expression of apoe3-hrp chimeric molecule in hek293

Annals of RSCB Vol. XIV, Issue 2

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EXPRESSION OF APOE3-HRP CHIMERIC MOLECULE IN HEK293 CELLS

Irina Cristina Florea, Violeta Georgeta Trusca, Ioana Madalina Fenyo, Anca Violeta Gafencu

INSTITUTE OF CELLULAR BIOLOGY AND PATHOLOGY "N. SIMIONESCU" BUCHAREST, ROMANIA

[email protected]

Summary The aim of this study is to obtain cells expressing a chimeric molecule containing human apolipoprotein E (apoE) linked to a molecule of horse radish peroxidase (HRP), that function as a reporter gene. To clone the chimeric molecule apoE-HRP, standard molecular biology techniques (PCR, RT-PCR, enzymatic restriction of DNA, ligation) were performed. HEK293 cells were transiently transfected with the obtained plasmid by calcium phosphate DNA precipitation or by lipofection. 48h after transfection, cells were analysed by RT-PCR, Western Blot, enzymatic activity assays, and light microscopy. The plasmid pLIONII-apoE3-HRP enclosing the chimera comprising human apoE linked to horseradish peroxidase was constructed in two steps: (i). construction of the plasmid containing apoE3 (pLIONII-apoE3); (ii). insertion of the sequence encoding HRP in the plasmid pLIONII apoE3. Expression of this plasmid in HEK293 cells showed that the chimera apoE-HRP is a protein of ~70kDa, being recognised by anti-apoE as well as anti-HRP antibodies. Moreover, HRP has enzymatic activity, revealing that this protein is functional. The plasmid pLIONII-apoE3-HRP encodes a chimeric molecule apoE3-HRP that can be express in mammalian cells. The secretion and the distribution of apoE in lipoprotein particles can be assessed by HRP, the reporter molecule.

Key words: apoE, HRP, chimeric molecule, pLIONII, transfection. Introduction

Apolipoprotein E (apoE), a glycoprotein of 34 kDa, is a major component of the lipoprotein transport system playing important roles in lipid metabolism, since it associates with chylomicrons remnant, VLDL, LDL and HDL (Shore and Shore 1973, Green et al., 1979, Danielsson et all, 1978, Schaefer et all, 1979, Wu et al., 1980). Deficiency in apoE results in atherosclerosis in humans as well as in animal models (Ghiselli et al., 1981, Plump et al., 1992). ApoE knockout mice represent the best-characterized model for experimental atherosclerosis (Greenow et al., 2005, Zhang et al., 1992, Reddick et al., 1994 Breslow 1996, Jawień et all, 2004). As a ligand for the LDL receptor expressed in the liver and in other tissues and for the apoE specific receptor (LDL receptor related protein) found also in the

liver (Kostner, 1989), apoE mediates lipoprotein clearance from plasma. A malfunction of these mechanisms involved in cholesterol clearance leads to accumulation of remnants in plasma, a process that is associated with premature atherosclerosis (Horejsí and Ceska, 2000). More recently, antioxidant and anti-inflammatory functions within the atherosclerotic plaque were attributed to apoE (Davignon, 2005, Ali et al., 2005). To follow apoE secretion and distribution in different lipoproteins particles, we designed a chimeric protein containing apoE and HRP, as a reporter molecule. The enzyme activity of this probe allows considerable signal amplification. This advantage is widely exploited in its use as an exogenous, fluid phase marker in the endocytic pathway (Graham and Karnovsky, 1966; Tooze and Hollinshead, 1991) and it allows its identification as an

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endogenous constituent within the exocytic pathway of cellular types that synthesize peroxidase (Bainton and Farquhar, 1970; Herzog and Miller, 1972). An important advantage of the HRP reaction product, when generated in the soluble phase within intracellular compartments, is that it remains contained within the lumen of the cisterna or of the vesicle in which the enzymes resides. A further, widely appreciated advantage of HRP a morphological probe is its ability to remain enzymatically active after being conjugation with a variety of protein ligands (Hopkins, 1985). In this paper, we report the construction of a lentiviral vector for the expression of the chimeric molecule apoE3-HRP. Expressed in mammalian cells, apoE3-HRP conserved the properties of the two proteins from which it is created. This chimera is useful for the study of apoE secreted from cells and incorporated in different forms of lipoproteins. Materials and methods

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Chemicals were obtained from the following sources: Pfu polymerase and the restriction enzymes BamH I, EcoR I, EcoR V were from Fermentas, Calf Intestinal Alkaline Phosphatase, M-MLV reverse transcriptase and Trizol from Invitrogen, phorbol 12-myristate 13-acetate (PMA) and LigaFast kit from Promega, Midiprep Kit QIAfilter Plasmid Purification from Qiagen, Dulbecco’s modified Eagle’s medium (DMEM) and fetal calf serum from Biochrom AG. The primers were from Metabion. The ECL kit for Western Blotting was purchased from Pierce. All other chemicals were from Sigma. The pRK5- ssHRP plasmid containing a synthetic form of HRP was kindly offered by Dr. D. F. Cutler (MRC Lab. Molec. Cell Biol., London). The construct pLIONII-apoE3-HRP was obtained by molecular cloning. For this, standard molecular biology techniques

(PCR, RT-PCR, enzymatic restriction of DNA, ligation) were performed. HEK293 cells, kindly provided by Dr. D. Kardassis (Univ. Crete, Greece) were cultured in DMEM supplemented with 10% FCS, and antibiotics (penicillin and streptomycin). One day before transfection, cells were seeded at a density of 5x105cells/cm2. HEK293 cells were transiently transfected by calcium phosphate DNA precipitation or by lipofection. 48h after from transfection, cells were analysed by RT-PCR, Western Blot, enzymatic activity, and optic microscopy.

Results and discussion

Plasmid Construction The aim of the work was to produce a chimeric protein containing human apoE linked to horseradish peroxidase (Figure 1A). The expression vector used for generating the protein was pLIONII, described in Figure 1B.

Figure 1. The apoE3-HRP construct and the lentiviral vector used for cloning. The construct contain the apoE signal sequence (ss-apoE) as well as the sequence encoding apoE, followed

by HRP sequence (A). The vector has T7 promoter followed by the multiple cloning site (MCS). Note that in MCS BamH I, EcoR I and

EcoR V are successive restriction sites (B).


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The cloning strategy of apoE3-HRP chimera described in the Figure 2, implies two steps: i. construction of the plasmid

containing apoE3 (pLIONII-apoE3); ii. insertion of the sequence encoding HRP in the plasmid pLIONII apoE3.

Figure 2. The cloning strategy of apoE3-HRP chimera. The cDNA sequence for apoE3 was obtained

by RT-PCR using RNA isolated from PMA activated THP-1 cells. A synthetic sequence encoding HRP was amplified by PCR from the plasmid pRK5-ssHRP. The cloning strategy included two steps:

(i). construction of the plasmid containing apoE3 (pLION-apoE3); (ii). Insertion of the sequence encoding HRP in the plasmid pLION-apoE3 and generation of the plasmid pLION-apoE3-HRP.

We have previously identified that the apoE isoform expressed in activated THP-1 cells is apoE3. Thus, we isolated RNA from THP-1 cells activated for 48h with 100nM PMA.

After reverstranscription, the obtained cDNA was used as template to amplify apoE3 encoding DNA by PCR, using primers containing sites for BamH I and EcoR V restriction enzymes at the 5’end, as described in the table 1.

Table 1. Primers used for amplification of apoE3 and HRP encoding sequences.

Molecule Primers Restriction site

Size of PCR product

F apoE 5’-cgggatccCACAGGCAGGAAGATGAAGG Bam H1 R apoE 5’-ggaattcGTGATTGTCGCTGGGCACAG EcoR I

977 bp

F HRP 5’- ggaattcCAGTTAACCCCTACATTCTACGAC EcoR I R HRP 5’- gggatatcTTAGTTGACCACTCTGCAGTTCAG EcoR V

925 bp


The PCR product and the plasmid pLION II were digested with BamH I and EcoR I restriction enzymes (Figure 3A), and the products were isolated after agarose gel electrophoresis. Digested insert (apoE) as well as digested vector (pLION) were subjected to ligation reaction. The ligation product was used for transformation in E.coli DH5, and the insertion of the apoE sequence in the plasmid pLION was tested in nine colonies (Figure 3B lanes 2-10). Eight colonies (Figure 3B lanes 2-7, 9,10) were positive for the insert. The colony represented in Figure 3B lanes 2, containing the plasmid pLION-apoE3 (Figure 3C) was consequently amplified and the plasmid of interest was isolated and used further in the cloning protocol.

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Figure 3. The construction of the plasmid pLION-apoE3. (A) The vector (pLION) and

the insert (apoE fragment obtained by RT-PCR) were digested with BamH I and EcoR I and

then subjected to ligation reaction. (B) Eight of the nine colonies resulted after transformation

were positive for apoE sequence insertion in the plasmid pLION (lane 2-7, 9 and 10) and one

was negative (lane 8); lane 1 represents molecular weight marker and lane 11 is the

empty vector. The HRP was amplified by PCR using as template the plasmid pRK5-ssHRP; the primers used contain EcoR I and EcoR V restriction sites at the 5’ end (Table 1). As presented in Figure 1, the PCR product as

well as the plasmid pLION II-apoE3 (the vector) were digested with EcoR I and EcoR V restriction enzymes (Figure 4A), and the products were isolated after agarose gel electrophoresis. The digested insert (HRP) and the digested vector (pLION-apoE3) were subjected to ligation reaction. The resulting mixture was used for transformation in E.coli DH5. We obtained one colony and it was positive for the HRP insert (EcoR I and EcoR V digestion, Figure 4B lanes 2) as well as for the apoE3-HRP construct (BamH I and EcoR V digestion, Figure 4B, lanes 3); Additional validation of the results was done with Apa I, Bgl II, and Pst I (Figure 4B lanes 3-5),when fragments of expected size were obtained.

Figure 4. The construction of the plasmid

pLION-apoE3-HRP. (A) The vector (pLION-apoE3) and the insert (HRP) were digested with EcoR I and EcoR V and subjected to ligation. (B) Validation of the plasmid pLION-apoE3- HRP: digestion with EcoR I and EcoR V gave one band of 925bp (HRP) and one of 7942bp; (lane 2), digestion with EcoR V and BamH I

gave one band of 1893bp (the apoE-HRP) construct and one band of 6974bp (lane 3), digestion with Apa I gave 3 bands (1388bp,

1692bp and 5786bp, lane 4); digestion with Bgl II gave 2 bands (3222bp and 5645bp, lane 5),

digestion with Pst I gave 5 bands (195bp, 330bp, 1000bp, 1601bp and 5712bp, lane 6); lane 1 represents molecular weight marker.


The sequence of the apoE-HRP construct is represented in the Figure 5. The sequence begins with the apoE encoding sequence, including the signal sequence and full-length cDNA (letters on black background). To link apoE and HRP

sequences, six nucleotides representing the EcoR I restriction site were added. These nucleotides form 2 codons that are translated in Glutamic acid (E) and Phenylalanine (F). The HRP sequence, has a STOP codon at the end.

Figure 5. The sequence of the apoE3-HRP construct. The letters on black background represent the apoE cDNA, followed by six nucleotides representing the restriction site for EcoR I, and subsequently

by the sequence of HRP, that has a STOP codon at the end. Expression of apoE3-HRP chimera HEK293 cells were transiently transfected with pLIONII-apoE3-HRP using the calcium phosphate DNA precipitation or lipofection. 48h after from transfection, cells were analysed by RT-PCR, Western blotting, enzymatic activity assays and optic microscopy. As showed in Figure 6A, mRNA for both apoE (977bp) and HRP (925bp) are expressed in cells transfected by lipofection (Figure 6A, lanes 2 and 6) or by calcium phosphate precipitation (Figure 6A, lanes 3 and 5). In fact, the primers used for cloning (described in the Tabel 1) recognize the same mRNA that linked apoE and HRP mRNAs. As positive control, the corresponding

fragments cloned in the plasmid pLION-apoE3-HRP were amplified (Figure 6A, lanes 1 and 7). Expression of this plasmid in HEK293 cells showed that the chimera apoE-HRP is a protein of ~70kDa being recognised by anti-human apoE as well as by anti-HRP antibodies (Figure 6B, lane 1 and lane 4, respectively). For the negative control, untransfected HEK293 cells were subjected to the same treatment as transfected cells. As showed in the Figure 6B, lanes 2 and 5, at this level of sensitivity there are no protein recognized by anti-apoE or anti-HRP antibodies expressed by native cells. As positive control, apoE secreted by HepG2 and HRP were used (Figure 6A, lanes 3 and 6, respectively).

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Figure 6. Expression of apoE3-HRP chimera in transiently transfected HEK 293 cells. A. RT-PCR for detection of mRNA for apoE (977bp) and HRP (925bp), expressed in cells transfected by lipofection (lanes 2 and 6) or by calcium phosphate precipitation (lanes 3 and 5); the corresponding

fragments cloned in the plasmid pLION-apoE3-HRP were amplified as positive controls (lanes 1 and 7); molecular weight marker is in the lane 4. B. Western Blot detection of apoE3-HRP chimera

expressed in transfected HEK293 cells revealed a protein of ~70kDa recognised by anti-human apoE as well as anti-HRP antibodies (lane 1 and lane 4, respectively); at this level of sensitivity, in

unransfected HEK293 cells are negative for any protein that is recognized by anti-apoE or anti-HRP antibodies (lanes 2 and 5); HepG2 secreted apoE by HRP were used as positive controls, (lanes 3 and

6). C. The peroxidase activity of the chimera apoE-HRP confirmed that the HRP fragment in the chimera is functional. D. As revealed by microscopy, the HRP reaction product appears in the cytoplasm of the transfected cells. E. No peroxidase product is present in non-transfected cells.

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pitate (Figure 6 E).

The peroxidase activity of the chimera apoE-HRP was determined by means of colorimetric methods using o-phenilamine dyhidrochloride and H2O2 as enzymatic substrate as described in Heltianu et all., 1997. Data showed that apoE-HRP maintain HRP enzymatic activity (Figure 6C), revealing that the HRP fragment of the chimera is functional. To evidentiate the HRP enzymatic activity product, transfected HEK 293 cells were fixed with 2,5% glutaraldehyde and the monolayer was incubated with a mix containing 3,3’-diaminobenzidine and H2O2 in Tris-HCl buffer in a dark ch r. As showed in the Figure 6 D, a dark product appeared in the

cytoplasm of the transfected cells. Untransfected cells did not present any preci

Conclusions

The plasmid pLIONII-apoE3-HRP encodes a chimeric molecule consisting of human apoE3 and synthetic HRP. The apoE3-HRP can be express in mammalian cells. Using this chimera, the expression, secretion and association of apoE in different particles can be revealed by HRP, the reporter molecule.

Acknowledgements: work supported by a grant from CNCSIS, Idea Programme (project 1307), and the Romanian Academy.


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expression of apoe3-hrp chimeric molecule in hek293

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