HMGB2186facilitates gene delivery into COS-7 cells To investigate HMGB2186-mediated gene transfer, the pCMS-eGFP-E2C-Luc reporter gene plasmid was used which encodes enhanced green fluore
Trang 1protein 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 HMGB2186 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 (TATPTD -HMGB2186), the nuclear localization sequence of the simian virus 40 (SV40) large T-antigen (SV40NLS-HMGB2186), or the importin-b-binding domain (IBB) of human importin-a (IBB-HMGB2186), chimeric fusion pro-teins were produced which displayed markedly improved transfection effi-ciency Addition of chloroquine strongly enhanced gene transfer by all four HMGB2186derivatives studied, indicating cellular uptake of protein–DNA complexes via endocytosis The IBB-HMGB2186 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.
Trang 2DNA-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 HMGB2186 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 HMGB2186 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-HMGB2186encodes 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 HMGB2186by Ni 2+ affinity chromatography HMGB2186was identified with Myc-tag-specific antibody 9E10 followed by horseradish peroxidase-coupled secondary antibody and chemiluminescent detection.
Trang 3Truncated human HMGB2186is 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 (HMGB2186) was derived
by PCR and inserted into the expression vector pSW5
[24] In the resulting pSW5-HMGB2186 plasmid,
cyto-plasmic expression of HMGB2186 fused to C-terminal
Myc and His-tags is controlled by an isopropyl
b-d-thio-galactopyranoside-inducible tac promoter (Fig 1A)
HMGB2186 protein was expressed in Escherichia coli
strain BL21(kDE3)trxB–[25] and purified from bacterial
lysates under native conditions by Ni2+-affinity
chro-matography as described in Experimental Procedures
In SDS⁄ PAGE analysis, purified HMGB2186 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 HMGB2186binds to plasmid DNA
and cell surfaces
Binding of HMGB2186 protein to plasmid DNA was
investigated in a gel retardation experiment (Fig 2A)
Increasing amounts of purified HMGB2186 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 HMGB2186, 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 HMGB2186 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 HMGB2186 complexes containing 2.8 lg or
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 HMGB2186 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 HMGB2186, and bound protein was detected with mAb 9E10 recognizing the Myc tag included in the molecule, followed by fluorescein isothiocyanate-conjugated or phycoerythrin-isothiocyanate-conjugated secondary anti-bodies As shown in Fig 2B, concentration-dependent and saturable binding of HMGB2186to the cell surface was detected, suggesting specific interaction with an as yet unidentified target molecule
HMGB2186facilitates gene delivery into COS-7 cells
To investigate HMGB2186-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 HMGB2186protein 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 HMGB2186-mediated gene delivery was found, which was more efficient than poly(l-lysine)-mediated transfection at an HMGB2186 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 HMGB2186, with luciferase activity eight times higher than in the poly (l-lysine) control Interestingly, in COS-7 cells, effi-ciency of HMGB2186-mediated gene transfer decreased again at higher HMGB2186 concentrations, possibly because of saturation of cell surface molecules occupied
by excess free HMGB2186protein
To investigate the possible involvement of the endo-cytic pathway in the internalization of HMGB2186– DNA complexes, cells were also transfected in the presence of the endosomolytic agent chloroquine [27,28] As shown in Fig 2D, the efficiency of HMGB2186-mediated gene transfer into COS-7 cells was substantially increased by chloroquine Luciferase
Trang 4activity was enhanced 15-fold and 55-fold for
com-plexes containing 43 and 64.5 lg HMGB2186,
respect-ively Taken together, these data show that HMGB2186
on its own is able to mediate nonviral gene delivery,
and strongly suggest that HMGB2186–DNA complexes
enter the cells through the endocytic pathway
Construction of HMGB2186derivatives containing the TAT PTD or the SV40 T-antigen NLS
To investigate whether HMGB2186-mediated gene transfer can be improved by including in the molecule
a cell-penetrating peptide, the PTD of the HIV-1 TAT
A
B
Fig 2 Functional characterization of purified HMGB2186 (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 HMGB2186to 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 HMGB2186(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 l M ) (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 HMGB2186with 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.
Trang 5protein (amino acids 47–57) flanked by additional
gly-cine residues was fused to the N-terminus of the
HMGB2186 fragment (Fig 3A; TATPTD 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 HMGB2186
-mediated gene transfer independent of cell-penetrating
activity, a similar HMGB2186 derivative was
construc-ted which carries the classical NLS of the SV40 large
T-antigen [30] at the N-terminus (Fig 3A; SV40NLS sequence shown in Table 1) TATPTD-HMGB2186 and SV40NLS-HMGB2186proteins were expressed in E coli and purified from bacterial lysates as described above for unmodified HMGB2186 As expected, in compari-son with HMGB2186, 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 HMGB2186derivatives carrying the TAT PTD (TATPTD; amino acids 47–57) or the NLS of SV40 large T-antigen (SV40NLS) (A) Schematic representation of the TATPTD-HMGB2186and SV40NLS-HMGB2186expression 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 SV40NLS-HMGB2186(lanes 1, 5) and TATPTD-HMGB2186(lanes 2, 4) in comparison with unmodified HMGB2186(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.
IBB-HMGB2186 MPRHHHHHH-AARLHRFKNKGKDSTEMRRRRIEVNVELRKAKKDDQMLKRRNVSSFPD-GT-GKGD 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).
Trang 6The DNA-binding activity of purified TATPTD
-HMGB2186 and SV40NLS-HMGB2186 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
TATPTD-HMGB2186and SV40NLS-HMGB2186
mediate gene transfer
To examine the gene-transfer activity of HMGB2186
fusion proteins, protein–DNA complexes were
pre-pared by mixing increasing amounts of HMGB2186,
TATPTD-HMGB2186 and SV40NLS-HMGB2186 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 TATPTD -HMGB2186 and SV40NLS-HMGB2186 was more efficient than HMGB2186-mediated transfection (Fig 4A,D) Maximal luciferase activity in COS-7 cells was achieved using 16.5 lg TATPTD-HMGB2186
(molar protein to DNA ratio of 1270 : 1), which was
38 times higher than after HMGB2186-mediated transfection with 13 lg of the unmodified protein (molar protein to DNA ratio of 1070 : 1), and still
A
-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 SV40NLS-HMGB2186or TATPTD -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.
Trang 7seven times higher than with 52 lg HMGB2186 (molar
protein to DNA ratio of 4200 : 1; Fig 4A, left and
right panels) In HepG2 cells, TATPTD-HMGB2186
was more than three times more effective than
HMGB2186 at the highest protein amounts used
(Fig 4D) Unexpectedly, luciferase activities measured
after SV40NLS-HMGB2186-mediated gene delivery into
COS-7 and HepG2 cells were very similar to those
obtained after TATPTD-HMGB2186-mediated
transfec-tion at comparable molar protein to DNA ratios For
gene delivery into HepG2 cells, SV40NLS-HMGB2186
was even slightly more effective than TATPTD
-HMGB2186(Fig 4A,D, middle panels)
As inclusion of the TATPTD domain did not
enhance transfection efficiency more than inclusion of
the SV40NLSdomain, the NLS function of TAT rather
than its membrane-translocating properties may be
responsible for the enhanced reporter gene expression
observed in comparison with HMGB2186 Therefore,
to analyze the possible involvement of the endocytic
pathway during TATPTD-HMGB2186-mediated and
SV40NLS-HMGB2186-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 HMGB2186
-mediated and SV40NLS-HMGB2186-mediated, but
also TATPTD-HMGB2186-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 TATPTD-HMGB2186 protein–DNA
com-plexes may indeed enter cells primarily via an
endo-somal pathway, rather than by direct membrane
translocation as originally hypothesized for TATPTD
-containing fusion proteins
An HMGB2186derivative carrying the IBB of
importin-a displays enhanced gene-delivery
activity
The viral TATPTD 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 HMGB2186
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
HMGB2186sequence (Fig 5A; IBB sequence shown in
Table 1) The resulting IBB-HMGB2186 fusion protein
was expressed in E coli as described above for
HMGB2186 After purification, IBB-HMGB2186 could
be identified as the major band on SDS⁄ PAGE and
immunoblot analysis, and DNA-binding activity sim-ilar to that of HMGB2186was confirmed in a gel retar-dation assay
The gene-transfer activity of IBB-HMGB2186 was investigated in transfection experiments as described above using COS-7 cells HMGB2186, SV40NLS -HMGB2186 and TATPTD-HMGB2186 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-HMGB2186 was clearly less efficient than SV40NLS-HMGB2186-mediated and TATPTD -HMGB2186-mediated transfection at similar protein to DNA ratios, and was comparable to gene delivery mediated by unmodified HMGB2186 (Fig 5B) In stri-king contrast, in the presence of chloroquine, IBB-HMGB2186-containing complexes were remarkably effective, with ensuing luciferase activities higher than those achieved after transfection with TATPTD -HMGB2186–DNA complexes (Fig 5C) Importantly, with the exception of HMGB2186, which in this experi-ment was used at suboptimal protein concentrations, gene transfer mediated by the recombinant HMGB2186 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-HMGB2186-mediated gene transfer in the presence
of chloroquine However, the differences were not dramatic, with luciferase activities measured after IBB-HMGB2186-mediated and PEI-mediated transfec-tion being of the same order of magnitude (less than fourfold difference; Fig 5C)
To analyze whether IBB-HMGB2186-mediated gene delivery is dependent on specific cell binding similar to that found for uncomplexed HMGB2186, competition experiments were performed COS-7 cells were pre-treated for 15 min with increasing amounts of IBB-HMGB2186 or unmodified HMGB2186 protein before IBB-HMGB2186–DNA complexes at an optimal pro-tein to DNA mass ratio of 8.2 were added Control cells were treated with IBB-HMGB2186–DNA com-plexes in the absence of competitor As shown in Fig 6, in the presence of an amount of free IBB-HMGB2186 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-HMGB2186 was doubled Free unmodified HMGB2186 also affected transfection effi-ciency of IBB-HMGB2186–DNA complexes, but to a lesser extent than identical molar concentrations of
Trang 8free IBB-HMGB2186 (reduction to 66% and 43% of
controls) These data suggest that IBB-HMGB2186–
DNA complexes bind to the cell surface primarily via
the HMGB2186 domain and to the same structures
recognized by uncomplexed HMGB2186derivatives
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-HMGB2186was again the most effective HMGB2186 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, SV40NLSand TATPTD 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-HMGB2186expression cassette also encodes a C-terminal Myc (M), and N-ter-minal and C-terN-ter-minal polyhistidine (H) tags, inserted into plasmid pSW5 Shown below are SDS ⁄ PAGE (lane 1) and immunoblot analysis (lane 3) of purified IBB-HMGB2186
in comparison with unmodified HMGB2186 (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-HMGB2186(lanes 2, 3) The positions of supercoiled and open circular DNA, and protein–DNA complexes are indicated by arrows (B, C) IBB-HMGB2186-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 -HMGB2186, or TATPTD-HMGB2186proteins 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.
Trang 9The 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 HMGB2186 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 HMGB2186 in E coli, and high yields of soluble recombinant protein could be obtained after purifica-tion from bacterial lysates under native condipurifica-tions [up to 4 mgÆ(L culture))1]
Purified HMGB2186 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 HMGB2186 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, HMGB2186 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 HMGB2186to defined target molecules rather than unspecific attachment to the cell mem-brane This also appears to be the case for HMGB2186 derivatives complexed with DNA, as transfection efficiency of preformed IBB-HMGB2186–DNA com-plexes was significantly reduced when free HMGB2186
or IBB-HMGB2186 proteins were added as competi-tors Likewise, gene transfer was decreased when HMGB2186 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 HMGB2186derivatives 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.
Trang 10In in vitro transfection experiments, treatment of
dif-ferent tumor cell lines with HMGB2186–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 HMGB2186 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 HMGB2186 or HMGB2186 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 HMGB2186derivatives 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 l M 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·.