Recombinant derivatives of the human high-mobility group protein HMGB2 mediate efficient nonviral gene delivery Arjen Sloots and Winfried S. Wels Chemotherapeutisches Forschungsinstitut, Georg-Speyer-Haus, Frankfurt am Main, Germany Virus-based vectors have been the gene delivery vehi- cles of choice in most gene therapy approaches to date, and use of these vectors has led to significant successes in a number of clinical trials [1]. Nevertheless, recent adverse events in patients treated with different viral vectors have revived interest in alternative, nonviral delivery systems for gene therapy [2,3]. Although still less efficient than most viral vectors, nonviral gene delivery vehicles are not usually associated with serious safety concerns. In addition to synthetic nonviral vectors such as lipids and polycationic reagents, certain natural peptides and proteins are able to bind and condense plasmid DNA, a prerequisite for the formation of transfection-competent complexes [4]. Consequently, cellular DNA-binding proteins including histones [5–9] and high-mobility group (HMG) proteins [10,11] have been investigated for their potential as nonviral gene delivery reagents. In these studies, DNA-binding pro- teins were extracted from tissues such as calf thymus, which requires large amounts of starting material and can yield heterogeneous protein fractions that display reduced DNA-binding activity because of exposure to acid during purification [12,13]. Therefore recombinant Keywords gene delivery; high-mobility group protein; importin-a; nuclear localization signal; protein transduction domain Correspondence W. S. Wels, Chemotherapeutisches Forschungsinstitut, Georg-Speyer-Haus, Paul-Ehrlich-Straße 42–44, D-60596 Frankfurt am Main, Germany Fax: +49 69 63395 189 Tel: +49 69 63395 188 Email: wels@em.uni-frankfurt.de (Received 13 April 2005, revised 23 June 2005, accepted 24 June 2005) doi:10.1111/j.1742-4658.2005.04834.x Certain natural peptides and proteins of mammalian origin are able to bind and condense plasmid DNA, a prerequisite for the formation of transfec- tion-competent complexes that facilitate nonviral gene delivery. Here we have generated recombinant derivatives of the human high-mobility group (HMG) protein HMGB2 and investigated their potential as novel protein- based transfection reagents. A truncated form of HMGB2 encompassing amino acids 1–186 of the molecule was expressed in Escherichia coli at high yield. This HMGB2 186 protein purified from bacterial lysates was able to condense plasmid DNA in a concentration-dependent manner, and medi- ated gene delivery into different established tumor cell lines more efficiently than poly(l-lysine). By attaching, via gene fusion, additional functional domains such as the HIV-1 TAT protein transduction domain (TAT PTD - HMGB2 186 ), the nuclear localization sequence of the simian virus 40 (SV40) large T-antigen (SV40 NLS -HMGB2 186 ), or the importin-b-binding domain (IBB) of human importin-a (IBB-HMGB2 186 ), chimeric fusion pro- teins were produced which displayed markedly improved transfection effi- ciency. Addition of chloroquine strongly enhanced gene transfer by all four HMGB2 186 derivatives studied, indicating cellular uptake of protein–DNA complexes via endocytosis. The IBB-HMGB2 186 molecule in the presence of the endosomolytic reagent was the most effective. Our results show that recombinant derivatives of human HMGB2 facilitate efficient nonviral gene delivery and may become useful reagents for applications in gene therapy. Abbreviations eGFP, enhanced green fluorescent protein; HMG protein, human high-mobility group protein; IBB, importin-b-binding domain; NLS, nuclear localization sequence; PEI, polyethyleneimine; PTD, protein transduction domain; SV40, simian virus 40. FEBS Journal 272 (2005) 4221–4236 ª 2005 FEBS 4221 DNA-binding proteins such as human histone H1 expressed in bacteria and rat HMGB1 produced in yeast cells are suitable alternatives [12,14]. On its own the ability of a nonviral vector to con- dense DNA is not sufficient to mediate gene delivery with high efficiency. Eukaryotic cells are protected against the uptake of exogenous nucleic acids by a series of cellular barriers that must be overcome before a delivered gene can be expressed in the target cell nucleus. In particular, ineffective escape from endo- somal compartments and poor nuclear trafficking are considered major limiting factors for many nonviral gene-transfer systems [15,16]. The production of pro- tein-based gene-delivery vectors in recombinant form in principle allows their activities to be modified by inclu- ding heterologous sequences that help to overcome these cellular barriers by improving cellular uptake, endosome escape, and intracellular routing [17]. Here we report the construction of recombinant derivatives of the human nonhistone chromatin protein HMGB2 and their functional characterization as non- viral gene delivery vectors. Vertebrate HMGB proteins such as HMGB1 and HMGB2 are composed of three structurally defined regions [18,19]. They contain two homologous but distinct DNA-binding motifs, termed HMG boxes, and an acidic C-terminal domain. In human HMGB2, the HMG boxes A and B, inter- spaced by basic amino acids, are connected by another basic region to a stretch of 22 acidic amino acids at the C-terminus of the protein [20,21] (schematically shown in Fig. 1A). These basic regions together with some basic amino-acid residues at the N-terminus of HMG box A have been suggested to function as a nuclear localization signal (NLS) [22]. We generated a truncated HMGB2 derivative which lacks the acidic tail previously reported to decrease the affinity of HMG proteins for DNA [23]. This bacterially expressed HMGB2 186 fragment formed complexes with plasmid DNA, and mediated gene delivery into different established tumor cell lines more efficiently than poly(l-lysine). Further- more, by including additional functional domains such as the HIV-1 TAT protein transduction domain (PTD), the NLS of the simian virus 40 (SV40) large T-antigen, or the importin-b-binding domain (IBB) of human importin-a2, alternative gene delivery vec- tors were produced that displayed markedly enhanced transfection efficiency. A B Fig. 1. Construction and bacterial expression of HMGB2 186 . (A) Schematic representation of the human HMGB2 protein and the expression construct encoding truncated HMGB2 186 . Full-length HMGB2 consists of HMG box A, a linker region (L), HMG box B, a joiner region (J) and an acidic C-terminal tail. The bacterial expression vector pSW5-HMGB2 186 encodes under the control of the isopropyl b-D-thiogalactopyrano- side-inducible tac promoter (tac) amino acids 1–186 of human HMGB2 fused to C-terminal Myc (M) and polyhistidine (H) tags. (B) SDS ⁄ PAGE (lanes 1–4) and immunoblot analysis (lanes 5–8) of bacterial lysate (lanes 1, 5), flow through (lanes 2, 6), wash (lanes 3, 7) and eluate fraction (lanes 4, 8) during purification of HMGB2 186 by Ni 2+ affinity chromatography. HMGB2 186 was identified with Myc-tag-specific antibody 9E10 followed by horseradish peroxidase-coupled secondary antibody and chemiluminescent detection. Recombinant HMGB2 mediates gene delivery A. Sloots and W. S. Wels 4222 FEBS Journal 272 (2005) 4221–4236 ª 2005 FEBS Results Truncated human HMGB2 186 is readily expressed in bacteria For bacterial expression of a truncated derivative of HMGB2 that lacks the acidic tail, a cDNA fragment encoding amino acids 1–186 (HMGB2 186 ) was derived by PCR and inserted into the expression vector pSW5 [24]. In the resulting pSW5-HMGB2 186 plasmid, cyto- plasmic expression of HMGB2 186 fused to C-terminal Myc and His-tags is controlled by an isopropyl b-d-thio- galactopyranoside-inducible tac promoter (Fig. 1A). HMGB2 186 protein was expressed in Escherichia coli strain BL21(kDE3)trxB – [25] and purified from bacterial lysates under native conditions by Ni 2+ -affinity chro- matography as described in Experimental Procedures. In SDS⁄ PAGE analysis, purified HMGB2 186 could be detected as a single band with an apparent molecular mass slightly larger than calculated from its sequence (24.2 kDa; Fig. 1B). Similar results were obtained in immunoblot analysis with mAb 9E10 specific for the Myc tag included in the molecule, further confirming the identity of the recombinant protein (Fig. 1B). Recombinant HMGB2 186 binds to plasmid DNA and cell surfaces Binding of HMGB2 186 protein to plasmid DNA was investigated in a gel retardation experiment (Fig. 2A). Increasing amounts of purified HMGB2 186 were incu- bated with 0.5 lg reporter gene plasmid pCMS-eGFP- E2C-Luc. The electrophoretic mobility of the resulting protein–DNA complexes in 1% agarose gel was then determined. In the absence of HMGB2 186 , plasmid DNA migrated as expected, with supercoiled and relaxed forms as separate bands (Fig. 2A, lane 1). The addition of 0.7 or 1.4 lg HMGB2 186 already retarded the plasmid DNA substantially (Fig. 2A, lanes 3, 4), and with 2.8 lg or more, maximal retardation of plas- mid DNA was achieved (Fig. 2A, lane 5). In contrast, BSA, included as a control protein, had no effect on the electrophoretic mobility of plasmid DNA. Plasmid DNA in HMGB2 186 complexes containing 2.8 lgor more purified protein similar to DNA in poly(l-lysine) complexes was completely protected against degrada- tion by DNase I (data not shown). Another important determinant of successful gene delivery is the binding of DNA–vector complexes to target cells. This can occur by direct interaction of transfection complexes with integral components of the cell membrane, or by binding to other molecules expressed on the cell surface [15,16]. Binding of recom- binant HMGB2 186 to target cells was investigated by FACS analysis using human HeLa cells and COS-7 African green monkey kidney cells as a model. The cells were incubated with increasing concentrations of purified HMGB2 186 , and bound protein was detected with mAb 9E10 recognizing the Myc tag included in the molecule, followed by fluorescein isothiocyanate- conjugated or phycoerythrin-conjugated secondary anti- bodies. As shown in Fig. 2B, concentration-dependent and saturable binding of HMGB2 186 to the cell surface was detected, suggesting specific interaction with an as yet unidentified target molecule. HMGB2 186 facilitates gene delivery into COS-7 cells To investigate HMGB2 186 -mediated gene transfer, the pCMS-eGFP-E2C-Luc reporter gene plasmid was used which encodes enhanced green fluorescent protein (eGFP) and an optimized form of firefly luciferase under the control of the SV40 enhancer ⁄ promoter and the cytomegalovirus immediate early promoter, respectively. Transfection complexes containing increasing amounts of purified HMGB2 186 protein and 2.3 lg pCMS-eGFP- E2C-Luc DNA were added to COS-7 cells in standard growth medium with serum, and left on the cells for 4 h before the medium was exchanged. Cells were lysed 40 h later, and luciferase activity was measured. For compar- ison, control cells were transfected with poly(l-lysine)– DNA complexes containing 2.3 lg reporter plasmid and a 60-fold molar excess of poly(l-lysine) as described [26]. The results are shown in Fig. 2C. Concentration- dependent HMGB2 186 -mediated gene delivery was found, which was more efficient than poly(l-lysine)- mediated transfection at an HMGB2 186 amount of 21.5 lg (representing a protein ⁄ DNA mass ratio of 9.5) or higher. In this experiment, maximal reporter gene expression was reached with 43 lg HMGB2 186 , with luciferase activity eight times higher than in the poly (l-lysine) control. Interestingly, in COS-7 cells, effi- ciency of HMGB2 186 -mediated gene transfer decreased again at higher HMGB2 186 concentrations, possibly because of saturation of cell surface molecules occupied by excess free HMGB2 186 protein. To investigate the possible involvement of the endo- cytic pathway in the internalization of HMGB2 186 – DNA complexes, cells were also transfected in the presence of the endosomolytic agent chloroquine [27,28]. As shown in Fig. 2D, the efficiency of HMGB2 186 -mediated gene transfer into COS-7 cells was substantially increased by chloroquine. Luciferase A. Sloots and W. S. Wels Recombinant HMGB2 mediates gene delivery FEBS Journal 272 (2005) 4221–4236 ª 2005 FEBS 4223 activity was enhanced  15-fold and 55-fold for com- plexes containing 43 and 64.5 lg HMGB2 186 , respect- ively. Taken together, these data show that HMGB2 186 on its own is able to mediate nonviral gene delivery, and strongly suggest that HMGB2 186 –DNA complexes enter the cells through the endocytic pathway. Construction of HMGB2 186 derivatives containing the TAT PTD or the SV40 T-antigen NLS To investigate whether HMGB2 186 -mediated gene transfer can be improved by including in the molecule a cell-penetrating peptide, the PTD of the HIV-1 TAT A B CD Fig. 2. Functional characterization of purified HMGB2 186 . (A) DNA binding was analyzed by agarose gel electrophoresis of 0.5 lg pCMS- eGFP-E2C-Luc plasmid DNA in the absence of protein (lane 1), or after incubation with the indicated amounts of recombinant HMGB2 186 (lanes 2–5). The positions of supercoiled and open circular DNA, and protein–DNA complexes are indicated by arrows. (B) Binding of HMGB2 186 to the surface of HeLa cells (left panel) and COS-7 cells (middle panel) was investigated by FACS analysis. Cells were incubated with the indicated concentrations of purified HMGB2 186 protein. Then bound protein was detected with Myc-tag-specific antibody 9E10 fol- lowed by fluorescein isothiocyanate-conjugated (HeLa) or phycoerythrin-conjugated (COS-7) anti-mouse IgG. Control cells were incubated with antibodies in the absence of HMGB2 186 (open area). Mean fluorescence values (arbitrary units) were calculated from the COS-7 FACS experiments and plotted against the protein concentrations used (16.5 n M to 18.59 lM) (right panel). HMGB2 186 -mediated gene transfer into COS-7 cells in the absence (C) or presence of 100 l M chloroquine (D). Cells were seeded at a density of 7 · 10 4 cells per well 24 h before transfection. Transfection complexes were formed by incubating the indicated amounts of purified HMGB2 186 with pCMS-eGFP-E2C-Luc reporter plasmid before addition to the cells in normal growth medium (2.3 lg DNA per well). Control cells were treated with poly( L-lysine) (pL)–DNA complexes (open bar). After 4 h the medium was exchanged and cells were grown for another 40 h before they were harvested for analysis. Luciferase activity is expressed in relative light units (RLU)Æ(mg total protein) )1 . Recombinant HMGB2 mediates gene delivery A. Sloots and W. S. Wels 4224 FEBS Journal 272 (2005) 4221–4236 ª 2005 FEBS protein (amino acids 47–57) flanked by additional gly- cine residues was fused to the N-terminus of the HMGB2 186 fragment (Fig. 3A; TAT PTD sequence shown in Table 1). This TAT fragment also includes a nonclassical NLS [29]. Therefore, to examine the pos- sible effect of a heterologous NLS on HMGB2 186 - mediated gene transfer independent of cell-penetrating activity, a similar HMGB2 186 derivative was construc- ted which carries the classical NLS of the SV40 large T-antigen [30] at the N-terminus (Fig. 3A; SV40 NLS sequence shown in Table 1). TAT PTD -HMGB2 186 and SV40 NLS -HMGB2 186 proteins were expressed in E. coli and purified from bacterial lysates as described above for unmodified HMGB2 186 . As expected, in compari- son with HMGB2 186 , a slight increase in the apparent molecular mass was found for the fusion proteins in SDS ⁄ PAGE and immunoblot analysis with antibody against HMGB2 (Fig. 3B). A B C Fig. 3. (A, B) Bacterial expression of HMGB2 186 derivatives carrying the TAT PTD (TAT PTD ; amino acids 47–57) or the NLS of SV40 large T-antigen (SV40 NLS ). (A) Schematic representation of the TAT PTD -HMGB2 186 and SV40 NLS -HMGB2 186 expression cassettes also encoding C-terminal Myc (M) and polyhistidine (H) tags, inserted into plasmid pSW5. (B) SDS ⁄ PAGE (lanes 1, 2) and immunoblot analysis (lanes 3–5) of purified SV40 NLS -HMGB2 186 (lanes 1, 5) and TAT PTD -HMGB2 186 (lanes 2, 4) in comparison with unmodified HMGB2 186 (lane 3). The pro- teins were identified with an HMGB2-specific antibody followed by horseradish peroxidase-coupled secondary antibody and chemilumines- cent detection. (C) DNA binding was analyzed by agarose gel electrophoresis of 0.5 lg pCMS-eGFP-E2C-Luc plasmid DNA in the absence of protein (lanes 1, 6), or after incubation with the indicated amounts of recombinant SV40 NLS -HMGB2 186 (lanes 2–5) or TAT PTD -HMGB2 186 (lanes 7–10). The positions of supercoiled and open circular DNA, and protein–DNA complexes are indicated by arrows. Table 1. N-terminal amino acid sequence and calculated isoelectric point of HMGB2 derivatives. HMGB2 derivative N-terminal sequence a Calculated IEP b HMGB2 186 MGT-GKGD 9.75 TAT PTD -HMGB2 186 MG-YGRKKRRQRRR-GT-GKGD 9.99 SV40 NLS -HMGB2 186 MPR-PKKKRKVEDP-GT-GKGD. 9.84 IBB-HMGB2 186 MPRHHHHHH-AARLHRFKNKGKDSTEMRRRRIEVNVELRKAKKDDQMLKRRNVSSFPD-GT-GK GD 9.99 a Sequences of functional peptide domains are underlined. Positively charged amino acids are indicated in bold. The first four residues of the HMGB2 1–186 fragment are shown in italics. The N-terminal Met residue, His-tag and sequences encoded by cloning linkers are also inclu- ded. b Isoelectric points were calculated using Expasy (http://www.expasy.org/cgi-bin/protparam). A. Sloots and W. S. Wels Recombinant HMGB2 mediates gene delivery FEBS Journal 272 (2005) 4221–4236 ª 2005 FEBS 4225 The DNA-binding activity of purified TAT PTD - HMGB2 186 and SV40 NLS -HMGB2 186 proteins was analyzed in gel retardation experiments as described above. As shown in Fig. 3C, in the presence of 1.7 or 1.8 lg of the proteins, 0.5 lg pCMS-eGFP-E2C-Luc plasmid DNA was markedly retarded in the agarose gels, indicating effective DNA binding and complex formation. TAT PTD -HMGB2 186 and SV40 NLS -HMGB2 186 mediate gene transfer To examine the gene-transfer activity of HMGB2 186 fusion proteins, protein–DNA complexes were pre- pared by mixing increasing amounts of HMGB2 186 , TAT PTD -HMGB2 186 and SV40 NLS -HMGB2 186 with pCMS-eGFP-E2C-Luc DNA (2.3 lg per well) as des- cribed above. Transfection complexes were added to COS-7 and human HepG2 cells in complete growth medium containing serum for 4 h. Luciferase activities were determined 40 h later. In both, COS-7 and HepG2 cells, gene delivery mediated by TAT PTD - HMGB2 186 and SV40 NLS -HMGB2 186 was more efficient than HMGB2 186 -mediated transfection (Fig. 4A,D). Maximal luciferase activity in COS-7 cells was achieved using 16.5 lg TAT PTD -HMGB2 186 (molar protein to DNA ratio of 1270 : 1), which was 38 times higher than after HMGB2 186 -mediated transfection with 13 lg of the unmodified protein (molar protein to DNA ratio of 1070 : 1), and still A BC DE Fig. 4. Gene transfer mediated by SV40 NLS - HMGB2 186 and TAT PTD -HMGB2 186 . COS-7 (A, B), HeLa (C) or HepG2 cells (D, E) were transfected with protein–DNA complexes containing 2.3 lg pCMS-eGFP-E2C-Luc reporter plasmid and the indicated amounts of purified SV40 NLS -HMGB2 186 or TAT PTD - HMGB2 186 proteins in the absence (A, D) or presence of 100 l M chloroquine (B, C, E) as described in the legend of Fig. 2. Protein– DNA complexes prepared with unmodified HMGB2 186 were included for comparison. Luciferase activity is expressed in relative light units (RLU)Æ(mg total protein) )1 . Recombinant HMGB2 mediates gene delivery A. Sloots and W. S. Wels 4226 FEBS Journal 272 (2005) 4221–4236 ª 2005 FEBS seven times higher than with 52 lg HMGB2 186 (molar protein to DNA ratio of 4200 : 1; Fig. 4A, left and right panels). In HepG2 cells, TAT PTD -HMGB2 186 was more than three times more effective than HMGB2 186 at the highest protein amounts used (Fig. 4D). Unexpectedly, luciferase activities measured after SV40 NLS -HMGB2 186 -mediated gene delivery into COS-7 and HepG2 cells were very similar to those obtained after TAT PTD -HMGB2 186 -mediated transfec- tion at comparable molar protein to DNA ratios. For gene delivery into HepG2 cells, SV40 NLS -HMGB2 186 was even slightly more effective than TAT PTD - HMGB2 186 (Fig. 4A,D, middle panels). As inclusion of the TAT PTD domain did not enhance transfection efficiency more than inclusion of the SV40 NLS domain, the NLS function of TAT rather than its membrane-translocating properties may be responsible for the enhanced reporter gene expression observed in comparison with HMGB2 186 . Therefore, to analyze the possible involvement of the endocytic pathway during TAT PTD -HMGB2 186 -mediated and SV40 NLS -HMGB2 186 -mediated gene transfer, the effect of chloroquine on transfection efficiency was investi- gated, with HeLa cells included in the analysis in addition to COS-7 and HepG2 cells. As shown in Fig. 4B,C,E, in all three cell lines not only HMGB2 186 - mediated and SV40 NLS -HMGB2 186 -mediated, but also TAT PTD -HMGB2 186 -mediated gene delivery was enhanced by chloroquine to a similar degree in com- parison with transfection by the respective proteins in the absence of an endosomolytic reagent. These results suggest that TAT PTD -HMGB2 186 protein–DNA com- plexes may indeed enter cells primarily via an endo- somal pathway, rather than by direct membrane translocation as originally hypothesized for TAT PTD - containing fusion proteins. An HMGB2 186 derivative carrying the IBB of importin-a displays enhanced gene-delivery activity The viral TAT PTD domain can function as a nonclassi- cal NLS by direct interaction with importin-b [29]. To investigate whether attachment of an endogenous cellu- lar importin-b binding sequence to HMGB2 186 enhan- ces its gene-delivery activity to a similar extent, the IBB of human importin-a2 (amino acids 11–58) together with an N-terminal His-tag was fused to the HMGB2 186 sequence (Fig. 5A; IBB sequence shown in Table 1). The resulting IBB-HMGB2 186 fusion protein was expressed in E. coli as described above for HMGB2 186 . After purification, IBB-HMGB2 186 could be identified as the major band on SDS ⁄ PAGE and immunoblot analysis, and DNA-binding activity sim- ilar to that of HMGB2 186 was confirmed in a gel retar- dation assay. The gene-transfer activity of IBB-HMGB2 186 was investigated in transfection experiments as described above using COS-7 cells. HMGB2 186 , SV40 NLS - HMGB2 186 and TAT PTD -HMGB2 186 containing com- plexes were included for comparison. As controls, gene-transfer complexes were also prepared with poly(l-lysine) and polyethyleneimine (PEI). Surpris- ingly, in the absence of chloroquine, gene transfer mediated by IBB-HMGB2 186 was clearly less efficient than SV40 NLS -HMGB2 186 -mediated and TAT PTD - HMGB2 186 -mediated transfection at similar protein to DNA ratios, and was comparable to gene delivery mediated by unmodified HMGB2 186 (Fig. 5B). In stri- king contrast, in the presence of chloroquine, IBB- HMGB2 186 -containing complexes were remarkably effective, with ensuing luciferase activities higher than those achieved after transfection with TAT PTD - HMGB2 186 –DNA complexes (Fig. 5C). Importantly, with the exception of HMGB2 186 , which in this experi- ment was used at suboptimal protein concentrations, gene transfer mediated by the recombinant HMGB2 186 derivatives was comparable to or more efficient than poly(l-lysine)-mediated transfection. Not unexpectedly, transfection of cells with PEI, which is considered to be one of the most efficient nonviral gene-delivery agents currently available, was still more effective than IBB-HMGB2 186 -mediated gene transfer in the presence of chloroquine. However, the differences were not dramatic, with luciferase activities measured after IBB-HMGB2 186 -mediated and PEI-mediated transfec- tion being of the same order of magnitude (less than fourfold difference; Fig. 5C). To analyze whether IBB-HMGB2 186 -mediated gene delivery is dependent on specific cell binding similar to that found for uncomplexed HMGB2 186 , competition experiments were performed. COS-7 cells were pre- treated for 15 min with increasing amounts of IBB- HMGB2 186 or unmodified HMGB2 186 protein before IBB-HMGB2 186 –DNA complexes at an optimal pro- tein to DNA mass ratio of 8.2 were added. Control cells were treated with IBB-HMGB2 186 –DNA com- plexes in the absence of competitor. As shown in Fig. 6, in the presence of an amount of free IBB- HMGB2 186 comparable to the amount of protein in the complex, transfection efficiency was only 37% of controls, and was reduced further to 22% if the con- centration of free IBB-HMGB2 186 was doubled. Free unmodified HMGB2 186 also affected transfection effi- ciency of IBB-HMGB2 186 –DNA complexes, but to a lesser extent than identical molar concentrations of A. Sloots and W. S. Wels Recombinant HMGB2 mediates gene delivery FEBS Journal 272 (2005) 4221–4236 ª 2005 FEBS 4227 free IBB-HMGB2 186 (reduction to 66% and 43% of controls). These data suggest that IBB-HMGB2 186 – DNA complexes bind to the cell surface primarily via the HMGB2 186 domain and to the same structures recognized by uncomplexed HMGB2 186 derivatives. The pCMS-eGFP-E2C-Luc reporter plasmid in addition to luciferase also encodes eGFP, which allows identification of transfected cells individually. In a separate experiment, COS-7 cells were incubated in the presence of chloroquine with protein–DNA complexes at a molar protein to DNA ratio of  1200 : 1 as des- cribed above. At 20 h after transfection, cells were first analyzed by fluorescence microscopy (Fig. 7A), fol- lowed another 20 h later by FACS analysis for quanti- fication of eGFP-expressing cells (Fig. 7B). Confirming the results obtained in the luciferase assays, in the presence of chloroquine, IBB-HMGB2 186 was again the most effective HMGB2 186 derivative resulting in suc- cessful transfection and measurable eGFP expression in 13% of the cells, which compares well with PEI- mediated transfection (19% of eGFP-positive cells). Taken together, these data suggest that IBB has no effect during uptake of protein–DNA complexes via the endocytic pathway, but upon release from endo- somes with the help of chloroquine may serve as a pure NLS, greatly improving transport of plasmid DNA to the nucleus and enabling efficient gene expres- sion. In contrast, SV40 NLS and TAT PTD may also con- tribute other activities that improve nonviral gene delivery, as indicated by their ability to enhance trans- fection efficiency already in the absence of an endo- somolytic reagent. A B C Fig. 5. (A) Bacterial expression of an HMGB2 186 derivative containing IBB (amino acids 11–58) of human importin-a2. The IBB-HMGB2 186 expression cassette also encodes a C-terminal Myc (M), and N-ter- minal and C-terminal polyhistidine (H) tags, inserted into plasmid pSW5. Shown below are SDS ⁄ PAGE (lane 1) and immunoblot analysis (lane 3) of purified IBB-HMGB2 186 in comparison with unmodified HMGB2 186 (lanes 2, 4). The proteins were identified with an HMGB2-specific antibody followed by horseradish peroxidase-coupled secon- dary antibody and chemiluminescent detec- tion. DNA binding was analyzed by agarose gel electrophoresis of 0.5 lg pCMS-eGFP- E2C-Luc plasmid DNA in the absence of protein (lane 1), or after incubation with the indicated amounts of recombinant IBB- HMGB2 186 (lanes 2, 3). The positions of supercoiled and open circular DNA, and protein–DNA complexes are indicated by arrows. (B, C) IBB-HMGB2 186 -mediated gene transfer. COS-7 cells were transfected with protein–DNA complexes containing 2.3 lg pCMS-eGFP-E2C-Luc reporter plas- mid and the indicated amounts of purified IBB-HMGB2 186 , or HMGB2 186 , SV40 NLS - HMGB2 186 ,orTAT PTD -HMGB2 186 proteins for comparison in the absence (B) or pres- ence of 100 l M chloroquine (C) as described in the legend of Fig. 2. Control cells were treated with poly( L-lysine) (pL)–DNA com- plexes, or PEI–DNA complexes at an N ⁄ P ratio of 10 (open bars). Luciferase activity is expressed in relative light units (RLU)Æ(mg total protein) )1 . Recombinant HMGB2 mediates gene delivery A. Sloots and W. S. Wels 4228 FEBS Journal 272 (2005) 4221–4236 ª 2005 FEBS Discussion The ability to condense DNA is essential for a nonviral vector to be successful as a gene-delivery reagent [4]. Although synthetic vectors are most commonly used for nonviral gene transfer, certain DNA-condensing proteins of mammalian origin have also been shown to facilitate cellular uptake of plasmid DNA. Previous studies on histones and HMG proteins as gene-delivery reagents mainly used full-length proteins purified or enriched from animal tissues [5–11], whereas only a few groups have so far attempted to utilize such proteins in recombinant form [12,14]. Here we have generated a novel recombinant derivative of the human HMG pro- tein HMGB2, which facilitates nonviral delivery of plasmid DNA into tumor cells. By complementing the DNA-binding activity of this HMGB2 186 variant with additional functional domains from heterologous proteins, we achieved a marked increase in protein- mediated transfection. HMGB2 is a member of the HMGB subfamily of nonhistone chromatin proteins, which also includes HMGB1 and the more recently discovered HMGB3 [31,32]. HMGB proteins have little or no sequence spe- cificity and bind preferentially to certain (distorted) DNA structures [32,33]. Thereby the acidic C-terminus appears to control DNA binding, as truncated HMGB1 and HMGB2 lacking this sequence displayed increased affinity for DNA [23,34–37]. Deletion of the acidic tail also largely abolished the differences in DNA binding between the three HMGB proteins [23]. Consequently, as a reagent for nonviral gene delivery, we constructed a truncated HMGB2 derivative that encompasses amino acids 1–186 of the human protein, but lacks the acidic C-terminal part. Whereas in a previous report bacterial expression of full-length HMGB1 had only resulted in very low amounts of recombinant protein [38], here we encountered no problems with regard to expression of truncated HMGB2 186 in E. coli, and high yields of soluble recombinant protein could be obtained after purifica- tion from bacterial lysates under native conditions [up to 4 mgÆ(L culture) )1 ]. Purified HMGB2 186 was able to condense plasmid DNA in a concentration-dependent manner, indicated by marked retardation of the resulting protein–DNA complexes in an agarose gel. Starting at a molar pro- tein to DNA ratio of 1050 : 1 (representing an HMGB2 186 protein to DNA mass ratio of 5.6), maxi- mum retardation of plasmid DNA was achieved. Pre- viously for recombinant full-length HMGB1 expressed in yeast, in a similar assay, a protein to DNA ratio of 7000 : 1 was required [12]. This suggests that removal of the acidic tail indeed facilitated enhanced DNA binding of the protein, even if general differences between HMGB1 and HMGB2 may have partially contributed to this effect. As shown by FACS analysis, HMGB2 186 also bound to the surface of established tumor cell lines in a concentration-dependent and satu- rable manner. Although at present the exact nature of this interaction remains unclear, our data suggest spe- cific binding of HMGB2 186 to defined target molecules rather than unspecific attachment to the cell mem- brane. This also appears to be the case for HMGB2 186 derivatives complexed with DNA, as transfection efficiency of preformed IBB-HMGB2 186 –DNA com- plexes was significantly reduced when free HMGB2 186 or IBB-HMGB2 186 proteins were added as competi- tors. Likewise, gene transfer was decreased when HMGB2 186 derivatives were present in too high amounts in transfection complexes. For HMGB2, in contrast with the related HMGB1 molecule, so far no extracellular activity has been reported [32,39]. Never- theless, owing to the high homology between these proteins (80% amino-acid sequence identity), HMGB2 may bind to the same or similar cell surface molecules as HMGB1, which include the receptor for advanced glycation end products (RAGE) [40] and syndecan-1 [41]. Interestingly, PEI–DNA complexes have also recently been found to be internalized by adherent cells after binding to syndecans [42]. Fig. 6. Effect of uncomplexed HMGB2 186 derivatives on transfec- tion efficiency of IBB-HMGB2 186 –DNA complexes. COS-7 cells were transfected in the presence of 100 l M chloroquine with protein–DNA complexes containing 2.3 lg pCMS-eGFP-E2C-Luc reporter plasmid and 18.8 lg purified IBB-HMGB2 186 protein (protein ⁄ DNA mass ratio of 8.2) as described in the legend of Fig. 2. Before transfection, cells were treated for 15 min with uncomplexed IBB-HMGB2 186 or HMGB2 186 proteins as indicated. A. Sloots and W. S. Wels Recombinant HMGB2 mediates gene delivery FEBS Journal 272 (2005) 4221–4236 ª 2005 FEBS 4229 In in vitro transfection experiments, treatment of dif- ferent tumor cell lines with HMGB2 186 –DNA com- plexes resulted in transient expression of luciferase and eGFP reporter genes. Depending on the protein amounts used, gene transfer was more efficient than with poly(l-lysine)–DNA complexes, and was not inhib- ited by the serum in the culture medium. Although in these experiments poly(l-lysine) was used at amounts favoring the formation of electroneutral complexes, and no attempt was made to optimize transfection by this control reagent, gene delivery by modified HMGB2 186 derivatives compared well with PEI-mediated transfec- tion at optimal N⁄ P ratios previously shown to be far superior to poly(l-lysine) [43]. At the concentrations tested, cell viability was not affected by protein–DNA complexes containing HMGB2 186 or HMGB2 186 fusion proteins (data not shown). Addition of the endo- somolytic reagent, chloroquine, strongly enhanced the A B Fig. 7. Analysis of eGFP expression after transfection with protein–DNA complexes containing HMGB2 186 derivatives. COS-7 cells were transfected with protein–DNA complexes containing 2.3 lg pCMS-eGFP-E2C-Luc reporter plasmid and the indicated amounts of purified HMGB2 186 , SV40 NLS -HMGB2 186 , TAT PTD -HMGB2 186 , or IBB-HMGB2 186 proteins in the presence of 100 lM chloroquine as described in the legend of Fig. 2. Control cells were treated with poly( L-lysine) (pL)–DNA complexes, or PEI–DNA complexes at an N ⁄ P ratio of 12. (A) Micro- scopic analysis of eGFP expressing cells 20 h after transfection. Corresponding representative fields after fluorescence and bright field micro- scopy are shown. (B) Quantification of eGFP-expressing cells by FACS analysis. At 40 h after transfection, cells were collected and analyzed by flow cytometry. Untreated COS-7 cells were used as a control. The cut-off for eGFP expression was set at the fluorescence intensity at which 99.84% of the control cells displayed a lower fluorescent signal. The bars represent the percentage of eGFP expressing cells (1 · 10 4 cells per well analyzed in duplicate). Magnification, 100·. Recombinant HMGB2 mediates gene delivery A. Sloots and W. S. Wels 4230 FEBS Journal 272 (2005) 4221–4236 ª 2005 FEBS [...]... functionality of the endogenous FEBS Journal 272 (2005) 42214236 ê 2005 FEBS Recombinant HMGB2 mediates gene delivery HMGB2 NLS could be compromised, as this sequence is part of the HMG boxes that are directly involved in DNA binding [22] Reconstitution or enhancement of nuclear tropism may therefore at least partly explain the enhanced gene- transfer activity of the SV40NLSHMGB2186 and TATPTD -HMGB21 86 proteins... NLS sequences to HMGB21 86 To further investigate the effect of a protein domain that strongly enhances nuclear localization, we generated an HMGB21 86 derivative carrying at its N-terminus the IBB of human importin-a2 The classical nuclear import pathway in eukaryotic cells is initiated by binding of an NLS to importin-a in the cytoplasm Via the IBB of importin-a, the resulting complex then interacts... benecial if HMGB2 derivatives are used as components of DNA vaccines where the protein could play a dual role as a DNA -delivery reagent and a vaccine adjuvant Our results demonstrate that bacterially expressed derivatives of human HMGB2 facilitate efcient nonviral gene delivery Endogenous DNA-condensing and cell-binding activities of HMGB2 could successfully be complemented by the addition of NLS from... (1%) in the presence of ethidium bromide to visualize the DNA Transfection of cells Construction of the reporter gene construct pCMS-eGFP-E2C-Luc For DNA mobility-shift assays and transfection experiments, the reporter gene construct pCMS-eGFP-E2C-Luc was used, which contains the eGFP-encoding gene under the control of the SV40 enhancer promoter, and the rey Photinus pyralis luciferase gene under the. .. restricted expression pattern of HMGB2 in the adult in comparison with HMGB1 [39], this may not be physiologically relevant Nevertheless, as this part of the protein is also present in HMGB21 86, it could be important for potential in vivo applications of HMGB2- based gene delivery vectors Although immunostimulatory activity may limit their application in cases where the induction of an immune response is... chloroquine Like HMGB21 86 and SV40NLS -HMGB21 86, TATPTD -HMGB21 86-mediated gene delivery was markedly enhanced by the endosomolytic reagent, indicating that TATPTD -HMGB21 86 utilizes predominantly the endosomal pathway to transport plasmid DNA into the cell Similarly, recent reports on gene delivery mediated by monomeric or oligomeric TATPTD peptides on their own suggest cellular uptake of peptide DNA complexes... similar protein to DNA ratios was found to be the most potent gene- delivery reagent of the four HMGB21 86 derivatives tested IBB, TATPTD and SV40NLS are cationic peptides and increase the overall positive charge of the corresponding HMGB21 86 fusion proteins, which can be expected to result in improved DNA binding Indeed, IBB -HMGB21 86, TATPTD -HMGB21 86 and SV40NLSHMGB2186 displayed a comparable increase in... plasmid DNA Hum Gene Ther 7, 13951404 15 Nishikawa M & Huang L (2001) Nonviral vectors in the new millennium: delivery barriers in gene transfer Hum Gene Ther 12, 861870 16 Wiethoff CM & Middaugh CR (2003) Barriers to nonviral gene delivery J Pharm Sci 92, 203217 FEBS Journal 272 (2005) 42214236 ê 2005 FEBS A Sloots and W S Wels 17 Uherek C & Wels W (2000) DNA-carrier proteins for targeted gene delivery Adv... active transport of the trimeric complex into the nucleus [53,54] Consequently, once in the cytoplasm of the target cell, IBB -HMGB21 86, which is similar to proteins such as HIV-1 TAT that contain a nonclassical NLS, can be expected to bind directly to importin-b, thereby bypassing the requirement to rst recruit importin-a Indeed, in the presence of chloroquine, IBB -HMGB21 86 at similar protein to DNA... increased gene transfer Recently, internalization of TAT fusion proteins via lipid raft-dependent macropinocytosis, a specialized form of endocytosis, has been demonstrated, questioning the possibility of TAT-mediated direct penetration of the lipid bilayer [48] Uptake of TATPTD -HMGB21 86DNA complexes by endocytosis is supported by the data obtained in transfection experiments in the presence of chloroquine . Recombinant derivatives of the human high-mobility group protein HMGB2 mediate efficient nonviral gene delivery Arjen Sloots and Winfried S. Wels Chemotherapeutisches. facilitate nonviral gene delivery. Here we have generated recombinant derivatives of the human high-mobility group (HMG) protein HMGB2 and investigated their