-terminal domain of the γ-1 chain of extracellular matrix laminin, and is involved in neuronal adhesion and neurite growth (Graner et al., 2000a, b). To understand further the biological functions of PrPc, binding molecules were sought extensively (reviewed by Martins et al., 2001). In an attempt to characterize a PrPc receptor, we (Martins et al., 1997) designed a peptide that was predicted to bind PrPc on the basis of the complementary hydropathy theory (see for example Bost et al., 1985; Brentani, 1988; Boquet et al., 1995). Theoretically, the peptide should mimic the docking site of PrPc in a ligand. Antibodies raised against this peptide recognize a 66 kDa cell surface antigen, which binds PrPc in vitro. The same antibody prevented toxicity of the human PrPc peptide comprising amino acids 106–126 towards neurons in culture, indicating that the 66 kDa protein may be a receptor for the infectious agent, involved in the pathogenesis of prion diseases. Conversely, the protein might also work in association with PrPc in normal cellular functions. Here, we report that the PrPc membrane ligand is stress-inducible protein 1 (STI1), a heat shock protein, first described in a macromolecular complex with Hsp70 and Hsp90 chaperone family proteins (Blatch et al., 1997; Lässle et al., 1997). Recombinant PrPc and STI1 showed specific and high affinity binding both in vitro and at the cellular level. The binding site in mouse PrPc confirmed our earlier prediction and spans amino acids 113–128. Furthermore, we identified a domain in the mouse STI1 molecule (amino acids 230–245) with the same hydropathy profile as the predicted PrPc-binding peptide (Martins et al., 1997). This peptide prevented the PrPc–STI1 interaction, indicating that it contains the binding site at the STI1 molecule. We have shown (Chiarini et al., 2002) that PrPc transduces neuroprotective signals when challenged with either the theoretically derived PrPc-binding peptide or with certain antibodies. Here we demonstrate that interactions of PrPc with either STI1 or the STI1 peptide that contains the PrPc-binding site induce neuroprotection. Results STI1 is the molecule recognized by antiserum raised against the predicted PrPc-binding peptide (PrR) To identify the previously characterized PrPc ligand, we ran two-dimensional gel electrophoresis of a 45–55% ammonium sulfate fraction from whole mouse brain extracts, which partially purifies this ligand (Martins et al., 1997). A 66 kDa doublet with pIs ranging from 6.2 to 6.4 is readily observed after Coomassie Blue staining (Figure 1A), which was also recognized in a western blot (Figure 1B) by antiserum against the predicted PrPc-binding peptide (Martins et al., 1997). Herein, this peptide is referred to as PrR. The same spots were excised from a parallel gel and separately subjected to mass spectrometry. The amino acid sequences deduced for the protein extracted from each of the spots displayed 93–96% identity with the mouse STI1 molecule (Blatch et al., 1997; Lässle et al., 1997). Figure 1C shows the mass spectrometry (MS) spectrum of a doubly charged tryptic peptide from the spot with higher pI. The two spots may represent differential phosphorylation of the molecules, as described (Lässle et al., 1997). Despite the obvious sequence differences, we also tested whether the 66 kDa protein corresponds to the previously reported PrPc-binding laminin receptor (Rieger et al., 1997), which has a similar molecular weight. The relevant spots did not react with a specific monoclonal antibody against the laminin receptor (a kind gift of Dr Sylvie Mènard, Institute of Pathology, Milan University), and its molecular weight was unaffected by treatment with concentrated hydroxylamine (not shown), which cleaves acetyl groups and converts the 67 kDa laminin receptor into the 37 kDa precursor (Buto et al., 1998). Figure 1.Two-dimensional gel analysis of the ammonium sulfate 45–55% saturation fraction from total brain extract. (A) Coomassie Blue-stained proteins. (B) Immunoblot of an identical gel reacted with mouse serum against the PrR peptide developed using peroxidase-labeled anti-mouse Ig. Two spots of 66 kDa and pIs of ∼6.2 and 6.4 are recognized specifically, and each was subjected to microsequencing analysis. (C) MS spectrum of a doubly charged tryptic peptide (MH22+ at m/z 744.9) from the spot with the higher pI. A series of fragment ions (b and y ions) were observed due to the breakage of peptide bonds during collision-induced dissociation. The peptide sequence is determined as LAYINPDLALEEK, identifying the protein as mouse stress-inducible protein 1 (accession No. 881485). Download figure Download PowerPoint Mouse recombinant STI1 protein was used to generate a rabbit polyclonal antiserum. Figure 2A shows that the recombinant protein has the expected molecular weight (lane 1) and is recognized by the antiserum (lane 2). Furthermore, recombinant STI1 is also recognized by the serum against the PrR peptide (lane 4) which, as shown before (Martins et al., 1997), recognizes a band with the same molecular weight in a 45–55% ammonium sulfate fraction from whole brain extracts (lane 6). Serum raised against STI1 also recognizes a band of similar molecular weight in the 45–55% ammonium sulfate fraction from whole brain extracts (lane 8). Controls with mouse or rabbit non-immune serum did not react in this assay (lanes 3, 5, 7 and 9). Figure 2.STI1 is the 66 kDa protein located at the cell membrane recognized by serum against the PrR peptide. (A) Western blot assay of recombinant mSTI1 (lanes 2–5) or ammonium sulfate fractions at 45–55% saturation from brain extracts (lanes 6–9), done with rabbit serum against recombinant mST11 (α-STI1, lanes 2 and 8), serum against PrR peptide (α-PrR, lanes 4 and 6) or non-immune serum (NI, lanes 3, 5, 7 and 9). The recombinant STI1 protein stained with Ponceau is shown in lane 1. (B) Western blot assay from purified membrane fractions from brain extracts (lanes 1 and 2) done with rabbit serum against recombinant mST11 (α-STI1, lane 1) or non-immune serum (NI, lane 2). Cell surface proteins from N2a cells were biotinylated followed by extract preparation and immunoprecipitation with anti-STI1 antibody (α-STI1, lane 3) or non-immune serum (NI, lane 4). The immunoprecipitated material was developed using streptavidin– peroxidase. Download figure Download PowerPoint STI1 was found either in the cytoplasm (Lässle et al., 1997) or in the Golgi apparatus and small vesicles (Honoré et al., 1992). However, we previously have described that a small fraction of the PrPc ligand was present at the cell surface (Martins et al., 1997). To approach protein localization further, we used membrane preparations from mouse brain and N2a cells, which together with mouse brain extracts were used in the previous study (Martins et al., 1997). Western blots of the mouse brain membrane fraction (Figure 2B, lane 1) showed a specific 66 kDa band recognized by anti-STI1 antibodies. In addition, N2a cell surface proteins were conjugated with biotin and cell lysates were immunoprecipitated with anti-STI1 antibodies. A 66 kDa biotinylated band was labeled with peroxidase-coupled streptavidin in immunoprecipi tated material (lane 3), similar to that recognized in crude brain membrane preparations (lane 1), strongly suggesting that at least part of the STI1 is located at the cell surface. PrPc binds STI1 with high affinity and independently of copper We next measured binding of 125I-labeled His6-STI1 to His6-PrPc. Figure 3 shows curves representative of at least four independent assays carried out with PrPc refolded in either the absence (Figure 3A) or presence of Cu2+ (Figure 3B). The presence of copper ions in the refolded protein was determined by atomic emission spectrometry (data not shown) and confirms the presence of ∼10 Cu2+ atoms bound per His6-PrPc molecule (Brown et al., 1999). PrPc binds STI1 with high affinity, in a saturable manner, and 1.2–1.5 times as much STI1 binds PrPc refolded in the absence than in the presence of Cu2+ ions, although the affinity constants were similar (Kd = 1.4 × 10−7 and 1.2 × 10−7 M in the absence and presence of Cu2+, respectively). Since the amount of His6-PrPc added in both experiments was the same, these data indicate that there is more PrPc able to bind STI1 when the refolding occurs in the absence of Cu2+. One possibility is that due to the extensive process to refold the protein (Wong et al., 2000), a small fraction is degraded. Figure 3.STI1 binds PrPc in a saturable and specific manner and independently of Cu2+ incorporation into the PrPc molecule. Representative curves of [125I]His6-STI1 binding to His6-PrPc refolded in the absence (A) or presence (B) of Cu2+. [125I]His6-STI1 was incubated with adsorbed His6-PrPc in the absence (total) or presence of unlabeled STI1 (non-specific). Non-specific binding (triangles) was subtracted from the total binding (squares) to yield His6-PrPc-specific binding to [125I]His6-STI1 (circles). Scatchard plots (inserts) gave Kds of 1.4 × 10−7 and 1.2 × 10−7 M for PrPc refolded in the absence or presence of Cu2+, respectively. Download figure Download PowerPoint STI1 interacts within amino acids 113–128 of PrPc To map the STI1-binding domain in PrPc, we constructed a deletion mutant lacking the region around the neurotoxic domain (Forloni et al., 1993), which contains the previously predicted binding site (Martins et al., 1997). The mouse PrPc mutant Δ105–128 (human 106–129) is unable to bind STI1, while PrPc without the copper-binding domain (PrPc Δ51–90) binds similarly to the wild-type molecule (Figure 4A). We then tested a series of peptides covering the entire PrPc molecule (Figure 4B). Peptide P10, which contains the amino acid sequence spanning residues 113–132 of the mouse sequence, was the most effective competitor of PrPc–STI1 binding, while peptide P9 (amino acids 103–122), which shares residues 113–122 with P10, was also inhibitory, albeit at a concentration 2.5 times higher than that used for P10 (data not shown). Moreover, the human neurotoxic peptide (NTX) (Forloni et al., 1993), which is equivalent to mouse PrPc amino acids 105–125, was able to compete for PrPc–STI1 interaction, but less efficiently than P10. Therefore, amino acids 126–131 present in P10 and absent in both P9 and the NTX peptide appear to be added to the 113–122 domain of PrPc in the interaction with STI1. Since the PrPc deletion mutant Δ105–128 is unable to bind STI1, the data are consistent with the hypothesis that the region comprising amino acids 113–128 of the mouse PrPc, which was the sequence used to draw the PrR peptide (Martins et al., 1997), is a unique binding site for STI1, independent of copper (Figure 4A). Figure 4.Mapping the STI1 binding site in PrPc using deletion mutants and synthetic PrPc peptides. (A) Wild-type PrPc and deletion mutants Δ51–90 and Δ105–128 were incubated with [125I]His6-STI1. The binding between wild-type His6-PrPc and [125I]His6-STI1 was set to 100% (control). The results for each PrPc mutant were expressed as percentage binding compared with wild-type. *P <0.01 versus control, single mean Student‘s t-test. (B) Twenty mouse PrPc peptides covering the PrPc (23–231) protein sequence were synthesized chemically as a 20mer with 10 overlapping residues. The scheme shows localization of the 20 peptides, the neurotoxic peptide (NTX) and the main PrPc domains: β1 and β2, β-sheet domains; H1, H2 and H3, α-helix domains; GPI, GPI anchor. (C) The synthetic peptides were pre-incubated with [125I]His6-STI1 followed by incubation with adsorbed His6-PrPc. Total binding between His6-PrPc and [125I]His6-STI1 was set to 100%. The results are expressed as the relative percentage of the binding produced by competition with each peptide. *P < 0.01 versus control, single mean Student’s t-test; #P < 0.01 NTX versus P10, Mann– Whitney test. Download figure Download PowerPoint PrPc interacts with an STI1 domain with a hydropathy profile identical to that of the PrR peptide Theoretically, the PrPc docking site within the STI1 molecule should display a similar hydropathic profile to that of the PrR (Martins et al., 1997). We searched for this region using the software HYDROLOG (S.G.Jaquieri, S.M.Zanata and R.R.Brentani, in preparation) that provides a hydropathy index profile of amino acid sequences and searches for a domain with a similar pattern in a given protein. Figure 5A shows the hydropathy profile of an STI1 region with high similarity to PrR. This STI1 peptide (STI1 pep.1), which spans amino acids 230–245, and two other STI1 peptides from either the N- (amino acids 61–76) or the C-terminus (amino acids 422–437), as well as PrR were tested for competition with the PrPc–STI1 interaction. Of the three STI1 peptides, only STI1 pep.1 inhibited the binding of PrPc–STI1 similarly to PrR (Figure 5B). Thus, the region covering amino acids 230–245 in STI1, which has the same hydropathy profile as PrR (Martins et al., 1997), seems to contain the binding site for PrPc. Figure 5.Mapping PrPc binding at the STI1 molecule using the complementary hydropathy theory and synthetic peptides. (A) Hydropathy plot of STI1 pep.1 (amino acids 230–245) (filled circles) and the PrR peptide (open squares). (B) Competition of His6-PrPc–[125I]His6-STI1 binding by increasing amounts of the synthetic peptides PrR, scrambled peptide, STI1 pep.1, STI1 N-terminus peptide (STI1 pep.N) or STI1 C-terminus peptide (STI1 pep.C). Total binding between His6-PrPc and [125I]His6-STI1 was set to 100% (control). The results are expressed as the relative percentage of the binding produced by competition with each peptide. #P < 0.04 and *P < 0.01 versus control, single mean Student's t-test. Download figure Download PowerPoint Cellular STI1 associates with recombinant PrPc To test whether recombinant PrPc binds STI1 at the cellular level, we used HEK 293T cells overexpressing green fluorescent protein (GFP)–STI1. PrPc binds to the surface of these cells but not to control cells expressing only GFP (Figure 6A). These results indicate that PrPc binding to the cell surface increases after STI1 ectopic expression. We did not detect PrPc binding to non-transfected cells (Figure 6A), probably due to competition between resident and recombinant PrPc for the surface ligands. In fact, recombinant PrPc binds to the surface of primary cultured fibroblasts from PrP0/0 mice (Figure 6B), indicating that the absence of cellular PrPc permits the binding of recombinant PrPc to surface ligands. We next performed pull-down experiments (Rohm et al., 2000; Zanata et al., 2002) using cultured primary fibroblasts from PrP0/0 mice (Büeler et al., 1992) to test whether STI1 was one of these PrPc ligands. Whole cells or cellular extracts were incubated with His6-PrPc followed by affinity chromatography with Ni-NTA–agarose, and resin- bound proteins were assayed by western blot with either anti-STI1 (Figure 6C, top panel) or anti-PrPc antibodies (Figure 6C, bottom panel). Recombinant PrPc binds to cellular STI1 both at the cell surface (lane 1) and in cell-free conditions (lane 2), while STI1 did not associate with the Ni-NTA–agarose resin in the absence of recombinant PrPc (lane 3). The membrane was also re-probed with antibodies against actin and we were unable to detect any reactivity (data not shown). These data indicate that recombinant PrPc specifically binds to cellular STI1. Figure 6.Cellular STI1 binds recombinant PrPc. (A) HEK 239T cells transfected with GFP–STI1 or GFP, or non-transfected (NT) were incubated in the absence or presence of 20 mg of His6-PrPc followed by incubation with mouse anti-PrPc or non-immune serum and anti-mouse R-phycoerythrin conjugate. Analyses were carried out using a Becton Dickinson FACScan Cytometer. The specific fluorescence intensity was determined by subtraction of the fluorescence obtained with non-immune serum from that produced with anti PrPc serum. *P <0.01, GFP–STI1 + 20 mg PrPc versus GFP–STI1 without PrPc and #P <0.03, GFP–STI1 + 20 mg PrPc versus GFP + 20 mg PrPc, Mann– Whitney test. (B) Primary fibroblast cultures from PrP0/0 animals were incubated in the absence (b) or presence (c) of His6-PrPc followed by incubation with mouse anti-PrPc (b and c) or non-immune serum (a). (C) Whole cells (lane 1) or cellular extracts (lane 2) from PrP0/0 mice fibroblasts were incubated with His6-PrPc. Whole cells were washed, lysed and the extracts incubated with Ni-NTA–agarose. Extracts from cells without His6-PrPc addition were also incubated with Ni-NTA–agarose (lane 3). The bound material was eluted off the beads and analyzed by western blot using anti-STI1 (α-STI1, upper panel) or anti-PrPc (α-PrPc, lower panel) serum. Download figure Download PowerPoint STI1 associates with cellular PrPc HEK 293T cells were transfected with vectors containing cDNA encoding the fusion proteins GFP–PrPc (Lee et al., 2001a) and GFP–STI1. The cellular STI1 (66 kDa) or the GFP–STI1 protein (96 kDa) were detected by western blots using anti-STI1 antibodies (Figure 7, lanes 1 and 2), while only GFP–PrPc was observed after the reaction with anti-PrPc antibody (Figure 7, lane 3). However, using flow cytometry assays, we observed that HEK 293T cells express PrPc at their surface (Figure 6A). In fact, PrPc is hardly observed in conventional western blots from cell lines (Scott et al., 1988; Cabral et al., 2002). Cells were co-transfected with GFP–PrPc and GFP–STI1 followed by conjugation of surface proteins with biotin and immunoprecipitation using anti-PrPc antibody. The blotting reaction of the immunoprecipitated material with strepta vidin–peroxidase revealed four major bands (lane 5): one 57–58 kDa band corresponding to GFP–PrPc, and bands of 60, 70 and 96 kDa. The latter band is not observed when GFP–PrPc is transfected alone (data not shown), and reacts with anti-STI1 antibodies (Figure 7, lane 6), indicating that the 96 kDa protein co-immunoprecipitated with PrPc corresponds to GFP–STI1. The blot in lane 6 was also re-probed with a pan antibody to cadherin, which recognizes a 110 kDa isoform, and no reaction was detected (data not shown), indicating specificity for the co-immunoprecipitation reaction. We did not detect the resident STI1, probably because of its low expression at the cell surface. The identity of the 70 kDa band is unknown, and the 60 kDa band is non-specific, since it was also present following immunoprecipitation with non-immune serum (Figure 7, lane 4). These data indicate that PrPc is associated with STI1 at the cellular level, and that at least part of the PrPc–STI1 binding occurs at the cell surface. Figure 7.PrPc co-immunoprecipitates with STI1 located at the cell membrane. HEK 293T cells were transfected with GFP–PrPc and/or GFP–STI1 as indicated. Cell extracts were resolved by SDS–PAGE and western blots were done using anti-STI1 (lanes 1 and 2) or anti-PrPc (lane 3) antibodies. Cell surface proteins from transfected cells were biotinylated (lanes 4–6) and immunoprecipitated with anti-PrPc (lanes 5 and 6) or non-immune serum (lane 4). The reactions were developed using streptavidin–peroxidase (lanes 4 and 5) or anti-STI1 antibody followed by anti-rabbit IgG–peroxidase (lane 6). Lane 6 is shown only from 60 kDa upwards, because the antibody used for immunoprecipi tation reacts with the secondary antibody used to develop the western blot. Download figure Download PowerPoi
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