Apo B100 similarities to viral proteins suggest basis for LDL-DNA binding and transfection capacity
2010; Elsevier BV; Volume: 51; Issue: 7 Linguagem: Inglês
10.1194/jlr.m003277
ISSN1539-7262
AutoresJuan Guevara, Nagindra Prashad, Boris S. Ermolinsky, John W. Gaubatz, Dongcheul Kang, Andrea E. Schwarzbach, David S. Loose, Juan Guevara,
Tópico(s)interferon and immune responses
ResumoLDL mediates transfection with plasmid DNA in a variety of cell types in vitro and in several tissues in vivo in the rat. The transfection capacity of LDL is based on apo B100, as arginine/lysine clusters, suggestive of nucleic acid-binding domains and nuclear localization signal sequences, are present throughout the molecule. Apo E may also contribute to this capacity because of its similarity to the Dengue virus capsid proteins and its ability to bind DNA. Synthetic peptides representing two apo B100 regions with prominent Arg/Lys clusters were shown to bind DNA. Region 1 (0014Lys-Ser0160) shares sequence motifs present in DNA binding domains of Interferon Regulatory Factors and Flaviviridae capsid/core proteins. It also contains a close analog of the B/E receptor ligand of apo E. Region 1 peptides, B1-1 (0014Lys-Glu0054) and B1-2 (0055Leu-Ala0096), mediate transfection of HeLa cells but are cytotoxic. Region 2 (3313Asp-Thr3431), containing the known B/E receptor ligand, shares analog motifs with the human herpesvirus 5 immediate-early transcriptional regulator (UL122) and Flaviviridae NS3 helicases. Region 2 peptides, B2-1 (3313Asp-Glu3355), and B2-2 (3356Gly-Thr3431) are ineffective in cell transfection and are noncytotoxic. These results confirm the role of LDL as a natural transfection vector in vivo, a capacity imparted by the apo B100, and suggest a basis for Flaviviridae cell entry. LDL mediates transfection with plasmid DNA in a variety of cell types in vitro and in several tissues in vivo in the rat. The transfection capacity of LDL is based on apo B100, as arginine/lysine clusters, suggestive of nucleic acid-binding domains and nuclear localization signal sequences, are present throughout the molecule. Apo E may also contribute to this capacity because of its similarity to the Dengue virus capsid proteins and its ability to bind DNA. Synthetic peptides representing two apo B100 regions with prominent Arg/Lys clusters were shown to bind DNA. Region 1 (0014Lys-Ser0160) shares sequence motifs present in DNA binding domains of Interferon Regulatory Factors and Flaviviridae capsid/core proteins. It also contains a close analog of the B/E receptor ligand of apo E. Region 1 peptides, B1-1 (0014Lys-Glu0054) and B1-2 (0055Leu-Ala0096), mediate transfection of HeLa cells but are cytotoxic. Region 2 (3313Asp-Thr3431), containing the known B/E receptor ligand, shares analog motifs with the human herpesvirus 5 immediate-early transcriptional regulator (UL122) and Flaviviridae NS3 helicases. Region 2 peptides, B2-1 (3313Asp-Glu3355), and B2-2 (3356Gly-Thr3431) are ineffective in cell transfection and are noncytotoxic. These results confirm the role of LDL as a natural transfection vector in vivo, a capacity imparted by the apo B100, and suggest a basis for Flaviviridae cell entry. 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Apolipoprotein E-low density lipoprotein receptor interaction: influences of basic residue and amphipathic α-helix organization in the ligand.J. Lipid Res. 2000; 41: 1087-1095Abstract Full Text Full Text PDF PubMed Google Scholar, 28.Lalazar A. Weisgraber K.H. Rall Jr., S.C. Giladi H. Innerarity T.L. Levanon A.Z. Boyles J.K. Amit B. Gorecki M. Mahley R.W. et al.Site-specific mutagenesis of human apolipoprotein E.J. Biol. Chem. 1988; 263: 3542-3545Abstract Full Text PDF PubMed Google Scholar) is present in the structural proteins of these viruses. In previous reports, we showed that human LDL binds DNA and RNA in vitro (18.Guevara, J. G., Jr., Hoogeveen, R. C., Moore, J. P. 2003. Lipoproteins as nucleic acid vectors. US Patent 6,635,623 B1.Google Scholar, 20.Guevara Jr., J.G. Kang Dc. Moore J.P. Nucleic acid-binding properties of low-density lipoproteins: LDL as a natural gene vector.J. Protein Chem. 1999; 18: 845-857Crossref PubMed Google Scholar) and that highly purified LDL can be used to transport and deliver plasmid DNA to the cell nucleus. We surmised that the capacity of LDL and VLDL to bind nucleic acids is likely based on the presence of regions in the apo B100 molecule that display sequence similarities to known nucleic acid-binding domains (29.Fujii Y. Shimizu T. Kusumoto M. Kyogoku Y. Taniguchi T. Hakoshima T. Crystal structure of an irf -DNA complex reveals novel DNA recognition and cooperative binding to a tandem repeat of core sequences.EMBO J. 1999; 18: 5028-5041Crossref PubMed Scopus (175) Google Scholar). Based on the location of arginine and lysine clusters and other motifs, five potential DNA binding domains, 11 potential KH domains of the heterogeneous nuclear ribonucleoprotein K (30.Braddock D.T. Baber J.L. Levens D. Clore G.M. Molecular basis of sequence-specific single-stranded DNA recognition by KH domains: solution structure of a complex between hnRNP K KH3 and single-stranded DNA.EMBO J. 2002; 21: 3476-3485Crossref PubMed Scopus (114) Google Scholar, 31.Grishin N.V. KH domain: one motif, two folds.Nucleic Acids Res. 2001; 29: 638-643Crossref PubMed Scopus (213) Google Scholar), and numerous bipartite nuclear localization signal sequences (NLS) were identified in the apo B100 primary structure (18.Guevara, J. G., Jr., Hoogeveen, R. C., Moore, J. P. 2003. Lipoproteins as nucleic acid vectors. US Patent 6,635,623 B1.Google Scholar, 20.Guevara Jr., J.G. Kang Dc. Moore J.P. Nucleic acid-binding properties of low-density lipoproteins: LDL as a natural gene vector.J. Protein Chem. 1999; 18: 845-857Crossref PubMed Google Scholar). One candidate DNA-binding region is contained in the first 100 N-terminal residues of the apo B100. Several sequence motifs also present in the DNA-binding domains of interferon regulatory factors (29.Fujii Y. Shimizu T. Kusumoto M. Kyogoku Y. Taniguchi T. Hakoshima T. Crystal structure of an irf -DNA complex reveals novel DNA recognition and cooperative binding to a tandem repeat of core sequences.EMBO J. 1999; 18: 5028-5041Crossref PubMed Scopus (175) Google Scholar), the interferon regulatory factor (irf-s), are located in this region. In this report, we explore the hypothesis that apo B100 and apo E have structural and functional similarities to viral DNA-binding proteins. We focus on two candidate nucleic acid-binding regions: N-terminus of apo B100, residues 0014Lys-Ala0096 (Region 1) and the section encompassing the known B/E-receptor ligand of apo B100, residues 3313Asp-Thr3431 (Region 2) (32.Yang C-Y. Chen S-H. Gianturco S.H. Bradley W.A. Sparrow J.T. Tanimura M. Li W-H. Sparrow D.A. DeLoof H. 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Our hypothesis compels us to consider that LDL and LDL-related particles, intermediate and very low density particles, are involved in transporting nucleic acids, and this capacity is imparted by specific regions of the apo B100 as well as apo E. Here we present additional evidence that LDL and VLDL have the capacity to bind DNA. Further, LDL can be used to transfect a variety of cell types in vitro and in vivo. Based on the results of experimental studies utilizing synthetic peptides from Regions 1 and 2 of apo B100, we conclude that this capacity is mediated by elements in the apo B100 primary structure similar to viral proteins. All chemicals were of highest quality available. Water of 18.2 MΩ·cm resistivity was used throughout these studies. Tap water was passed through a mixed-resin filter, distilled, and filtered using a Barnstead E-pure system. Molecular biology grade Agarose was purchased from Fisher Scientific, Inc. PBS was obtained from MediaTech, Inc., Herndon VA, or other chemical suppliers. Tris-acetate (TA) buffer, DNA sequence grade, was obtained from TECNOVA, Inc. Nucleic acid sample loading buffer was from Bio-Rad, Inc. BOBO1TM-iodide (B-3582) and Cell TrackerTM CM-DiI (C-7001) fluorescence dyes, and LipofectinTM were purchased from Invitrogen, Inc. Synthetic peptides, of 95% or greater purity by HPLC analysis, were purchased from GenScript, Corp., Plasmid vectors, pCMV β-Gal, pEGFP-N1, and pEGFP-N2, were from ClonTech, Inc. Plasmids, phMGFP, pGL2-Control, pCMVTNT®, and restriction enzymes StuI and HindIII were obtained from Promega, Inc. Cell culture media, DMEM, and HyQ-RPMI 1640 were obtained from MediaTech, Inc. and Thermo Fisher Scientific, Inc. Transfection Reagent 1, DOTAP: DOPE (1:1) was purchased from Avanti Polar Lipids, Inc. QIAamp DNA Mini kit was obtained from QIAGEN, Inc. PCR reagents were obtained from Roche Biochemicals, Inc. DNA Molecular Weight standards were purchased from Roche Applied Science, Inc. Highly purified preparations of human plasma LDL were obtained from Invitrogen, Inc. and Athens Research and Technology, Athens, GA. LDL was also isolated from human plasma by sequential ultracentrifugation in NaBr solutions yielding the 1.019–1.05 g/ml density range of LDL fraction (6.Chapman M.J. Laplaud P.M. Luc G. Forgez P. Bruckert E. Goulinet S. LaGrange G. Further resolution of the low density lipoprotein spectrum in normal human plasma: physicochemical characteristics of discrete subspecies separated by density gradient ultracentrifugation.J. Lipid Res. 1988; 29: 442-458Abstract Full Text PDF PubMed Google Scholar). Single donor plasma with sodium EDTA added at time of collection was obtained from Innovative Research. Typically, purified preparations of LDL were dialyzed in PBS containing 10 mM MgCl2. LDL was than evaluated for purity using SDS-PAGE, and tested for DNA binding capacity using electrophoretic mobility shift assay (EMSA). Once DNA binding capacity of the LDL preparation was confirmed, it was frozen drop-wise in liquid nitrogen and stored in aliquots of about 200 µL in liquid nitrogen until needed. Highly purified, delipidated apo E was purchased from Athens Research and Technology, Inc. Female Sprague-Dawley rats, 8–10 weeks old, were obtained from Harland Laboratories, Inc. Animals were housed in the National Institutes of Health-accredited facilities in the University of Texas Health Science Center and Baylor College of Medicine. All animals were treated in accordance with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH publication no. 85-23, revised in 1996). All animal protocols were approved by the Animal Welfare Committees at the University of Texas Health Science and Baylor College of Medicine. Rat plasma was used to purify LDL. To obtain blood, animals were first sedated using inhalation anesthetic metaphane. This was followed by an injection of a combination anesthetic containing ketamine (42.8 mg/ml), xylazine (8.6 mg/ml), and acepromazine (1.4 mg/ml) in PBS. Blood was then collected by heart puncture using a 5 ml syringe containing 50 µl of 100 mM EDTA. Approximately 4–5 ml of blood was collected per animal. Samples were pooled and centrifuged at 400 rpm for 20 min. LDL was isolated using equilibrium ultracentrifugation in preformed gradient of KBr. Collected plasma, 9–10 ml from six rats, was diluted to 12 ml with saline, then the density of the sample was adjusted to 1.21 g/ml with KBr. Plasma was transferred to centrifuge tubes of SW 40 Ti swinging bucket rotor (Beckman), 4 ml per tube, overlaid with KBr solutions of the following densities: 1.063 g/ml (3.0 ml), 1.02 g/ml (3.0 ml), and 1.006 g/ml (2.5 ml). Samples were next centrifuged at 39,000 rpm for 60 h at 4°C. Seven fractions were collected from each tube, starting from the top of the centrifuge tube, and similar fractions from different centrifuge tubes were pooled then dialyzed against PBS. Protein concentration was routinely determined using modified Lowry method (18.Guevara, J. G., Jr., Hoogeveen, R. C., Moore, J. P. 2003. Lipoproteins as nucleic acid vectors. US Patent 6,635,623 B1.Google Scholar), and SDS-PAGE (2–12%) was performed. LDL fraction was identified by presence of apo B100 band. An aliquot of plasmid DNA digested using restriction enzymes was placed at the bottom of a microfuge tube; next, a buffer solution containing 25 mM TA, pH 7.6, and 10 mM MgCl2 was added. An aliquot of purified lipoprotein (LDL, VLDL, apo E) or synthetic peptides was then added; the cocktail was stirred gently for less than 5 s and incubated for 30 min at 37°C. Sample loading buffer (Bio-Rad) was then added at a 1:5 (v/v) ratio to the polypeptide-DNA mix. Next, each sample was mixed and subjected to electrophoresis using 0.5–0.8% agarose gels in TA buffer at 100 V. Aliquots of nucleic acid, synthetic peptides, and lipoprotein were each analyzed in separate lanes as controls. DNA bands were visualized using ethidium bromide. Peptides and proteins in lipoprotein particles were visualized using Coomassie brilliant blue R-250 (CBB). All cell types used in these studies were obtained from the American Type Culture Collection Organization. Cells were kept in liquid nitrogen until needed and then grown according to American Type Culture Collection protocols. Several cell types, NIH/3T3 (mouse embryonic fibroblast cell line, CRL-1658TM), CHO (chinese hamster ovary, CCL-61TM), MCF7 (human breast adenocarcinoma cell line, HTB-22TM), Hep G2 (human hepatocyte carcinoma cells, HB-8065TM), and HeLa (human cervix epithelial adenocarcinoma cell line, CCL-2TM), were used for LDL- and apo B100 synthetic peptide-mediated transfection. Cells were grown and maintained in media as follows: HeLa, Hep G2, MCF7, and NIH/3T3 cell types were in DMEM supplemented with 10% FBS, 100 units penicillin G-sodium, 100 units/ml streptomycin sulfate, and 250 ng/ml amphotericin B. CHO cells were in RPMI-1640 containing 10% FBS, l-glutamine, and 100 units penicillin G-sodium, 100 units/ml streptomycin sulfate, and 250 ng/ml amphotericin B. Cells were maintained at 37°C in an atmosphere of 5% CO2 in a humidified incubator. Typically, cells were grown in culture plates (with or without glass cover slip in wells) to 60–70% confluence (20.Guevara Jr., J.G. Kang Dc. Moore J.P. Nucleic acid-binding properties of low-density lipoproteins: LDL as a natural gene vector.J. Protein Chem. 1999; 18: 845-857Crossref PubMed Google Scholar). Prior to transfection, the medium was removed, and cells were washed thrice with PBS. Cells were then incubated for a minimum of 2 h but not more than 4 h in FBS-free medium. Dual label experiments were conducted using Hep G2 cells. The cells were grown overnight as described above on FBS-coated cover slips to enhance attachment. Cells were next washed with PBS and incubated in FBS-free medium for 4 h, then incubated for 3 h in 200 µl of transfection solution containing FBS-free DMEM, 10 mM MgCl2, and preformed complexes of BOBO-1-labeled pCMV β-Gal plasmid DNA (3 µg), and either 15 µL of CM-DiI-labeled LipofectinTM or 60 µg of CM-DiI-labeled LDL. The cell-coated cover slips were then removed, washed in PBS, and fixed in 4% paraformaldehyde for 10 min at 4°C. Each cover slip was then inverted over a well of a hanging drop slides containing PBS and viewed using an Olympus Model BH-2 fluorescent microscope. Similar methods were used to study LDL-mediated transfection of CHO, NIH3T3, and HeLa cells using BOBO-1-labeled plasmid DNA. CHO and NIH3T3 cells were grown on cover slips and transfected using LDL complexed to BOBO-1-pEGFP-N1 DNA as described above, except LDL was not labeled. The cover slips were recovered at different periods and were inverted over on well slides containing PBS without paraformaldehyde treatment. Cell images were obtained using a LUMAMTM EPI-Fluorescent microscope. Similarly, HeLa cells were grown in Costar® multi-well, flat bottom polystyrene plates and transfected using solutions containing LDL complexed to BOBO-1-pCMVTNT DNA. Results of HeLa cell transfection were documented using a Zeiss Axiovert 25 microscope. Nonlabeled LDL complexed to the nonlabeled pEGFP-N1 plasmid was used to transfect HeLa, MCF 7, CHO, and NIH/3T3 cell types as described above, and green fluorescence protein (GFP) expression was documented using a LUMAMTM fluorescence microscope with a GFP filter. HeLa cells were used also to ascertain the transfection capacity of two sets of synthetic peptides. Cells were grown as routine, preconditioned in FBS-free DMEM for 4 h, washed thrice with PBS, and 400 µl PBS supplemented with 10 mM MgCl2, containing either the synthetic peptides or peptide/DNA complexes was added. Cells were incubated at 37°C as described in Methods for 30 min, then washed, and Trypan Blue dye in PBS was added. Labeling of DNA with BOBO-1TM-iodide was accomplished according to the methods described by the vendor (Molecular Probes dimeric cyanine nucleic acid stains) with minor modification. Briefly, 3 µl of 10 µM dye (1 mM stock solution diluted 1:100 with ethanol) was added to 10 μg of DNA at a concentration of 0.5 μg/μl in PBS, and the solution was incubated at ambient temperature for 1 h. Labeling of LipofectinTM and LDL with Cell TrackerTM CM-DiI was performed according to the methods described by the vendor (Molecular Probes ). Transfection agents were labeled by adding 1 µl of 20 µg CM-DiI/ml in ethanol (stock solution) to 100 µL of Lipofectin (undiluted reagent) or LDL (1.5 mg/ml by protein), and the mixture was incubated for 1 h at ambient temperature. Unreacted dye was removed using a Sephadex G-25 column. Transfection Reagent 1 (10 mg, Avanti Polar Lipids, Inc.) was dissolved in 2 ml of a sterile buffer solution of 0.9% NaCl, 5.0% glucose, and 10% sucrose. The suspension was placed in a 37°C water bath for 10 min and vortexed to disperse the opaque lipid vesicles. Small unilamellar vesicles (SUV) were then formed by sonicating the mix for about 3 min. The transparent SUV mix was concentrated to approximately 11 mg/ml using an Amicon/Microcon filter concentrator with a YM-3 membrane (Sigma, Inc.). The solution was then sterilized through a 0.22 micron filter (Millipore, Inc.). All LDL preparations used in this study were shown to bind DNA in EMSA prior to cell transfection experiments. Plasmids pCMV β-Gal, pEGFP-N1, pEGFP-N2, phMGFP, pGL2-Control, and pCMVTNT® bound LDL in a similar manner. For cell transfection, purified LDL was added to the microfuge tube containing DNA and PBS with 10 mM MgCl2. The mixtures were then incubated at 37°C for 30 min before use. Typically, 20–40 µg of LDL protein was complexed with 1.0 µg DNA. Ten microliters of reagent in 100 µl culture medium was combined with 100 µl culture medium containing 10 µg DNA. The solution was then stirred gently and incubated for at least 30 min at room temperature. Transfection Reagent 1 was prepared as described above. The SUV preparation was combined with plasmid DNA (10:1) in TE buffer and incubated at ambient temperature for 5 min before use. Amino acid sequences of the peptides are listed in Table 1 (Region 1) and Table 3 (Region 2). Peptides were received in lyophilized form and 1 mg/ml solutions were prepared in PBS/10 mM MgCl2 by vortexing. Solutions were frozen drop wise in liquid nitrogen and stored at −80°C until use. Aliquots were thawed and sterilized using a 0.2 micron filter immediately prior to use. In EMSA, plasmid DNA was mixed with 3, 6, and 12 µg of peptide individually or in a mixture representing a region of apo B100. Peptides were first mixed in equal amounts (v/v) and incubated at 37°C for 30 min. The peptide mix was then added to the DNA in a microtube after which PBS/MgCl2 buffer was adde
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