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• W2024646520 abstract "The heparin-binding fibroblast growth factor (FGF) prototypes lack a classical signal sequence, yet their presence is required in the extracellular compartment for the activation of cell-surface receptor-dependent signaling. Early studies with FGF-1 demonstrated its presence in bovine brain as a novel high molecular weight complex, and subsequent studies identified a second heparin-binding protein that co-purified with FGF-1. Polypeptide sequence analysis revealed that this heparin-binding protein corresponded to the extravesicular domain of bovine synaptotagmin (Syn)-1, a transmembrane component of synaptic vesicles involved in the regulation of organelle traffic. Since FGF-1 is released in response to heat shock as a mitogenically inactive Cys-30 homodimer, we sought to determine whether this heparin-binding protein was involved in the release of FGF-1. We report that a proteolytic fragment of the extravesicular domain of Syn-1 is associated with FGF-1 in the extracellular compartment of FGF-1-transfected NIH 3T3 cells following temperature stress. By using heparin-Sepharose affinity to discriminate between the monomer and homodimer forms of FGF-1 and resolution by conventional and limited denaturant gel shift immunoblot analysis, it was possible to identify FGF-1 and Syn-1 as potential components of a denaturant- and reducing agent-sensitive extracellular complex. It was also possible to demonstrate that the expression of an antisense-Syn-1 gene represses the release of FGF-1 in response to heat shock. These data indicate that FGF-1 may be able to utilize the cytosolic face of conventional exocytotic vesicles to traffic to the inner surface of the plasma membrane where it may gain access to the extracellular compartment as a complex with Syn-1. The heparin-binding fibroblast growth factor (FGF) prototypes lack a classical signal sequence, yet their presence is required in the extracellular compartment for the activation of cell-surface receptor-dependent signaling. Early studies with FGF-1 demonstrated its presence in bovine brain as a novel high molecular weight complex, and subsequent studies identified a second heparin-binding protein that co-purified with FGF-1. Polypeptide sequence analysis revealed that this heparin-binding protein corresponded to the extravesicular domain of bovine synaptotagmin (Syn)-1, a transmembrane component of synaptic vesicles involved in the regulation of organelle traffic. Since FGF-1 is released in response to heat shock as a mitogenically inactive Cys-30 homodimer, we sought to determine whether this heparin-binding protein was involved in the release of FGF-1. We report that a proteolytic fragment of the extravesicular domain of Syn-1 is associated with FGF-1 in the extracellular compartment of FGF-1-transfected NIH 3T3 cells following temperature stress. By using heparin-Sepharose affinity to discriminate between the monomer and homodimer forms of FGF-1 and resolution by conventional and limited denaturant gel shift immunoblot analysis, it was possible to identify FGF-1 and Syn-1 as potential components of a denaturant- and reducing agent-sensitive extracellular complex. It was also possible to demonstrate that the expression of an antisense-Syn-1 gene represses the release of FGF-1 in response to heat shock. These data indicate that FGF-1 may be able to utilize the cytosolic face of conventional exocytotic vesicles to traffic to the inner surface of the plasma membrane where it may gain access to the extracellular compartment as a complex with Syn-1. Fibroblast growth factor (FGF) 1The abbreviations used are: FGFfibroblast growth factorRT-PCRreverse transcription-polymerase chain reactionPAGEpolyacrylamide gel electrophoresisSynsynaptotagminCMconditioned mediumDMEMDulbecco's modified Eagle's mediumDTTdithiothreitolHUVEChuman umbilical vein endothelial cells2-ME2-mercaptoethanol.1The abbreviations used are: FGFfibroblast growth factorRT-PCRreverse transcription-polymerase chain reactionPAGEpolyacrylamide gel electrophoresisSynsynaptotagminCMconditioned mediumDMEMDulbecco's modified Eagle's mediumDTTdithiothreitolHUVEChuman umbilical vein endothelial cells2-ME2-mercaptoethanol.-1 (acidic) and FGF-2 (basic) are the prototype members of the FGF gene family, and these proteins have been extensively characterized as potent angiogenic and neurotrophic factors (1Burgess W. Maciag T. Annum. Rev. Biochem. 1989; 58: 575-606Crossref PubMed Google Scholar). The FGF prototypes lack a conventional signal peptide sequence for secretion through the endoplasmic reticulum-Golgi-mediated pathway, yet their interaction with high affinity tyrosine kinase receptors on the cell surface implies the function of an alternative secretion pathway to mediate their release. fibroblast growth factor reverse transcription-polymerase chain reaction polyacrylamide gel electrophoresis synaptotagmin conditioned medium Dulbecco's modified Eagle's medium dithiothreitol human umbilical vein endothelial cells 2-mercaptoethanol. fibroblast growth factor reverse transcription-polymerase chain reaction polyacrylamide gel electrophoresis synaptotagmin conditioned medium Dulbecco's modified Eagle's medium dithiothreitol human umbilical vein endothelial cells 2-mercaptoethanol. We have previously reported that FGF-1 is actively released from FGF-1-transfected NIH 3T3 cells by a transcription- and translation-dependent mechanism in response to temperature stress (2Jackson A. Friedman S. Zhan X. Engleka K. Forough R. Maciag T. Proc Natl. Acad. Sci. U. S. A. 1992; 89: 10691-10695Crossref PubMed Scopus (226) Google Scholar). Analysis of the effects of brefeldin A on the release of FGF-1 in this system demonstrated the unconventional nature of the FGF-1 release pathway since disruption of endoplasmic reticulum-Golgi communication (3Lippincott-Schwartz J. Yuan L.C. Bonifacino J.S. Klausner R.D. Cell. 1989; 56: 801-813Abstract Full Text PDF PubMed Scopus (1309) Google Scholar) did not inhibit the appearance of extracellular FGF-1 in response to heat shock (4Jackson A. Tarantini F. Gamble S. Friedman S. Maciag T. J. Biol. Chem. 1995; 270: 33-36Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). In addition, we have also reported the following: (i) FGF-1 is released as a latent homodimer with reduced affinity for immobilized heparin (5Tarantini F. Gamble S. Jackson A. Maciag T. J. Biol. Chem. 1995; 270: 29039-29042Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar); (ii) the FGF-1 homodimer can be activated by treatment with either (NH4)2SO4 (2Jackson A. Friedman S. Zhan X. Engleka K. Forough R. Maciag T. Proc Natl. Acad. Sci. U. S. A. 1992; 89: 10691-10695Crossref PubMed Scopus (226) Google Scholar) or reducing agents (4Jackson A. Tarantini F. Gamble S. Friedman S. Maciag T. J. Biol. Chem. 1995; 270: 33-36Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar); (iii) FGF-1 homodimer formation utilizes residue Cys-30, and dimer formation is important for FGF-1 release (5Tarantini F. Gamble S. Jackson A. Maciag T. J. Biol. Chem. 1995; 270: 29039-29042Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar); (iv) FGF-1 contains a phosphatidylserine-binding domain between amino acid residues 114 and 137 (5Tarantini F. Gamble S. Jackson A. Maciag T. J. Biol. Chem. 1995; 270: 29039-29042Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar); (v) the release of FGF-1 involves the function of a carboxyl-terminal domain which is not present in FGF-2 (6Shi J. Friedman S. Maciag T. J. Biol. Chem. 1997; 272: 1142-1147Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar); and (vi) the FGF-1 secretion pathway does not restrict the release of high molecular weight forms of FGF-1 since an FGF-1:β-galactosidase chimera is released as a structurally intact protein (6Shi J. Friedman S. Maciag T. J. Biol. Chem. 1997; 272: 1142-1147Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). The ability of FGF-1 to interact with phosphatidylserine (5Tarantini F. Gamble S. Jackson A. Maciag T. J. Biol. Chem. 1995; 270: 29039-29042Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), an important component of the inner leaflet of the plasma membrane (7Zachowski A. Biochem. J. 1993; 294: 1-14Crossref PubMed Scopus (697) Google Scholar), implies that this interaction may play a role in its release. Finally, analysis of FGF-1 release using FGF-1:FGF-2 chimeric constructs demonstrated that FGF-1, but not FGF-2, is released in response to temperature stress (6Shi J. Friedman S. Maciag T. J. Biol. Chem. 1997; 272: 1142-1147Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Early studies on the purification of FGF-1 from bovine brain tissue demonstrated the presence of high and low molecular weight forms of the protein; the high molecular weight form of FGF-1 represents an acid-sensitive complex (8Burgess W.H. Mehlman T. Friesel R. Johnson W. Maciag T. J. Biol. Chem. 1985; 260: 11389-11392Abstract Full Text PDF PubMed Google Scholar). During the characterization of brain-derived FGF-1 as a heparin-binding protein, a second polypeptide was identified that co-eluted with FGF-1 from immobilized heparin (8Burgess W.H. Mehlman T. Friesel R. Johnson W. Maciag T. J. Biol. Chem. 1985; 260: 11389-11392Abstract Full Text PDF PubMed Google Scholar). This protein was isolated, sequenced, and identified as the extravesicular portion of synaptotagmin (Syn)-1, an integral component of synaptic vesicles (9Perin M.S. Brose N. Jahn R. Sudhof T.C. J. Biol. Chem. 1991; 266: 623-629Abstract Full Text PDF PubMed Google Scholar). Because Syn-1 has been implicated in the regulation of exocytotic (9Perin M.S. Brose N. Jahn R. Sudhof T.C. J. Biol. Chem. 1991; 266: 623-629Abstract Full Text PDF PubMed Google Scholar) and endocytotic organelle trafficking (10Zhang J.Z. Davletov B.A. Sudhof T.C. Anderson R.G.W. Cell. 1994; 78: 751-760Abstract Full Text PDF PubMed Scopus (431) Google Scholar), we questioned whether Syn-1 may also be involved in the temperature-dependent release of FGF-1. We now report the presence of denaturant- and reducing agent-sensitive forms of FGF-1 and Syn-1 in media conditioned by heat shock from FGF-1-transfected NIH 3T3 cells and have identified a p40 fragment of Syn-1 as a potential component of the extracellular FGF-1 complex. Stable NIH 3T3 FGF-1 cell transfectants were obtained as described previously (2Jackson A. Friedman S. Zhan X. Engleka K. Forough R. Maciag T. Proc Natl. Acad. Sci. U. S. A. 1992; 89: 10691-10695Crossref PubMed Scopus (226) Google Scholar) and maintained in Dulbecco's modified Eagle's medium (DMEM, Life Technologies, Inc.) on human fibronectin (10 μg/cm2)-coated dishes containing 10% (v/v) bovine calf serum (HyClone), 1× antibiotic/antimycotic (Life Technologies, Inc.), and 400 μg/ml G418 (Life Technologies, Inc.). A confluent monolayer of transfected cells was subjected to heat shock (42 °C for 2 h) in serum-free DMEM, containing 4 units/ml heparin (The Upjohn Co.), as described previously (2Jackson A. Friedman S. Zhan X. Engleka K. Forough R. Maciag T. Proc Natl. Acad. Sci. U. S. A. 1992; 89: 10691-10695Crossref PubMed Scopus (226) Google Scholar). Human umbilical vein endothelial cells were grown as described previously (11Zimrin A.B. Pepper M.S. McMahon G.A. Nguyen F. Montesano R. Maciag T. J. Biol. Chem. 1996; 271: 32499-32502Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Conditioned medium was collected after temperature stress (42 °C for 2 h), processed by filtration, and activated with either (NH4)2SO4 (2Jackson A. Friedman S. Zhan X. Engleka K. Forough R. Maciag T. Proc Natl. Acad. Sci. U. S. A. 1992; 89: 10691-10695Crossref PubMed Scopus (226) Google Scholar) or 0.1% (w/v) dithiothreitol (DTT, Sigma) (4Jackson A. Tarantini F. Gamble S. Friedman S. Maciag T. J. Biol. Chem. 1995; 270: 33-36Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The DTT-treated conditioned medium was adsorbed directly to a 1-ml heparin-Sepharose CL-6B column (Amersham Pharmacia Biotech), previously equilibrated with 50 mmTris, pH 7.4, containing 10 mm EDTA (TEB), and the column was washed with TEB and eluted with TEB containing increasing concentrations of NaCl. The elution fractions were concentrated using a Centricon 10 concentrator (Amicon, Inc.) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, as described previously (4Jackson A. Tarantini F. Gamble S. Friedman S. Maciag T. J. Biol. Chem. 1995; 270: 33-36Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The (NH4)2SO4-treated conditioned medium (90% saturation) was recovered by centrifugation (9,000 ×g, 40 min), resuspended in TEB, and processed over a 4-ml heparin-Sepharose column previously equilibrated with TEB. The column was washed with TEB and batch-eluted as 10-ml fractions with TEB containing increasing concentrations of NaCl. Each post-heparin elution fraction was divided into two samples to be analyzed by immunoblotting with either the anti-FGF-1 or the anti-Syn-1 antibodies. Each sample was divided again into two fractions of equal volume, concentrated using a Centricon 10 concentrator (Amicon, Inc.) to a final volume of 200 μl. Of the first 200 μl, half was resuspended in an equal volume of SDS-PAGE sample buffer (125 mm Tris, 2% SDS, 10% glycerol, and 0.1% bromphenol blue) containing 2-mercaptoethanol (2-ME) and resolved by 15% (w/v) conventional SDS-PAGE, and half was resuspended in SDS-PAGE sample buffer without 2-ME and resolved by 15% conventional SDS-PAGE. The second 200 μl were also divided in half, resuspended in an equal volume of SDS-PAGE sample buffer with and without 2-ME, and resolved using a limited denaturant electrophoretic gel shift system (15% (w/v) acrylamide), where SDS was present in the sample and running buffers but eliminated from the resolving gel (limited SDS-PAGE). Immunoblot analysis of the samples resolved by conventional and limited SDS-PAGE was performed as described previously (2Jackson A. Friedman S. Zhan X. Engleka K. Forough R. Maciag T. Proc Natl. Acad. Sci. U. S. A. 1992; 89: 10691-10695Crossref PubMed Scopus (226) Google Scholar). Briefly, after transfer to nitrocellulose filters, the filters were incubated in 24 mm Tris, pH 7.4, containing 136 mm NaCl, 2 mm KCl, and 0.1% (v/v) Tween 20 (TCB) which also contained 5% (w/v) milk for 2 h at 42 °C and were then washed 3 times with TCB. The filters were probed with either a rabbit anti-human FGF-1 antibody (12Sano H. Forough R. Maier J.A. Case J.P. Jackson A. Engleka K. Maciag T. Wilder R.L. J. Cell Biol. 1990; 110: 1417-1426Crossref PubMed Scopus (161) Google Scholar) or with a rabbit anti-rat Syn-1 antibody, at a concentration of 1 μg/ml, for 1 h at room temperature and then washed 3 times with TCB. The proteins were detected using the ECL detection system (Amersham Pharmacia Biotech) following the manufacturer's instructions. A rat Syn-1 construct (p65IIs) encoding amino acid residues 96–421 in the expression vector pGEX-KG and two additional constructs, p65–123 (encoding amino acid residues 96–265) and p65–345 (encoding amino acid residues 248–421), as glutathione S-transferase fusion proteins were kindly provided by Dr. Richard Scheller (13Bennett M.K. Miller K.G. Scheller R.H. J. Neurosci. 1993; 13: 1701-1707Crossref PubMed Google Scholar) and used for the preparation of antisera. The fusion proteins were generated and purified as described (13Bennett M.K. Miller K.G. Scheller R.H. J. Neurosci. 1993; 13: 1701-1707Crossref PubMed Google Scholar) except that a 50 mm Tris buffer, pH 8.8, containing 10 mm EDTA, 10 mm glucose, and 10 μg/ml lysozyme was used to lyse BL21 Escherichia coli. The thrombin-cleaved proteins were further purified by adsorption to heparin-Sepharose previously equilibrated in 50 mm phosphate buffer, pH 7.5, and eluted with a 0–1.5m NaCl gradient in 50 mm phosphate buffer, pH 7.5. Female, 6-month-old, New Zealand White rabbits (Hazelton Research Animals) were injected intradermally with 1 mg of protein suspended in Freund's complete adjuvant (Calbiochem), boosted with 200 μg of protein suspended in incomplete Freud's adjuvant (Calbiochem), and the antibodies purified by incubating the serum with immobilized p65IIs on a Problot polyvinylidene difluoride membrane (Applied Biosystems, Inc.) previously blocked at 42 °C with TCB containing 5% (w/v) bovine serum albumin. The membrane was washed 3 times with 0.05% (v/v) Triton X-100 in 50 mm Tris, pH 7.4, containing 150 mmNaCl, and the antibody was eluted from the membrane with 0.2m glycine, pH 2.8, and neutralized with 1 mK2HPO4, pH 7.4. For the analytical study of the recombinant Syn-1 heparin-binding affinity, 1 μg of thrombin-cleaved, p65IIs recombinant protein was adsorbed to a 1-ml heparin-Sepharose column, previously equilibrated with TEB. The column was washed with TEB and batch-eluted with 2.5 ml of TEB containing increasing concentrations of NaCl. The post-heparin elution fractions were concentrated by Centricon to a final volume of 50 μl and analyzed by conventional SDS-PAGE, under reducing conditions. The protein was detected by immunoblot analysis with a rabbit anti-rat Syn-1 antibody using the ECL detection system. Total RNA from FGF-1-transfected NIH 3T3 cells and human umbilical vein endothelial cells (HUVEC) was isolated using the guanidinium isothiocyanate method (14Samuel C.E. Pharmacol. Ther. 1992; 54: 307-317Crossref PubMed Scopus (18) Google Scholar). As described previously (15Garfinkel S. Haines D.S. Brown S. Wessendorf J. Gillespie D.H. Maciag T. J. Biol. Chem. 1992; 267: 24375-24378Abstract Full Text PDF PubMed Google Scholar), cDNA was obtained from 1 μg of total RNA and diluted to a final volume of 500 μl with H2O. PCR was performed on 5 μl of cDNA in a 10 mm Tris buffer, pH 8.3, containing 1 mmMgCl2, 50 mm KCl, 0.2 mm dNTPs, 2 units of Taq polymerase, and 0.5 μg/each of sense (5′-CCATTGCCACCGTGGGCCTT-3′) and antisense (5′-TCCAAAACAGTTACCACCAC-3′) oligonucleotide primers designed to recognize the human and the mouse Syn-1 sequences. PCR was performed for 35 cycles as follows: 1 min at 94 °C, 2 min at 54 °C, and 3 min at 72 °C. The PCR products were separated from the oligonucleotide primers, unincorporated nucleotides, and Taq polymerase using a Qiagen tip-5 column according to the manufacturer's instructions (Qiagen PCR purification kit, Qiagen Inc.) and cloned into a pCR 2.1 vector (TA cloning kit, Invitrogen) following the manufacturer's instructions. The identity of the PCR fragments was then confirmed by sequencing using the dideoxy sequencing method (Sequenase, U. S. Biochemical Corp.). For the Syn-1 antisense gene construct, a rat Syn-1 gene into the PCB1 vector (a gift from T. C. Sudhof (16Perin M.S. Fried V.A. Mignery G.A. Jahn R. Sudhof T.C. Nature. 1990; 345: 260-263Crossref PubMed Scopus (648) Google Scholar)) was used to obtain the Syn-1 open reading frame that was cloned into a pCRTM II cloning vector (Invitrogen) by PCR method, using the following primers: 5′-GGGAGACACGGGATCCTTACTTCTTGACAGCCAG-3′ (sense oligonucleotide, containing a BamHI restriction site) and 5′-GCGACAGGAGCATATGGTGAGTGCCAGTCATCC-3′ (antisense oligonucleotide, containing a NdeI restriction site). A fragment encompassing the AUG initiator codon and the first 640 nucleotides of the rat Syn-1 sequence was obtained from the Syn-1/pCRTM II construct by digestion with the restriction enzyme EcoRI, filling with DNA polymerase I large (Klenow) fragment (Boehringer Mannheim) to obtain blunt ends, and a second digestion with KpnI restriction enzyme. The product was purified by agarose gel electrophoresis and ligated (T4 ligase, Life Technologies, Inc.) in an antisense direction into HpaI/KpnI compatible ends of the eukaryotic expression vector p3′SS (LAC switch inducible mammalian expression system, Stratagene). The anti-Syn-1/p3′SS vector was transfected into NIH 3T3 FGF-1 transfectants using the calcium phosphate precipitation method. DMEM, containing 200 μg/ml hygromycin (Boehringer Mannheim) and 20% bovine calf serum, was used to select hygromycin-resistant colonies. Colonies were screened for the expression of the anti-Syn-1 gene by Northern blot analysis using the anti-Syn-1 fragment as probe. The FGF-1 transfectants and the FGF-1 and anti-Syn-1 co-transfectants were subjected to temperature stress as described previously (2Jackson A. Friedman S. Zhan X. Engleka K. Forough R. Maciag T. Proc Natl. Acad. Sci. U. S. A. 1992; 89: 10691-10695Crossref PubMed Scopus (226) Google Scholar). Conditioned media were treated with 0.1% DTT and adsorbed over a 1-ml heparin-Sepharose column previously equilibrated with TEB. The column was washed with TEB and the protein eluted with TEB containing 1.5m NaCl. The elution fractions were concentrated by Centricon, analyzed by conventional SDS-PAGE under reducing conditions, and immunoblotted with anti-FGF-1 antibody as described previously. The proteins recognized by the antibody were detected with an125I-protein A probe. For the growth assay experiments, FGF-1 transfectants and FGF-1 and anti-Syn-1 co-transfectants were plated on 6-well cell culture dishes at a concentration of 2 × 103 cells/well in DMEM, containing increasing concentrations of bovine calf serum (0–20%). When the effect of FGF-1 on the growth curve was analyzed, 5 ng/ml recombinant FGF-1 and 4 units/ml heparin were added to the culture medium. At day 7 the cells were trypsinized and counted with a Coulter counter. FGF-1 was purified by heparin-Sepharose affinity from bovine brain as described previously (8Burgess W.H. Mehlman T. Friesel R. Johnson W. Maciag T. J. Biol. Chem. 1985; 260: 11389-11392Abstract Full Text PDF PubMed Google Scholar). The presence of a second heparin-binding protein within the FGF-1 fraction, which eluted with FGF-1 at 1.0 m NaCl, was detected by reversed phase high pressure liquid chromatography. The co-eluted protein was characterized as a 40-kDa polypeptide by SDS-polyacrylamide gel electrophoresis and subjected to automated Edman degradation. Interestingly, the amino-terminal sequence of the p40 fragment corresponded to the extravesicular domain of synaptotagmin-1 (Syn-1) (9Perin M.S. Brose N. Jahn R. Sudhof T.C. J. Biol. Chem. 1991; 266: 623-629Abstract Full Text PDF PubMed Google Scholar), starting in the α-helical stretch proximal to its transmembrane domain (Fig.1 A). Syn-1 has been described as a neuronal-specific, integral component of synaptic vesicles (9Perin M.S. Brose N. Jahn R. Sudhof T.C. J. Biol. Chem. 1991; 266: 623-629Abstract Full Text PDF PubMed Google Scholar). It is considered to be the calcium sensor of the regulated neurotransmitter secretory pathway, and it displays potential docking properties through its syntaxin-binding domain (17Geppert M. Goda Y. Hammer R.E. Li C. Rosahl T.W. Stevens C.F. Sudhof T.C. Cell. 1994; 79: 717-727Abstract Full Text PDF PubMed Scopus (1207) Google Scholar, 18Sollner T. Whiteheart W.W. Brunner M. Erdjument-Bromage H. Geromanos S. Tempst P. Rothman J. Nature. 1993; 362: 318-324Crossref PubMed Scopus (2611) Google Scholar). Interestingly, Syn-1 is also able to interact with the clathrin AP-2 adaptor, participating in the clathrin-coated pit endocytotic pathway (10Zhang J.Z. Davletov B.A. Sudhof T.C. Anderson R.G.W. Cell. 1994; 78: 751-760Abstract Full Text PDF PubMed Scopus (431) Google Scholar). Since Syn-1 expression is considered to be neuron-specific, we examined two non-neuronal cell culture systems for the presence of the Syn-1 transcript. Analysis of human umbilical vein endothelial cells and FGF-1 NIH 3T3 cell transfectants by reverse transcription-polymerase chain reaction (RT-PCR) using human and murine Syn-1 primers demonstrated the presence of the Syn-1 transcript in both cell types (Fig. 1 B). Sequence analysis of the human and murine RT-PCR products confirmed the identity of these fragments as human and murine Syn-1 (data not shown). In order to define further the mechanism utilized by FGF-1 for release, we employed stable FGF-1-transfected NIH 3T3 cells to determine whether the p40 proteolytic fragment of Syn-1 was present in medium conditioned by temperature stress. FGF-1 and Syn-1 immunoblot analysis was performed on heparin-Sepharose fractions of medium conditioned by FGF-1 transfectants during heat shock (42 °C, 2 h). Since FGF-1 is released as a FGF-1 Cys-30 homodimer (4Jackson A. Tarantini F. Gamble S. Friedman S. Maciag T. J. Biol. Chem. 1995; 270: 33-36Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), the conditioned medium was initially treated with 0.1% w/v dithiothreitol (DTT) and resolved by heparin affinity-based chromatography prior to conventional immunoblot analysis under reducing conditions. As shown in Fig.2 A, the FGF-1 monomer was detected in the high NaCl elution fractions (0.6 to 1.5 mNaCl). Because the FGF-1 homodimer has been shown to elute from immobilized heparin near 0.4 m NaCl (19Engleka K.A. Maciag T. J. Biol. Chem. 1992; 267: 11307-11315Abstract Full Text PDF PubMed Google Scholar) and FGF-1 was undetectable in the low NaCl elution fractions (Fig. 2 A), we suggest that DTT treatment of media conditioned by heat shock was effective in reducing the FGF-1 homodimer to monomer. In addition, Syn-1 immunoblot analysis of the same heparin-Sepharose elution fractions by conventional SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions also resolved the presence of a p40 fragment of Syn-1 in the high NaCl elution fractions (0.8 to 1.0m NaCl) (Fig. 2 B). These data suggest that p40 Syn-1 and FGF-1 were both present in DTT-treated heat shock conditioned medium from FGF-1-transfected NIH 3T3 cells and define their respective heparin-Sepharose elution profiles. Since Syn-1 has been previously described as a heparin-binding protein with very high heparin affinity, eluting from immobilized heparin near 1.5 m NaCl (20Nishiki T. Kamata Y. Nemoto Y. Omori A. Ito T. Takahashi M. Kozaki S. J. Biol. Chem. 1994; 269: 10498-10503Abstract Full Text PDF PubMed Google Scholar), we sought to confirm its heparin-binding characteristics. A rat p40 Syn-1 fragment encoding amino acid residues 96–421, which is very similar to the extravesicular domain of the p40 Syn-1 fragment isolated from bovine brain (Fig. 1), was expressed as a prokaryotic recombinant protein, and its heparin affinity was examined. As shown in Fig.3, the recombinant form of rat Syn-1 eluted from heparin-Sepharose between 0.6 and 0.8 m NaCl (Fig. 3). These data demonstrate that treatment of heat shock conditioned medium with DTT generates a p40 fragment of Syn-1 with heparin binding affinity similar to the prokaryotic recombinant p40 fragment. It has been demonstrated previously that the latent, dimeric form of FGF-1 released into conditioned medium under temperature stress is not biologically active in mitogenic assays and requires activation with either DTT or (NH4)2SO4 to restore its mitogenic potential (4Jackson A. Tarantini F. Gamble S. Friedman S. Maciag T. J. Biol. Chem. 1995; 270: 33-36Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Since DTT treatment of medium conditioned by heat shock is able to generate a p40 Syn-1 fragment with similar heparin affinity as that exhibited by the recombinant p40, we sought to determine the heparin elution profile of extracellular FGF-1 and p40 Syn-1 when medium conditioned by temperature stress was treated with (NH4)2SO4. The samples obtained from heparin-Sepharose elution after treatment of conditioned medium with (NH4)2SO4 were independently examined for the presence of FGF-1 (Fig.4) and Syn-1 (Fig.5) by immunoblot analysis. Whereas immunoblot analysis performed after conventional SDS-PAGE under reducing conditions revealed the presence of monomeric FGF-1 in the low and high NaCl elution fractions (Fig. 4 A), the appearance of p40 Syn-1 was limited to the low NaCl elution fractions (Fig.5 A). In contrast, FGF-1 (Fig. 4 B) immunoblot analysis performed after conventional SDS-PAGE under non-reducing conditions demonstrated that the presence of the FGF-1 monomer was restricted to the high NaCl elution fractions and revealed the presence of high molecular weight FGF-1 immunoreactive bands in the low NaCl elution fractions (Fig. 4 B). Similarly, Syn-1 immunoblot analysis of the heat shock-conditioned, post-heparin-Sepharose samples resolved by conventional SDS-PAGE under non-reducing conditions (Fig.5 B) also detected the presence of high molecular weight bands in the low NaCl elution fractions. These data indicate the following: (i) a p40 fragment of Syn-1, as well as FGF-1, is detected in the low NaCl elution samples of conditioned media treated with (NH4)2SO4, but not DTT, and (ii) the electrophoretic mobility of both FGF-1 and p40 Syn-1 present in the low salt elution fractions is sensitive to the presence of reducing agent. Thus, it is likely that the monomeric forms of FGF-1 and Syn-1 detected as high affinity heparin-binding proteins in the high salt elution fractions of conditioned medium following DTT treatment may represent the reduced products of the low affinity heparin-binding forms of FGF-1 and Syn-1 present in the low NaCl elution fractions from medium treated with (NH4)2SO4.Figure 5Syn-1 immunoblot analysis of reducing agent- and denaturant-sensitive forms of Syn-1 in medium conditioned by heat shock after (NH4)2SO4 treatment and heparin-Sepharose affinity chromatography. Heat shock CM from NIH 3T3 FGF-1 transfectants was collected, treated with (NH4)2SO4, and processed over a heparin-Sepharose column as described under “Experimental Procedures.” Fractions were collected, divided into two samples, and concentrated by Centricon to a final volume of 200 μl as described under “Experimental Procedures.” The first sample" @default.
• W2024646520 created "2016-06-24" @default.
• W2024646520 creator A5001998019 @default.
• W2024646520 creator A5002729616 @default.
• W2024646520 creator A5022595479 @default.
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• W2024646520 date "1998-08-01" @default.
• W2024646520 modified "2023-09-30" @default.
• W2024646520 title "The Extravesicular Domain of Synaptotagmin-1 Is Released with the Latent Fibroblast Growth Factor-1 Homodimer in Response to Heat Shock" @default.
• W2024646520 cites W135922689 @default.
• W2024646520 cites W1510595339 @default.
• W2024646520 cites W1528843291 @default.
• W2024646520 cites W1529696685 @default.
• W2024646520 cites W1545699847 @default.
• W2024646520 cites W1576276646 @default.
• W2024646520 cites W1762647466 @default.
• W2024646520 cites W1768439806 @default.
• W2024646520 cites W1967817609 @default.
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