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• W2038559352 abstract "ALG-2, a prototypic member of the penta-EF-hand protein family, interacts with Alix at its C-terminal Pro-rich region containing four tandem PXY repeats. Human phospholipid scramblase 3 (PLSCR3) has a similar sequence (ABS-1) in its N-terminal region. In the present study, we found that ALG-2 interacts with PLSCR3 expressed in HEK293 cells in a Ca2+-dependent manner by co-immunoprecipitation, pulldown with glutathione S-transferase (GST) fused ALG-2 and an overlay assay using biotin-labeled ALG-2. The GST fusion protein of an alternatively spliced isoform of ALG-2, GST-ALG-2ΔGF122, pulled down green fluorescent protein (GFP)-fused PLSCR3 but not GFP Alix. Deletion of a region containing ABS-1 was not sufficient to abrogate the binding. A second ALG-2-binding site (ABS-2) was essential for interaction with ALG-2ΔGF122. Real-time interaction analyses with a surface plasmon resonance biosensor using synthetic oligopeptides and recombinant proteins corroborated direct Ca2+-dependent binding of ABS-1 to ALG-2 and that of ABS-2 to ALG-2 as well as to ALG-2ΔGF122. The sequence of ABS-2 contains multiple prolines and two phenylalanines, among which Phe49 was found to be critical, because its substitution with Ala or Tyr caused a loss of binding ability by pulldown assays using oligopeptide-immobilized beads. ALG-2-interacting proteins were classified into two groups based on binding ability to ALG-2ΔGF122: (i) isoform-non-interactive (ABS-1) types, including Alix, annexin A7, annexin A11, and TSG101 and (ii) isoform-interactive (ABS-2) types including PLSCR3, PLSCR4 and Sec31A. GST-pulldown assays using single amino acid-substituted ALG-2 mutants revealed differences in binding specificities between the two groups, suggesting structural flexibility in ALG-2-ligand complex formation. ALG-2, a prototypic member of the penta-EF-hand protein family, interacts with Alix at its C-terminal Pro-rich region containing four tandem PXY repeats. Human phospholipid scramblase 3 (PLSCR3) has a similar sequence (ABS-1) in its N-terminal region. In the present study, we found that ALG-2 interacts with PLSCR3 expressed in HEK293 cells in a Ca2+-dependent manner by co-immunoprecipitation, pulldown with glutathione S-transferase (GST) fused ALG-2 and an overlay assay using biotin-labeled ALG-2. The GST fusion protein of an alternatively spliced isoform of ALG-2, GST-ALG-2ΔGF122, pulled down green fluorescent protein (GFP)-fused PLSCR3 but not GFP Alix. Deletion of a region containing ABS-1 was not sufficient to abrogate the binding. A second ALG-2-binding site (ABS-2) was essential for interaction with ALG-2ΔGF122. Real-time interaction analyses with a surface plasmon resonance biosensor using synthetic oligopeptides and recombinant proteins corroborated direct Ca2+-dependent binding of ABS-1 to ALG-2 and that of ABS-2 to ALG-2 as well as to ALG-2ΔGF122. The sequence of ABS-2 contains multiple prolines and two phenylalanines, among which Phe49 was found to be critical, because its substitution with Ala or Tyr caused a loss of binding ability by pulldown assays using oligopeptide-immobilized beads. ALG-2-interacting proteins were classified into two groups based on binding ability to ALG-2ΔGF122: (i) isoform-non-interactive (ABS-1) types, including Alix, annexin A7, annexin A11, and TSG101 and (ii) isoform-interactive (ABS-2) types including PLSCR3, PLSCR4 and Sec31A. GST-pulldown assays using single amino acid-substituted ALG-2 mutants revealed differences in binding specificities between the two groups, suggesting structural flexibility in ALG-2-ligand complex formation. ALG-2 is a 22-kDa calcium-binding protein possessing five serially repetitive EF-hand motifs, and it belongs to the penta-EF-hand (PEF) 2The abbreviations used are:PEFpenta-EF-handABSALG-2-binding sitebio-biotin-labeledBSAbovine serum albuminCBBCoomassie Brilliant BlueGFPgreen fluorescent proteinGSTglutathione-S-transferasePLSCRphospholipid scramblaseSPRsurface plasmon resonancepAbpolyclonal antibodymAbmonoclonal antibodyFc1-2, -3, Flow cell nos. 1–3TBSTris-buffered salineCaMcalmodulin. protein family (1Maki M. Narayana S.V. Hitomi K. Biochem. J. 1997; 328: 718-720PubMed Google Scholar, 2Maki M. Kitaura Y. Satoh H. Ohkouchi S. Shibata H. Biochim. Biophys. Acta. 2002; 1600: 51-60Crossref PubMed Scopus (157) Google Scholar). Based on differences in the primary structures of EF1, mammalian PEF proteins are classified into two groups: group I (ALG-2 and peflin) and group II (typical calpains, including the small subunit, sorcin and grancalcin) (2Maki M. Kitaura Y. Satoh H. Ohkouchi S. Shibata H. Biochim. Biophys. Acta. 2002; 1600: 51-60Crossref PubMed Scopus (157) Google Scholar). Homologs of group I PEF proteins have been found not only in vertebrates but also widely in eukaryotes, including lower animals, plants, fungi, and protists (3Ohkouchi S. Nishio K. Maeda M. Hitomi K. Adachi H. Maki M. J. Biochem. 2001; 130: 207-215Crossref PubMed Scopus (25) Google Scholar). On the other hand, group II PEF proteins are restricted to the animal kingdom and are thought to have diverged during the evolution of animals (4Jekely G. Friedrich P. J. Mol. Evol. 1999; 49: 272-281Crossref PubMed Scopus (37) Google Scholar). ALG-2 forms a homodimer or a heterodimer with peflin through their EF5 regions (5Kitaura Y. Matsumoto S. Satoh H. Hitomi K. Maki M. J. Biol. Chem. 2001; 276: 14053-14058Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 6Kitaura Y. Satoh H. Takahashi H. Shibata H. Maki M. Arch. Biochem. Biophys. 2002; 399: 12-18Crossref PubMed Scopus (32) Google Scholar). Despite the original report of a pro-apoptotic function of ALG-2 in T cell hybridomas (7Vito P. Lacana E. D'Adamio L. Science. 1996; 271: 521-525Crossref PubMed Scopus (457) Google Scholar), ALG-2-deficient mice develop normally with no obvious abnormalities in the immune system (8Jang I.K. Hu R. Lacana E. D'Adamio L. Gu H. Mol. Cell. Biol. 2002; 22: 4094-4100Crossref PubMed Scopus (60) Google Scholar). Nonetheless, potential physiological roles of ALG-2 in regulation of ER-stress-induced apoptosis (9Rao R.V. Poksay K.S. Castro-Obregon S. Schilling B. Row R.H. del Rio G. Gibson B.W. Ellerby H.M. Bredesen D.E. J. Biol. Chem. 2004; 279: 177-187Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar), neuronal cell death during development (10Mahul-Mellier A.L. Hemming F.J. Blot B. Fraboulet S. Sadoul R. J. Neurosci. 2006; 26: 542-549Crossref PubMed Scopus (72) Google Scholar), and cancer (11la Cour J.M. Mollerup J. Winding P. Tarabykina S. Sehested M. Berchtold M.W. Am. J. Pathol. 2003; 163: 81-89Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 12Subramanian L. Polans A.S. Biochem. Biophys. Res. Commun. 1999; 322: 1153-1165Crossref Scopus (30) Google Scholar) have been reported. Alix (also named AIP1) was the first protein identified as an ALG-2-interacting protein (13Missotten M. Nichols A. Rieger K. Sadoul R. Cell Death Differ. 1999; 6: 124-129Crossref PubMed Scopus (212) Google Scholar, 14Vito P. Pellegrini L. Guiet C. D'Adamio L. J. Biol. Chem. 1999; 274: 1533-1540Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). This cytoplasmic 95-kDa protein is now recognized as a multifunctional protein involved in various cellular functions, including endosomal sorting, retrovirus budding, actin cytoskeleton assembly, signal transduction, and apoptosis (see Refs. 15Dikic I. BioEssays. 2004; 26: 604-607Crossref PubMed Scopus (16) Google Scholar, 16Morita E. Sundquist W.I. Annu. Rev. Cell Dev. Biol. 2004; 20: 395-425Crossref PubMed Scopus (549) Google Scholar, 17Odorizzi G. J. Cell Sci. 2006; 119: 3025-3032Crossref PubMed Scopus (146) Google Scholar, 18Sadoul R. Biol. Cell. 2006; 98: 69-77Crossref PubMed Scopus (54) Google Scholar for reviews). penta-EF-hand ALG-2-binding site biotin-labeled bovine serum albumin Coomassie Brilliant Blue green fluorescent protein glutathione-S-transferase phospholipid scramblase surface plasmon resonance polyclonal antibody monoclonal antibody -2, -3, Flow cell nos. 1–3 Tris-buffered saline calmodulin. We and others previously identified an ALG-2-binding site in the C-terminal proline-rich region in Alix (19Shibata H. Yamada K. Mizuno T. Yorikawa C. Takahashi H. Satoh H. Kitaura Y. Maki M. J. Biochem. 2004; 135: 117-128Crossref PubMed Scopus (63) Google Scholar, 20Trioulier Y. Torch S. Blot B. Cristina N. Chatellard-Causse C. Verna J.M. Sadoul R. J. Biol. Chem. 2004; 279: 2046-2052Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Results of analyses of amino acid-substituted mutants and deletion mutants of Alix suggested that a potential polyproline II-helix containing four tandem PXY repeats is an ALG-2-interacting core motif (801-PPYPTYPGYPGY-812), in which Ala substitutions of either all four Pro or Tyr residues and Phe substitutions of all four Tyr residues abolished the binding ability (19Shibata H. Yamada K. Mizuno T. Yorikawa C. Takahashi H. Satoh H. Kitaura Y. Maki M. J. Biochem. 2004; 135: 117-128Crossref PubMed Scopus (63) Google Scholar). Generally, Pro-rich regions often serve as domains for either specific protein-protein interaction or rapid but nonspecific interaction via their sticky arms extending out from the rest of the protein molecules (21Williamson M.P. Biochem. J. 1994; 297: 249-260Crossref PubMed Scopus (842) Google Scholar, 22Kay B.K. Williamson M.P. Sudol M. FASEB J. 2000; 14: 231-241Crossref PubMed Scopus (1050) Google Scholar). Both sorcin and ALG-2 bind to the presumably extended or disordered N-terminal Pro-rich regions of annexins A7 and A11 (23Brownawell A.M. Creutz C.E. J. Biol. Chem. 1997; 272: 22182-22190Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 24Satoh H. Shibata H. Nakano Y. Kitaura Y. Maki M. Biochem. Biophys. Res. Commun. 2002; 291: 1166-1172Crossref PubMed Scopus (58) Google Scholar, 25Satoh H. Nakano Y. Shibata H. Maki M. Biochim. Biophys. Acta. 2002; 1600: 61-67Crossref PubMed Scopus (63) Google Scholar). While exact binding sites for ALG-2 have not been determined yet, the N-terminal ∼30 residues that are rich in Pro, Gly and Tyr but have no charged residues in annexin A11 are sufficient for binding to sorcin (23Brownawell A.M. Creutz C.E. J. Biol. Chem. 1997; 272: 22182-22190Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Neither annexin A7 nor annexin A11, however, possesses the four tandem PXY repeats found in Alix. Instead, both annexins have GYPP repeats in sorcin-binding regions. One conspicuous difference between sorcin and ALG-2 for annexin A7 binding is the requirement of N-terminal non-PEF regions: the N-terminal domain of sorcin is essential (26Verzili D. Zamparelli C. Mattei B. Noegel A.A. Chiancone E. FEBS Lett. 2000; 471: 197-200Crossref PubMed Scopus (44) Google Scholar), but that of ALG-2 is dispensable (25Satoh H. Nakano Y. Shibata H. Maki M. Biochim. Biophys. Acta. 2002; 1600: 61-67Crossref PubMed Scopus (63) Google Scholar). ALG-2 binding motifs remain to be clarified. In this study, we found that phospholipid scramblase 3 (PLSCR3) is a novel ALG-2-interacting protein, and we identified two binding sites, designated ABS (ALG-2-binding site)-1 and ABS-2, in its N-terminal Pro-rich region. Although ABS-1 resembles the Alix PXY-repeat motif, ABS-2 does not conform to the canonical sequence and does not contain Tyr. ABS-2 possesses a unique binding property: it interacts with an alternatively spliced ALG-2 isoform, ALG-2ΔGF122, and other single amino acid-substituted ALG-2 mutants, with which ABS-1 and Alix do not interact. An ABS-2-oligopeptide-immobilized column has been proved to be useful for rapid one-step affinity purification of untagged recombinant ALG-2 proteins, including ALG-2ΔGF122. Furthermore, we demonstrated that ALG-2-interacting proteins can be classified into two groups based on the binding ability to ALG-2ΔGF122. Differences in binding specificities to other ALG-2 mutants suggest structural flexibility in ALG-2-ligand complex formation. Materials—Untagged, biotin-tagged, and phosphorylated synthetic oligopeptides were obtained from Biosynthesis (Lewis-ville, TX). Rabbit anti-GFP polyclonal antibody (pAb) and anti-apoptosis inducing factor pAb were purchased from Abcam (#ab1998, Cambridge, UK). Mouse monoclonal antibodies (mAbs) of anti-GFP (B2) and anti-annexin VII (A-1) and goat anti-annexin XI pAb (N-17) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse mAbs of anti-α-tubulin (clone DM1A), anti-glyceraldehyde-3-phosphate dehydrogenase, and anti-Lamp-1 were from Sigma, Chemicon, and Pharmingen, respectively. Anti-TSG101 mAb (4A10) and anti-Sec31A mAb (clone 32) were from Gene Tex (San Antonio, TX) and BD Transduction Laboratories (San Diego, CA), respectively. Streptavidin-peroxidase was obtained from Rockland Immunochemicals (Philadelphia, PA). Preparations of rabbit anti-ALG-2 pAb and anti-Alix pAb were described previously (27Maki M. Yamaguchi K. Kitaura Y. Satoh H. Hitomi K. J. Biochem. 1998; 124: 1170-1177Crossref PubMed Scopus (53) Google Scholar, 28Shibata H. Suzuki H. Yoshida H. Maki M. Biochem. Biophys. Res. Commun. 2007; 353: 756-763Crossref PubMed Scopus (66) Google Scholar). Anti-PLSCR3 antiserum was raised in rabbits using GST-fused PLSCR3 N-terminal Pro-rich region protein as antigen. Sulfosuccinimidyl N-(d-biotinyl)-6-aminohexanoate was purchased from Dojin (Kumamoto, Japan). Plasmid Constructions—Human cDNAs for PLSCR1, PLSCR3, and PLSCR4 were cloned from a human skeletal muscle cDNA library (Clontech) (for PLSCR3) or a Human Fetus Marathon-Ready™ cDNA library (Clontech) (for PLSCR1 and PLSCR4) by the PCR method using a proofreading thermostable PfuTurbo DNA polymerase (Stratagene) (see supplemental materials). pGFP-Alix was described previously (29Katoh K. Shibata H. Suzuki H. Nara A. Ishidoh K. Kominami E. Yoshimori T. Maki M. J. Biol. Chem. 2003; 278: 39104-39113Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). Alanine-substituted mutants of GST-fused human ALG-2 at various amino acid residues were created by the PCR-based mutagenesis method (see supplemental materials). Construction of an Escherichia coli expression plasmid of N-terminally truncated ALG-2 that lacks the hydrophobic and Pro/Gly-rich region, pET3d-des3-23ALG-2 (previous name: pET3d-ALG-2ΔN23), was described previously (25Satoh H. Nakano Y. Shibata H. Maki M. Biochim. Biophys. Acta. 2002; 1600: 61-67Crossref PubMed Scopus (63) Google Scholar), and the produced protein was re-named des3-23ALG-2 in this study. Construction of a plasmid for GST-fused protein of an alternatively spliced ALG-2 isoform that lacks Gly121-Phe122 (designated GST-ALG-2ΔGF122) was described previously (30Katoh K. Suzuki H. Terasawa Y. Mizuno T. Yasuda J. Shibata H. Maki M. Biochem. J. 2005; 391: 677-685Crossref PubMed Scopus (63) Google Scholar). Other constructs are described in the supplemental materials. Establishment of PLSCR3-expressing Cell Line—A PLSCR3 cDNA fragment was inserted into the BamHI site of pIRES1neo (Clontech), and the resultant expression vector was used for transfection of HEK293 subcloned cells designated YS14 (28Shibata H. Suzuki H. Yoshida H. Maki M. Biochem. Biophys. Res. Commun. 2007; 353: 756-763Crossref PubMed Scopus (66) Google Scholar). G418-resistant cells were isolated by cylinder cloning and further screened for constant expression of PLSCR3 by Western blotting using anti-PLSCR3 antibody. Subcellular fractionation by differential centrifugation was performed essentially as described previously (28Shibata H. Suzuki H. Yoshida H. Maki M. Biochem. Biophys. Res. Commun. 2007; 353: 756-763Crossref PubMed Scopus (66) Google Scholar). GST-ALG-2 Pulldown, Bio-ALG-2 Overlay, and Co-immunoprecipitation—Pulldown assays using GST-ALG-2 and its mutant proteins were performed as described previously (28Shibata H. Suzuki H. Yoshida H. Maki M. Biochem. Biophys. Res. Commun. 2007; 353: 756-763Crossref PubMed Scopus (66) Google Scholar, 30Katoh K. Suzuki H. Terasawa Y. Mizuno T. Yasuda J. Shibata H. Maki M. Biochem. J. 2005; 391: 677-685Crossref PubMed Scopus (63) Google Scholar) using the cleared lysate of HEK293 cells untransfected or transfected with respective expression vectors of GFP-fused proteins. An overlay assay using bio-ALG-2 was carried out as described previously (30Katoh K. Suzuki H. Terasawa Y. Mizuno T. Yasuda J. Shibata H. Maki M. Biochem. J. 2005; 391: 677-685Crossref PubMed Scopus (63) Google Scholar) using the immunoprecipitates of anti-GFP pAb from the cleared lysate of HEK293 cells that had been transfected with pGFP-PLSCR3 or an empty pEGFP vector. Alternatively, immunoprecipitation was performed in the presence or absence of Ca2+, and immunoprecipitates were subjected to Western blotting with anti-ALG-2 pAb essentially as described previously (19Shibata H. Yamada K. Mizuno T. Yorikawa C. Takahashi H. Satoh H. Kitaura Y. Maki M. J. Biochem. 2004; 135: 117-128Crossref PubMed Scopus (63) Google Scholar). Specific conditions for incubation with antibodies are described in the supplemental materials. Pulldown Assay of des3-23ALG-2 with Mutant Oligopeptide-immobilized Sepharose Beads—Each synthetic oligopeptide (0.2 mg) dissolved in 0.1 ml of the coupling buffer (0.2 m NaHCO3, pH 8.3, 0.5 m NaCl) was immobilized to a 0.2-ml bed volume of NHS-activated Sepharose 4 Fast Flow beads (GE Healthcare/Amersham Biosciences) according to the manufacturer's instructions. A binding assay was performed by incubating 10 μl of beads in a total volume of 50 μl in binding buffer (20 mm Tris-HCl, pH 7.5, 50 mm NaCl, 5 mm 2-mercaptoethanol, 0.5 mm CaCl2, 0.5% Triton X-100) containing 10 μg each of des3-23ALG-2, bovine serum albumin (BSA), ovalbumin, and α-lactalbumin by mixing with MicroMixer E-36 (Taitec, Japan) at room temperature for 30 min. Then the mixture was centrifuged at 3000 rpm for 3 min, and pelleted beads were washed with 0.4 ml of binding buffer three times. The unbound fraction (first supernatant) and bound fraction (beads) were subjected to SDS-PAGE. After electrophoresis, gels were stained with Coomassie Brilliant Blue R-250 (CBB) and relative amounts of proteins were estimated by densitometric analysis using the free imaging software (Scion Image Beta 4.02) available from Scion. Affinity Purification of Recombinant ALG-2 Proteins—Lys-tagged ABS-2 oligopeptide (k-ABS-2: kQVPAPAPGFALFP-SPGPVA; k, extraneous Lys for facilitating cross-linking) was immobilized to a HiTrap NHS-activated HP column (GE Healthcare/Amersham Biosciences). Affinity purification of recombinant ALG-2 proteins from E. coli was carried out by a method similar to that used for purification of recombinant PEF domain proteins of the calpain subunits (31Yang H.Q. Ma H. Takano E. Hatanaka M. Maki M. J. Biol. Chem. 1994; 269: 18977-18984Abstract Full Text PDF PubMed Google Scholar, 32Takano E. Ma H. Yang H.Q. Maki M. Hatanaka M. FEBS Lett. 1995; 362: 93-97Crossref PubMed Scopus (73) Google Scholar) with some modifications (supplemental materials). Surface Plasmon Resonance Measurements—Real-time interaction analysis was performed at 25 °C using an SPR biosensor, BIAcore2000 system (BIAcore, Uppsala, Sweden). N-terminally biotin-labeled oligopeptides of ABS-1 (bio-ABS-1: bio-qGYAPSPPPPYPVTPGYPEPA; q, extraneous Gln for adjusting N-terminal residue with bio-ABS-2) and ABS-2 (bio-ABS-2: bio-QVPAPAPGFALFPSPGPVA) that had been dissolved in water (10 mg/ml) were diluted to 0.1 mg/ml with HBS-EP (10 mm HEPES-NaOH, pH 7.4, 150 mm NaCl, 3 mm EDTA, 0.005% surfactant P20), and each peptide was captured on flow cell no. 2 (Fc2) and no. 3 (Fc3) of a streptavidin (SA)-immobilized sensor chip, respectively, by running over the respective flow cell at the flow rate of 10 μl/min for 120 s. Flow cell no. 1 (Fc1) was used as a reference. For interaction analysis, the flow rate was maintained at 50 μl/min. A solution of analyte (full-length or truncated ALG-2 protein of either wild-type or ΔGF122 isoform) was diluted with HBS-CP (10 mm HEPES-NaOH, pH 7.4, 150 mm NaCl, 0.1 mm CaCl2, 0.005% surfactant P20) to 100 nm and injected. After keeping the flow over the immobilized sensor surface for 180 s, the sensor surface was washed with the running buffer for 180 s. To regenerate the sensor surfaces after each measuring cycle, analytes were completely dissociated from the immobilized ligand by injecting 50 μl of HBS-EP supplemented with EDTA to 10 mm. For analysis of Ca2+ concentration dependence, HBS-P (10 mm HEPES-NaOH, pH 7.4, 150 mm NaCl, 0.005% surfactant P20) was used as a running buffer. Analytes, diluted with HBS-P, were adjusted to varying concentrations of CaCl2. COINJECT mode was used to avoid transient wash of the sensor surface with HBS-P after the end of analyte injection. Plate Assay of ALG-2-binding to Phosphorylated ABS-1 Oligopeptide—Varying amounts (11–183 pmol) of ABS-1 (KGYAPSPPPPYPVTPGYPEPA) and Thr21-phosphorylated ABS-1 (KGYAPSPPPPYPV-pT-PGYPEPA, ABS-1-pT) oligopeptides were dissolved in 0.1 ml of 100 mm Na2CO3 buffer (pH 9.6) and immobilized to a 96-well Nunc Immobilizer™ Amino plate (Nunc A/S, Denmark) according to the provided instruction manual for 1 h at room temperature. The unreacted surface was inactivated with 10 mm ethanolamine (pH 8.3) for 1 h, followed by washing with TBS (10 mm Tris-HCl, pH 7.5, 0.15 m NaCl) and blocking with TBS containing 3% BSA. Purified recombinant ALG-2 was labeled with biotin in vitro using sulfosuccinimidyl N-(d-biotinyl)-6-aminohexanoate (Biotin-AC5 Sulfo-OSu) as described previously (33Wormmeester J. Stiekema F. Groot C. Methods Enzymol. 1990; 184: 314-319Crossref PubMed Scopus (4) Google Scholar). A solution (0.1 ml) of 120 nm biotin-labeled ALG-2 (bio-ALG-2) in binding buffer TBS-C (10 mm Tris-HCl, pH 7.5, 0.15 m NaCl, 0.1 mm CaCl2) containing 0.1% Tween 20 and 0.1% BSA was added to the wells, and the mixture was incubated at room temperature for 1 h. After the wells had been washed with 0.3 ml of TBS-C containing 0.1% Tween 20, bound bio-ALG-2 was reacted with SA-peroxidase for 1 h. The wells were washed once with TBS-C containing 0.1% Tween 20 and three times with TBS-C. Colorimetric peroxidase reaction was carried out using 0.4 mg/ml o-phenylenediamine and 0.003% H2O2 in 0.1 ml of 50 mm sodium citrate-100 mm sodium phosphate buffer (pH 5.0), and the reaction products were measured with a plate reader (BiotrakII reader, GE Healthcare/Amersham Biosciences) at 492 nm. PXY Repeat Sequence in the Pro-rich Region of PLSCR3—The ALG-2-binding sequence in Alix (Alix-ABS) contains four tandem repeats of PXY (Fig. 1). To identify novel ALG-2-binding proteins, we searched a human protein data base (SwissProt) with the BLAST program “Search for Short, Nearly Exact Matches” on the Internet (www.ncbi.nlm.nih.gov/BLAST/) using PPYPTYPGYPGY (an essential sequence in Alix for efficient ALG-2 binding) as a query sequence. The top ten high score sequences were derived from the following proteins (scores and SwissProt accession numbers indicated in parentheses): PDCD6-interacting protein (Alix/AIP1/Hp95) (45.2, Q8WUM4), transmembrane BAX inhibitor motif-containing protein 1 (RECS1 protein homolog) (30.3, Q969X1), annexin A11 (27.4, P50995), PLSCR3 (27.4, Q9NRY6), annexin A7 (25.7, P20073), homeobox protein Nkx-2.5 (25.7, P52952), NFATc4 (25.2, Q14934), EYA2 (25.2, O00167), TSG101 (24.8, Q99816), and SCAMP4 (24.8, Q969E2). In this study, we selected PLSCR3 as the most possible candidate protein for in vitro ALG-2 binding analyses because of the absence of a gap in the aligned PXY-repeat sequence (Fig. 1B). Analyses of Interaction between ALG-2 and PLSCR3—We performed a co-immunoprecipitation assay by overexpressing GFP-fused PLSCR3, GFP-Alix (positive control), and GFP (negative control), respectively, in HEK293 cells. The expressed proteins were immunoprecipitated with anti-GFP pAb. As shown in Fig. 2A, specific bands corresponding to ALG-2 were detected with anti-ALG-2 pAb in the immunoprecipitates of GFP-Alix and GFP-PLSCR3 in the presence of 10 μm Ca2+ but not in the presence of a Ca2+-chelator, EGTA (2 mm). No ALG-2 bands were detected in the immunoprecipitates of GFP in either condition. In a separate experiment, we performed an overlay assay using biotin-labeled ALG-2 (bio-ALG-2) as a probe. As shown in Fig. 2B, the immunoprecipitate of GFP-PLSCR3, but not that of GFP, presented a specific band, suggesting a direct physical interaction between ALG-2 and PLSCR3. To investigate whether ALG-2 associates with endogenous PLSCR3, human lymphoma cell lines such as Raji and Jurkat and rat fat cells were first analyzed for expression of PLSCR3. Endogenous PLSCR3 protein, however, was not detected with our rabbit anti-PLSCR3 antiserum. Then, we established a PLSCR3-expressing HEK293 cell line (HEK293/Scr3) as shown in Fig. 2C. Using this stable cell line as an alternative source of endogenous PLSCR3, we performed subcellular fractionation by the differential centrifugation method to obtain PLSCR3-enriched fraction. Fig. 2D shows a representative result of the fractionation in the presence of 10 μm CaCl2 under incomplete cell disruption condition as indicated by the presence of anti-apoptosis inducing factor (mitochondrial inter-membrane space protein), Lamp-1 (late endosome/lysosome protein), and glyceraldehyde-3-phosphate dehydrogenase (cytosolic protein) in the 600 × g pellets (P0.6) in addition to their expected fractions of either 10,000 × g pellets (P10) or 100,000 × g supernatant (S100). In agreement with previous studies (5Kitaura Y. Matsumoto S. Satoh H. Hitomi K. Maki M. J. Biol. Chem. 2001; 276: 14053-14058Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 28Shibata H. Suzuki H. Yoshida H. Maki M. Biochem. Biophys. Res. Commun. 2007; 353: 756-763Crossref PubMed Scopus (66) Google Scholar), ALG-2 was recovered mostly in the P0.6 fraction when the lysis buffer containing Ca2+ was used, but faint bands corresponding to ALG-2 were detected in the P10 and P100 fractions. PLSCR3 was detected more in the P10 fraction than in the P0.6 fraction. PLSCR3 was extracted from the P10 fraction with the buffer-containing detergent (0.2% Nonidet P-40) and either 100 μm CaCl2 or 2 mm EGTA, and then the supernatant was subjected to a co-immunoprecipitation assay using anti-ALG-2 pAb for immunoprecipitation and anti-PLSCR3 pAb for Western blotting. As shown in Fig. 2E, PLSCR3 was detected in the immunoprecipitates by anti-ALG-2 pAb (α-ALG-2) but not by control IgG. Two ALG-2-binding Sites in the Pro-rich Region of PLSCR3— To further analyze the interaction between human ALG-2 and PLSCR3, GST-pulldown assays were performed using GST-fused ALG-2 of wild-type and previously constructed mutants. ALG-2E47A/114A is a calcium-binding defective mutant (34Lo K.W. Zhang Q. Li M. Zhang M. Biochemistry. 1999; 38: 7498-7508Crossref PubMed Scopus (72) Google Scholar), which was shown to bind neither Alix (19Shibata H. Yamada K. Mizuno T. Yorikawa C. Takahashi H. Satoh H. Kitaura Y. Maki M. J. Biochem. 2004; 135: 117-128Crossref PubMed Scopus (63) Google Scholar) nor TSG101 (30Katoh K. Suzuki H. Terasawa Y. Mizuno T. Yasuda J. Shibata H. Maki M. Biochem. J. 2005; 391: 677-685Crossref PubMed Scopus (63) Google Scholar). In Western blot analysis using anti-GFP mAb, a GFP-PLSCR3 band was not detected in the pelleted bead fraction (pulldown products) of GST-ALG-2E47A/114A in the presence or absence of Ca2+ in the binding buffer (Fig. 3A). ALG-2ΔGF122 corresponds to a naturally occurring alternatively spliced isoform of human ALG-2 lacking Gly121Phe122, which was first reported in mice (35Tarabykina S. Moller A.L. Durussel I. Cox J. Berchtold M.W. J. Biol. Chem. 2000; 275: 10514-10518Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Consistent with the previous report that the isoform did not interact with Alix by the yeast two-hybrid assay (35Tarabykina S. Moller A.L. Durussel I. Cox J. Berchtold M.W. J. Biol. Chem. 2000; 275: 10514-10518Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), GST-ALG-2ΔGF122 did not pull down GFP-Alix, but, surprisingly, it pulled down GFP-PLSCR3 (Fig. 3B). Because the initially predicted ABS in the Pro-rich region of PLSCR3 has a PXY repeat and resembles the Alix-ABS, we speculated that PLSCR3 contains an additional ABS that is capable of binding to ALG-2ΔGF122. To corroborate this hypothesis, we constructed a deletion mutant of GFP-PLSCR3 lacking residues 12–27 in the Pro-rich region (designated GFP-PLSCR3ΔABS-1) and performed the pulldown assay. Not only GST-ALG-2 but also GST-ALG-2ΔGF122 efficiently pulled down GFP-PLSCR3ΔABS-1 (Fig. 3C). Next, a mutant lacking residues 43–58 (designated GFP-PLSCR3ΔABS-2) was constructed because of the notable presence of two phenylalanine residues surrounded by proline residues. GFP-PLSCR3ΔABS-2 was pulled down by GST-ALG-2 but not by GST-ALG-2ΔGF122. When both ABS-1 and ABS-2 were deleted, the mutant (designated GFP-PLSCR3ΔABS-1/2) was not essentially pulled down anymore. Affinity Purification of ALG-2 Proteins with an ABS-2 Oligopeptide-immobilized Column—The nature of Ca2+-dependent specific interaction of PEF proteins with target peptide sequences has been utilized for affinity purification of recombinant PEF domain proteins of the calpain large subunit and the small subunit (previous name: calmodulin-like domain) using calpastatin subdomain oligopeptides that were immobilized to Sepharose beads (31Yang H.Q. Ma H. Takano E. Hatanaka M. Maki M. J. Biol. Chem. 1994; 269: 18977-18984Abstract Full Text PDF PubMed Google Scholar, 32Takano E. Ma H. Yang H.Q. Maki M. Hatanaka M. FEBS Lett. 1995; 362: 93-97Crossref PubMed Scopus (73) Google Scholar). A similar approach was taken to purify untagged recombinant ALG-2 proteins expressed in E. coli. A Lys-tagged synthetic oligopeptide of ABS-2 (k-ABS-2: kQVP" @default.
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• W2038559352 date "2008-04-01" @default.
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• W2038559352 title "Identification of Alix-type and Non-Alix-type ALG-2-binding Sites in Human Phospholipid Scramblase 3" @default.
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