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Originally published In Press as doi:10.1074/jbc.M703367200 on June 12, 2007

J. Biol. Chem., Vol. 282, Issue 32, 23402-23409, August 10, 2007
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A Novel Approach for Enzyme Replacement Therapy

THE USE OF PHENYLALANINE HYDROXYLASE-BASED FUSION PROTEINS FOR THE TREATMENT OF PHENYLKETONURIA*

Ronen Eavri and Haya Lorberboum-Galski1

From the Department of Cellular Biochemistry and Human Genetics, Faculty of Medicine, Hebrew University, Ein-Kerem, Jerusalem 91120, Israel

Received for publication, April 23, 2007 , and in revised form, June 11, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Metabolic diseases arise from mutations in key enzymes of major metabolic pathways. One promising approach for the treatment of such diseases is based on the administration of a wild-type enzyme to substitute the activity of the impaired enzyme by the use of enzyme replacement therapy, yet it is important to deliver this enzyme to the specific deficient tissue. We suggest a new concept for the treatment of metabolic diseases using fusion proteins. We examined the feasibility of this concept in the well characterized metabolic disease, phenylketonuria (PKU), which results from a mutation in the liver enzyme phenylalanine hydroxylase (PAH). PAH is a key enzyme in the metabolic pathway of phenylalanine. Deficiency in PAH leads to high and persistent levels of this amino acid in the plasma of PKU patients, causing permanent neurological damage. Currently a low protein diet is still considered the only effective treatment for most PKU patients. To restore PAH activity in the liver of PKU patients, we constructed PAH-based fusion proteins with delivery moieties based on the HIV-transactivator of transcription peptide, and fragments of human hepatocyte growth factor aiming to specifically target PAH to the liver. We show that these new fusion proteins can be delivered into a variety of human liver cell lines and retain PAH activity after being internalized. We also show that plasma phenylalanine levels were dramatically lowered in mice treated with PAH-based fusion proteins after intravenous administration. We therefore suggest an alternative concept for the treatment of PKU using targeted fusion proteins, which may also be applied to the treatment of other metabolic diseases.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Phenylketonuria (PKU)2 is a metabolic disorder resulting from an abnormal form of the enzyme phenylalanine hydroxylase (PAH, EC 1.14.16.1 [EC] ). PAH catalyzes the irreversible hydroxylation of the amino acid phenylalanine to tyrosine in the liver. This step is considered the rate-limiting step in the catabolic pathway of Phe (1). Up until now, more than 500 mutations have been found in the PAH gene, most of them leading to a dysfunctional enzyme. The disease is inherited in a classic recessive way, with an incidence of 1 out of 10,000 new-borns (Online Mendelian Inheritance in Man (OMIM) 261600 [OMIM] ). PAH is a liver-specific enzyme with a crucial role in the maintenance of a low and steady level of Phe in the plasma. Untreated PKU patients suffer serious neurological damage due to sustained high Phe levels in the blood. Children diagnosed with PKU are immediately transferred to a low protein diet for life, to lower Phe levels in their plasma and maintain this normal level (2).

New approaches for treatment have been suggested in recent years, including gene therapy (3, 4) and enzyme replacement therapy using the yeast-derived enzyme PAL, which converts Phe into a harmless metabolite (5). These new ideas are considered promising, but still remain outside clinical practice, leaving reliance on the low protein diet as the only treatment used today for PKU.

In this report we present a novel approach for the treatment of PKU. This approach is based on directing the PAH enzyme to the liver using either a common peptide or a selective homing ligand, both aimed at delivering the enzyme to the liver and its fast clearance from the blood.

Recently new mechanisms for protein delivery have been suggested. One new promising approach is fusion with small peptides called protein transduction domains. These small peptides have been found to facilitate the penetration of fused proteins through the membranes of a large variety of eukaryotic cells. Though the mechanism of action involved in this internalization process is not clearly understood, involving neither receptor and clatherin-mediated endocytosis nor phagocytosis, these new protein transduction domains are considered promising delivery tools. Out of many protein transduction domains discovered so far, the HIV transactivator of transcription (TAT) peptide has been most widely studied. TAT fusion proteins were shown to be delivered efficiently into cultured cells, intact tissue, and live tissues when injected into mice (6, 7). Proteins delivered by this peptide retained their activity after internalization. A recent study conducted on the pharmacokinetics of TAT-beta-galactosidase fusion protein, revealed that the liver is the main target for this fusion protein (8). Based on these notions we first constructed a fusion protein containing TAT fused to human PAH.

To achieve even better selectivity toward delivery of the enzyme to the liver, the natural location and the site of biological activity of PAH, we also constructed a different set of PAH-based fusion proteins using human hepatocyte growth factor (HGF) as the specific delivering sequence. HGF is a potent paracrine growth factor with mitogenic and morphogenic activities that is synthesized by mesenchymal cells. Liver is the main target of HGF and biological responses to this growth factor are mediated by the tyrosine kinase receptor encoded by the Met oncogene (cMet) (9, 10). It is the most potent mitogen for hepatocytes, and was considered to be the major growth factor responsible for driving hepatic regeneration (10). Research conducted on the binding properties of HGF revealed that the {alpha}-chain alone (including the N terminus and four kringle domains) can bind to the cMet receptor. This fragment however, will not induce phosphorylation of the cMet receptor, hence mitogenic, motogenic, and morphogenic responses will not be activated (11). Therefore we constructed PAH fusion proteins with the HGF-{alpha}-chain as the delivering sequence. We used various fragments of the {alpha}-chain to construct NK1-PAH, NK2-PAH, NK3-PAH, and {alpha}HGF-PAH. These constructs contain the N terminus fragment and kringle domains 1, 1 and 2, and 1–3 and full-length {alpha}-HGF, respectively.

All the PAH-based fusion proteins were produced in bacterial cells. We show here that both TAT- and HGF-derived leading peptides/proteins can be used to deliver functional PAH into liver cells. Most importantly, we show that the Phe plasma level of C57BL normal mice treated with TAT-PAH was lowered as soon as 15 min after the intravenous administration of the fusion protein. Phe plasma levels remained low compared with untreated mice for several hours. This work demonstrates that fusion proteins could be potential candidates for treatment of metabolic diseases such as PKU.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Construction of Plasmids Encoding TAT-PAH and PAH—To construct the pET28-PAH plasmid with a His tag sequence at the 5' side of the coding sequence, we first cut the plasmid containing the cDNA sequence of human PAH (a gift from Dr. Shwartz, Sheba Hospital, Tel Hashomer, Israel) with SpeI. The 5' sticky end was removed with mung bean nuclease. This fragment was then cut with EcoRI generating a 1358-bp insert that was ligated into the pET28a vector (Novagen, Madison, WI) cut with HindIII, followed by a mung bean nuclease reaction and a second cut with EcoRI. Escherichia coli strain DH5 (Stratagene, La Jolla, CA) was used for all plasmid transformations. Restriction and modifying enzymes were obtained from Roche Applied Science (Mannheim, Germany) or New England Biolabs (Ipswich, MA).

The TAT-PAH plasmid was constructed using the 5' primer: 5'-TTT GAA TTC GTC CAC TGC GGT CCT GGA A-3'; and the 3' primer: 5'-TTT GTC GAC TTA CTT TAT TTT CTG GAG GGC A-3'. The PCR insert cut with EcoRI and SalI was ligated into the pTAT2.1 vector cut with the same enzymes (pTAT2.1 was a gift from Dr. S. Dowdy (University of California-San Diego, La Jolla, CA)).

Construction of Plasmids Encoding the NK1-PAH, NK2-PAH, NK3-PAH, and {alpha}HGF-PAH Fusion Proteins—The PAH plasmid was cut with EcoRI and ClaI restriction enzymes and ligated with an oligonucleotide linker containing a BstEII recognition site downstream to the PAH sequence. Linker sense: 5'-AAT TCG TTA ACA TGT GGG TGA CCG AT-3'; antisense: 5'-CGA TCG GTC ACC CA ATG TTA ACG-3'. This plasmid served as an intermediate plasmid. Total RNA was isolated from fresh human placenta with the TriPure Isolation reagent (Roche Applied Science) and then reverse transcribed into first strand cDNA using a reverse transcription system (Promega, Madison, WI).

The NK1, NK2, NK3, and {alpha}HGF sequences, flanked by ClaI restriction sites, were generated by PCR, using total human placenta cDNA, and the following synthetic oligonucleotide primers: HGF CT: 5'-GGA TCG ATT CGC AAT TGT TTC GT-3'; HGF NT: 5'-AGA TCG ATA TGT GGG TGA CCA AA-3'; PK1L: 5'-CCA TCG ATT TCA ACT TCT GAA CAC TG-3'; PK2L: 5'-CCA TCG ATT TCA GTT GTT TCC AAA GG-3'; and PK3L: 5'-CCA TCG ATA TCT TGT CCA TGT GAC AT-3'. These primers covered different regions of the {alpha} chain coding region of the HGF protein. HGF CT and HGF NT primers were used to construct the {alpha}HGF construct; HGF NT and PK1L primers for the NK1 construct; HGF NT and PK2L primers for the NK2 construct; and HGF NT and PK3L primers for the NK3 construct. The intermediate plasmid and PCR products were then cut by BstEII and ClaI and ligated to produce NK1-PAH, NK2-PAH, NK3-PAH, and {alpha}HGF-PAH carrying plasmids (Ptrhp4, Ptrhp5, Ptrhp6, and Ptrhp7, respectively).

Protein Expression and Purification of PAH and TAT-PAH Proteins—E. coli strain BL21(DE3), which carries a T7 RNA polymerase gene in a lysogenic and inducible form, was used for the expression of all fusion proteins. Cells carrying the hPAH-based plasmids were grown in LB medium containing either ampicillin (100 µg/ml) or kanamycin (100 µg/ml) according to the expression vector.

After reaching an A600 value of 0.8–1, the cultures were induced for 24 h at 22 °C with 1 mM isopropyl-1-thio-D-galactopyranoside (Sigma-Aldrich) and supplemented with 0.02 mM FeNH4(SO4)2 (Sigma-Aldrich). The cells were collected by centrifugation, and the pellet was stored at -70 °C for several hours. The frozen pellet was thawed and suspended in PBS with 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich), followed by sonication and centrifugation at 35,000 x g for 30 min. Both the pellet (insoluble fraction) and the supernatant (soluble fraction) were collected. The soluble fraction was then filtered and loaded on HiTrap Chelating HP columns (Amersham Biosciences) pre-loaded with NiSO4. The proteins were eluted with 80 mM histidine using theÁKTA fast-protein liquid chromatography system (Amersham Biosciences). The purified TAT-PAH fusion protein and the PAH protein were eluted from the column and dialyzed against PBS, pH 7.4, containing 0.02 mM FeNH4(SO4)2 (final concentration).

Protein Expression and Purification of HGF-based Proteins—The NK1-PAH, NK2-PAH, NK3-PAH, and {alpha}HGF-PAH fusion proteins were expressed as described above for the TAT-PAH fusion proteins. Expressing cells were then sub-fractionized and both the supernatant (soluble fraction), and the pellet (insoluble fraction) were collected. The soluble fraction was used for further analysis.

Western Blot Analysis—Samples from the various protein fractions (5–20 µg of protein/lane) were loaded on 12% (w/v) SDS-PAGE gels. The proteins were electrotransferred onto Immobilon-P transfer membrane (Millipore, Billerica, MA), and blotted either with mouse anti-human PAH antibody (Chemicon, Temecula, CA, 1:10,000), or mouse anti-His-tag antibody (Amersham Biosciences, 1:5,000). Antibody binding was detected by enhanced chemiluminescence (ECL) as described by the manufacturer (Amersham Biosciences).

Phenylalanine Hydroxylation Assay—L-[U-14C]Phe and L-[U-14C]Tyr were obtained from Amersham Biosciences. The PAH-based fusion proteins were tested for their enzymatic ability to hydroxylate [14C]Phe to [14C]Tyr in an in vitro cell-free assay (12) modified by the use of 40 µM tetrahydrobiopterin, 400 µM FeNH4(SO4)2, 4 mg/ml catalase, and 20 mM dithiothreitol (all obtained from Sigma) in a 100 mM Na-Hepes, pH 7, buffer. The reaction mixture was incubated for 2 h at 30 °C and then loaded onto 3-mm chromatography paper (Schleicher & Schuell, Dassel, Germany), and separated with 73% butanol and 27% acetic acid as the mobile phase. Overnight film exposure revealed the relative presence of [14C]Phe and of [14C]Tyr in the reaction. Relative concentrations were calculated by processing the film with ImageJ software (National Institutes of Health, Bethesda, MA).

Cell Lines—All cell lines were obtained from the American type Culture Collection (ATCC, Manassas, VA). Hepatocarcinoma HepG2 and HuH7 cell lines were maintained in Dulbecco's modified Eagle's medium (Biological Industries, Beit-Haemek, Israel). The colon adenocarcinoma Colo205 cell line was maintained in RPMI 1640 medium (Biological Industries). All media were supplemented with 10% fetal bovine serum (HyClone, Logan, UT), 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin (Biological Industries), cultured in 100-mm Petri dishes, and grown in a humidified atmosphere of 5% CO2, 95% air at 37 °C. All cell cultures were tested for mycoplasma contamination and found to be negative.

Primary Hepatocytes—Mice primary hepatocytes were obtained by perfusion of liver from C57BL female mice, age 7–8 weeks according to the method described by Berry et al. (13) with the following modifications: liver perfusion rate was 5 ml/min without recirculation of the perfusate. Cells were maintained under the same conditions as HepG2 cells.

Protein Delivery into Cultured Cells—The human cell lines HepG2, HuH7, and Colo205 were incubated with PAH-based fusion proteins for various time periods, the cells were collected by centrifugation and washed twice with cold PBS. The cells were centrifuged at 500 x g for 5 min at 4 °C, and the packed cell volume was determined. The packed cells were resuspended in two packed cell volumes of buffer A (10 mM Hepes, pH 7.5, 1.5 mM MgCl2, 5 mM KCl) and allowed to swell on ice for 15 min. The cells were then lysed by rapidly pushing them through a narrow pipette tip 20 times. The cell homogenate was centrifuged at 10,000 x g for 5 min to obtain the cytoplasmic supernatant (cytoplasmic fraction). The internalized fusion proteins were detected in the cytoplasmic fraction by assessing cytoplasmic PAH enzymatic activity.

For analysis of cells treated with PAH-based fusion proteins by confocal microscopy, HepG2 cells grown on coverslips to 50–70% confluency were treated with TAT-PAH (20 µg/ml final concentration) and NK1-PAH and NK2-PAH (2 µg/ml final concentration) for various time periods. Cells were than washed with PBS, and with 0.05% trypsin for 2 min at 37 °C, fixed in 3.7% formaldehyde in PBS for 10 min at room temperature, and then permeabilized with 0.2% Triton X-100 for 30 min. Following a second wash, cells were incubated with mouse anti-human PAH antibody (PH8, 1:200, Chemicon) and then with a fluorescein isothiocyanate-conjugated goat anti-mouse antibody (1:250, Jackson ImmunoResearch, Cambridgeshire, UK). Cells were then treated with RNase (20 µg/ml) and then with propidium iodide (5 µg/ml) to visualize cell nuclei. Cells were analyzed with a confocal laser scanning microscope (C1, Nikon Corp., Tokyo, Japan).

Cell Proliferation Assay—HuH7 and HepG2 cells were used to investigate the proliferative effect of PAH-based fusion proteins. 5 x 104 cells were grown in 96-well plates for 24 h prior to incubation with the proteins. TAT-PAH (20 µg/ml) and NK1-PAH and NK2-PAH (2 µg/ml) proteins were added to cells for a period of 72 h, after which Cell Titer Blue® reagent (Promega) was used according to the manufacturer's instructions to analyze cell survival.

HPLC Analysis of Plasma Phenylalanine and Tyrosine Concentrations—3 C57BL female mice, 7–8 weeks old were, used for each time point. Mice were injected with 200 µl of 100 µg/ml TAT-PAH and sacrificed at various times after the injection. 0.5-ml blood samples from the mice at each time point were collected in heparin tubes (Belliver Industrial Estate, Plymouth, UK). The tubes were centrifuged at 7,000 x g for 5 min at 4 °C to separate the plasma. 6% trichloroacetic acid was then added to the supernatant, the tubes were centrifuged again at 20,000 x g for an additional 5 min, and the supernatant was then collected and diluted 1:10 in mobile phase for analysis. Phe levels were measured in mouse plasma by HPLC analysis similar to that described by Atherton and Green (14) with the following modifications: the mobile phase was 7.5% acetonitrile (J. T. Baker, Deventer, Netherlands) 20 µl/liter octylamine (Sigma-Aldrich), and 800 µl/liter 11.6 M perchloric acid (Merck, Darmstadt, Germany). Samples were separated using a C18 column (150-mm octadecylsilane, 4.6-mm pore size, Knauer, Berlin, Germany). Phe levels were detected with the Spectroflow 773 absorbance detector at 193 nm (Kratos, Kyoto, Japan) and the SP4400 integrator (Spectra Physics, San Jose, CA). Analysis was performed on a Merck HPLC system.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Construction, Expression, Purification, and Characterization of the Fusion Proteins—We constructed plasmids encoding the PAH-based fusion proteins TAT-PAH, NK1-PAH, NK2-PAH, NK3-PAH, {alpha}HGF-PAH, and PAH alone. TAT-PAH contains the delivering moiety TAT, which possesses the ability to penetrate a wide variety of eukaryotic membranes but is mainly taken up by the liver. NK1-PAH, NK2-PAH, NK3-PAH, and {alpha}HGF-PAH consist of various fragments of the human {alpha}HGF sequence as the delivering moiety, to selectively target PAH to liver cells. PAH, without a delivering domain yet with a protease cleavage sequence, was used as a control protein. The coding sequence of the proteins was confirmed by restriction enzyme analysis and DNA sequence analysis (results not shown). Following transformation of E. coli BL21(DE3) cells with one of the plasmids, expression of the fusion gene was controlled by the bacteriophage T7 late promoter. A schematic representation of the new fusion proteins is shown in Fig. 1a.


Figure 1
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  		FIGURE 1.
Schematic representation of PAH-based proteins (a) and their expression and purification (b–d). a, schematic representation of PAH-based proteins (PAH (control protein), TAT-PAH, NK1-PAH, NK2-PAH, NK3-PAH, and {alpha}HGF-PAH; *, amino acid (aa) sequence of PAH before proteolytic cleavage of the thrombin site on the N terminus). b, SDS-PAGE analysis of the purified fractions of TAT-PAH (1) and PAH (2). These proteins were purified using affinity chromatography as described under "Experimental Procedures." c, Western blot analysis of purified: TAT-PAH (1) and PAH (2) using antibodies against PAH. d, Western blot analysis of NK1-PAH (1), NK2-PAH (2), NK3-PAH (3), and {alpha}HGF-PAH (4), using antibodies against PAH. W, whole cell fraction of E. coli; S, soluble fraction; I, insoluble fraction. PAH, human phenylalanine hydroxylase; TAT, transactivator of transcription peptide; NK1, NK2, and NK3, N' terminal amino acid sequences of the {alpha} subunit of the human hepatocyte growth factor including kringles 1, 1 and 2, and 1–3, respectively; {alpha}HGF, {alpha} subunit of the human hepatocyte growth factor.

 
Analysis of the soluble fraction of E. coli-expressing cells on SDS-PAGE gels revealed a major band of the expected molecular mass of 53 kDa corresponding to PAH and TAT-PAH proteins (Fig. 1b). These bands reacted with anti PAH antibodies (Fig. 1c) and with antibodies against the His tag (data not shown). Immunoblotting analysis of the soluble fractions of E. coli cells expressing NK1-PAH and NK2-PAH using anti-PAH antibodies revealed bands of 76 and 87 kDa, respectively (Fig. 1d). Extracts of BL21 cells expressing the NK3-PAH (97 kDa) and the {alpha}HGF-PAH (109 kDa) fusion proteins revealed that these proteins were found mainly in the insoluble fraction (Fig. 1d). The soluble fraction of TAT-PAH and PAH was loaded on a nickel chelating column to affinity-purify these PAH-based proteins. Both the TAT-PAH and control PAH proteins were purified in a single step. Eluted proteins showed a major band of the expected size and were >95% pure, as determined by SDS-PAGE analysis (Fig. 1b).

Enzymatic Activity of the Proteins—TAT-PAH- and control PAH-purified proteins were tested for their PAH activity by an in vitro cell-free assay. The proteins were incubated in a reaction mixture containing the cofactors and reagents necessary for activity, and the PAH activity was monitored by following the conversion of [14C]Phe substrate into [14C]Tyr. The soluble fraction of the PAH-based proteins was found to be active in vitro, exhibiting the enzymatic activity of converting Phe to Tyr (Fig. 2). PAH and TAT-PAH proteins show a dose-dependent activity when the protein concentration is >40 ng/ml. However, at the concentration range of 40–4000 ng/ml, PAH exhibited a higher enzymatic activity than that of TAT-PAH (Fig. 2a). Further analysis conducted on both proteins revealed a higher Vmax value for the PAH enzyme (3.98 µM·min-1·µg-1 ± 0.63) than for TAT-PAH (1.76 µM·min-1·µg-1 ± 0.80). However, the Km values as calculated from this plot were the same for both proteins (9.55 µM ± 1.81 for PAH, and 9.47 µM ± 0.94 for TAT-PAH) (Fig. 2b). Thus, changes that decrease the specific activity were, most probably, induced in the PAH enzyme upon fusion with the TAT peptide. These changes however, did not cause a change in the affinity of the fusion protein to its substrate. It should be pointed out that the PAH-based proteins showed PAH enzymatic activity only in the soluble subcellular fraction. This is despite multiple efforts to denature and properly refold the highly enriched insoluble subfraction under various conditions.

An in vitro enzymatic activity test of the four HGF-targeted clones revealed that only the NK1-PAH and NK2-PAH fusion proteins retained the enzymatic activity of the PAH enzyme in the soluble fraction (Fig. 2c, lines 1 and 2). The enzymatic activity of these proteins was highly specific and dose-dependent. The soluble fraction of E. coli cells expressing NK3-PAH and {alpha}HGF-PAH did not show any activity (Fig. 2c, lines 3 and 4). As for the TAT-PAH protein, attempts made to denature and properly refold the four clones from the insoluble fraction failed and ended in a dysfunctional enzyme. Consequently, all subsequent experiments were performed with highly purified PAH-based proteins from the soluble subfraction of cells expressing TAT-PAH and PAH, or from the enriched soluble fraction of cells expressing our chimeric proteins NK1-PAH and NK2-PAH.

Internalization of PAH-based Proteins Assessed by Confocal Microscopy—HepG2 and HuH7 hepatocarcinoma cells were incubated with 20 µg/ml (final concentration) TAT-PAH and 2 µg/ml (final concentration) of NK1-PAH and NK2-PAH proteins for various times. A series of experiments using fluorescein isothiocyanate-conjugated anti-PAH antibodies and confocal microscopy were performed to visualize the internalization of the TAT-PAH protein into cells.

The images of treated cells revealed TAT-PAH within the cytoplasm of both HepG2 (Fig. 3) and HuH7 cells (data not shown) after 30-min incubation. TAT-PAH was distributed evenly throughout the cytoplasm of treated cells, and showed a stronger signal with time, as compared with control cells (Fig. 3). Images of HepG2 cells treated with TAT-PAH showed a stronger signal after 2-h incubation than that of HuH7 cells treated for the same period of time (data not shown), most probably indicating a more efficient delivery of the fusion proteins into HepG2 cells.


Figure 2
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  		FIGURE 2.
In vitro enzymatic activity of PAH-based proteins. a, PAH and TAT-PAH dose-dependent activity, based on intensity of the [14C]Tyr signals of an in vitro enzymatic activity assay of 2 h (as described under "Experimental Procedures"). b, Lineweaver-Burk plot of PAH and TAT-PAH (determination of the Km and Vmax of PAH and TAT-PAH: 1 µg of either PAH or TAT-PAH proteins were incubated in the presence of increasing concentrations of [14C]Phe: 2 µM, 4 µM, and 10 µM for time points: 5, 10, and 20 min. Formation of [14C]Tyr was quantified for each reaction and used to calculate the V0 for each substrate concentration. Once V0 values were determined, they were used to calculate the Vmax and Km values. c, chromatogram showing conversion of [14C]Phe to [14C]Tyr by whole cell (W) and soluble (S) fractions of E. coli expressing NK1-PAH (1), NK2-PAH (2), NK3-PAH (3), {alpha}HGF-PAH (4), and unrelated protein (5). M, equal amounts of [14C]Phe and [14C]Tyr.

 
We used two hepatocyte cell lines to test the ability of HGF targeting to deliver the PAH-based proteins into real liver target cells expressing cMet. HepG2 and HuH7 cell lines were first analyzed by reverse transcription-PCR for expression of cMet, and both of these lines were found to express this receptor in high levels (data not shown). Analysis of confocal images of HepG2 cells treated with NK1-PAH and NK2-PAH showed that these fusion proteins internalize into the cells within 10 min and that this internalization increases with time (Fig. 3). No increase in PAH was observed in the cells treated with the control PAH protein lacking a delivering domain (Fig. 3).


Figure 3
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  		FIGURE 3.
Internalization of PAH-based fusion proteins visualized by confocal microscopy. HepG2 cells were treated with TAT-PAH (20 µg/ml final concentration) and NK1-PAH and NK2-PAH (2 µg/ml final concentration) for 10 min, 30 min, 1 h, and 2 h. Cells were permeabilized and incubated with anti-PAH antibody followed by fluorescein isothiocyanate-conjugated secondary antibody and analyzed by a confocal laser scanning microscope (C1, Nikon). All images are at 1000x magnification.

 
Activity of PAH-based Proteins within Treated Cells—To assess the PAH activity of the fusion proteins after they were delivered into the cells, we lysed the treated cells with a hypotonic buffer. The reaction mixture containing the cofactors and [14C]Phe were then added to the cell lysates. This assay was based on the assumption that cell lysates of treated cells would show stronger PAH activity than untreated cells in an in vitro cell-free system. We found that lysates of cells treated with PAH-based proteins contained active PAH (Fig. 4). We found that both HuH7 and HepG2 cells had background PAH activity in the cell-free system assay. However, this activity was amplified in cells treated with TAT-PAH. The amplification of Tyr formation from Phe was observed in the homogenates of cells treated with TAT-PAH as after only 30-min incubation (Fig. 4a). The strongest effect of TAT-PAH on both HuH7 and HepG2 cells was seen after an incubation period of 3 h with an almost 10-fold decrease in Phe concentration. This decrease was observed for as long as 6 h. Longer incubation periods seem to decrease this effect (Fig. 4a). Similar results were observed in Colo205 cells treated with TAT-PAH (data not shown).


Figure 4
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  		FIGURE 4.
PAH enzymatic activity within extracts of cells treated with PAH-based proteins, "within-cell" activity. Cells were treated with TAT-PAH (20 µg/ml final concentration) for various times. Treated cells were then washed and homogenized. Cell homogenates were assayed for PAH activity by conversion of [14C]Phe to [14C]Tyr (as described under "Experimental Procedures"). a, line graphs of relative amounts of Phe (as compared to PBS-treated controls) in HuH7 and HepG2 cell extracts after 10-min to 72-h incubation with TAT-PAH (results are expressed as means ± S.E. of three independent repeats for each time point). b, PAH activity within extracts of HuH7 cells treated with PBS, PAH, and TAT-PAH (20 µg/ml final concentration) and NK1-PAH and NK2-PAH (2 µg/ml final concentration) for 3 h. These graphs are based on the analysis of the amount of [14C]Phe that remained in the cell extracts following a 2-h in vitro activity assay (as described under "Experimental Procedures"). Results are expressed as means ± S.E.; overall significance was determined by one-way analysis of variance with least significant difference test used for post hoc comparisons. Groups that share letters are not significantly different.

 
HepG2 and HuH7 are both hepatocarcinoma cell lines, strongly expressing the HGF receptor cMet. We next compared the decrease of [14C]Phe in the extracts of cells treated with the various proteins. A significant decrease in the [14C]Phe signal was observed in the lysates of cells treated with TAT-PAH (20 µg/ml final concentration) and NK1-PAH and NK2-PAH (2 µg/ml final concentration). Treatment with NK1-PAH had a greater effect (20-fold) on the decrease of [14C]Phe than did NK2-PAH (10-fold) (Fig. 4b, p < 0.01). We found no significant decrease in the [14C]Phe signal of the lysates of cells that were treated with the PAH protein lacking any delivering moiety as compared with PBS-treated control cells (Fig. 4b). These results confirmed the specific activity of the targeted fusion proteins.

To test the internalization and activity of our fusion proteins in primary cells, we isolated mouse primary hepatocytes and incubated them with TAT-PAH. Treated cells showed an almost 2-fold increase in radioactive Tyr formation from radioactive Phe as compared with control cells (Fig. 5a). Thus, TAT-PAH is internalized into primary mouse hepatocytes as well as into cells of established lines.


Figure 5
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  		FIGURE 5.
Effect of TAT-PAH on mouse primary cell lines and in vivo. a, PAH activity in extracts of mouse primary hepatocytes treated with PBS and TAT-PAH (20 µg/ml final concentration) for 3 h. Results are expressed as means ± S.E. of three independent repeats. b, plasma Phe levels relative to PBS-treated control mice. Plasma Phe concentrations were measured by HPLC after intravenous injection of TAT-PAH (20 µg) (as described under "Experimental Procedures"). Three C57Bl mice were used for each time point. Results are expressed as means ± S.E.

 
Toxicity and Proliferation—The delivery of fusion proteins into cells can cause undesired effects such as nonspecific toxicity or cell proliferation. Moreover, because HGF is known to induce proliferation of cells, it was important to rule out this possibility in cells treated with HGF-based fusion proteins. Therefore, PAH-based proteins were tested for their effect on the viability of HuH7 and HepG2 cells exposed to these proteins for 72 h. We used a proven toxic fusion molecule GnRH-PE as a positive control (15). We found no significant difference in the survival rate of HuH7 cells treated with 2 µg/ml NK1-PAH or NK2-PAH as compared with PBS-treated control cells (data not shown). Thus, HGF-PAH fusion proteins did not cause any toxic effect to the cells and did not contribute to the proliferation of the treated cells.

HPLC Analysis of Phe and Tyr Concentrations in Plasma of Mice Treated with TAT-PAH—PAH-based proteins should have the ability to reduce blood Phe concentration. To test the effect of TAT-PAH administration on blood Phe levels, three mice at each time point were administered 20 µg of highly purified TAT-PAH intravenously. Plasma was collected and analyzed by HPLC to determine Phe and Tyr levels. Phe was dramatically reduced with a decline starting after 15 min. The lowest Phe concentration was measured 1 h following TAT-PAH injection and stayed <20% of control for as long as 6 h (Fig. 5b). No significant rise in Tyr concentration was observed in these plasma samples.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We present here a novel approach for the treatment of PKU, by targeting a functional human PAH enzyme into liver cells. This enzyme will replace the activity of the mutant PAH found in the liver of PKU patients. To test the hypothesis that an active human PAH enzyme can be delivered into liver cells, we constructed the TAT-PAH fusion protein using the HIV protein transduction domain TAT as a delivering moiety. Moreover, to achieve better selectivity toward the liver, the primary target for the PAH enzyme, we constructed a series of liver-targeted fusion proteins, including NK1-PAH, NK2-PAH, NK3-PAH, and {alpha}HGF-PAH, using the human HGF as the targeting moiety.

By fusing human PAH to TAT and to fragments of HGF we were able to produce and purify enzymatically active fusion PAH-based proteins in bacterial cells. We demonstrated that TAT-PAH protein can be delivered to a variety of human cell lines and retain PAH activity for at least 6 h after internalization, and longer in some cases (see below). We were also able to demonstrate that by fusing PAH to NK1 and NK2 of human HGF, these proteins can enter human hepatocytes. Once inside the cells, these proteins sustained PAH activity. Finally, we treated C57BL mice with TAT-PAH proteins and showed that plasma Phe levels were lowered in the treated mice 15 min after intravenous administration, and remained low for several hours.

Our PAH-based fusion proteins were designed and constructed as independent PAH monomers. The PAH enzyme is naturally produced as a protease-resistant tetramer consisting of four independent monomers, each with the capability of hydroxylating Phe into Tyr. The PAH tetramer is formed by combining four monomers through a tetramerization domain at the C terminus of the enzyme (16). Thus, internalized PAH-based fusion proteins, designed as monomers, could be subjected to degradation by cellular proteases. However, we specifically constructed our PAH-based fusion proteins as monomers with the C-terminal free. This may enable these proteins to form protease-resistant tetramers once they enter the cell. Indeed, analysis of semi-native Western blots of TAT-PAH revealed bands corresponding to high molecular weights (data not shown). It is tempting to speculate that fusion of TAT together with an His6 tag at the N terminus of human PAH might still enable the formation of tetramers through the C terminus domain of this enzyme. Bands corresponding to higher molecular weights were also observed in Western blots of NK1-PAH and NK2-PAH (results not shown).

We found that TAT-PAH, NK1-PAH, and NK2-PAH fusion proteins were rapidly internalized into cells, and once inside the cells, these proteins were distributed throughout the cytoplasm (Fig. 3). Although the final concentrations of the NK1-PAH and NK2-PAH proteins were low and ~1/10 of the final concentration of TAT-PAH, the intensity of florescent signal was stronger in cells treated with NK1-PAH and NK2-PAH. This suggests that delivery of PAH is more efficient using fragments of the {alpha}HGF as the delivering moiety, although further analysis needs to be performed to confirm this idea.

Analysis of lysates from cells treated with TAT-PAH, NK1-PAH, and NK2-PAH retrieved from the soluble fraction showed a decrease in [14C]Phe and an increase in [14C]Tyr formation. The lysates of cells treated with the insoluble fractions did not show any additional PAH activity. Therefore, the increase in radioactive Tyr formation can only be explained by the contribution of PAH from the active fusion proteins that entered the cells. We were able to assess the stability of these proteins inside the cells by conducting the PAH enzymatic assay in a cell-free system after these proteins were internalized into the cells. TAT-PAH activity was observed in HuH7 cells up to 24 h. This activity lasted even longer in HepG2 cells, as long as 48 h (Fig. 4a). This difference in stability of TAT-PAH can be explained by a possible variation in the expression pattern of cellular proteases between these cell lines.

Because the NK1-PAH and NK2-PAH constructs are based on the HGF {alpha} subunit it was important to assess if addition of these sequences did not induce proliferation of the liver cells tested. We found no change in proliferation of liver cells treated with HGF-based fusion proteins, as assessed by a cell viability assay (data not shown). This is consistent with previous studies conducted on the binding properties of HGF, which revealed that {alpha}HGF and other smaller fragments of this factor can enter the cell without causing other downstream responses related to HGF (17, 18). This enables the use of the HGF-{alpha} subunit as a targeting moiety, while avoiding undesired biological responses that are induced by the growth factor itself.

The ultimate test in the treatment of PKU is to maintain stable low plasma Phe levels. Normal plasma Phe levels are 60 µmol/liter and are constant around this level from childhood until maturity. Children or adults displaying persistent values of Phe above 120 µmol/liter are defined as having hyperphenylalaninemia. Subsequent to the cell culture assays, we conducted an in vivo experiment to test whether the injection of PAH-based fusion proteins can affect plasma Phe levels of mice. Surprisingly, plasma Phe levels started to decrease 15 min after intravenous TAT-PAH injection and had decreased dramatically by 30 min. Phe levels remained <20% of the levels in control untreated mice for as long as 6 h. It is important to point out that this decrease of Phe levels was seen in healthy C57BL mice. This effect would probably be stronger in the PKU mouse model because plasma Phe levels of this mouse are much higher, and the main objective in treatment is lowering and maintaining a normal Phe concentration.

The possibility of targeting a protein to a specific tissue or cell has existed for some time. However, until now this approach has been specifically aimed at destroying a specific pathogenic cell population. In this report we present for the first time the possibility of targeting an active enzyme to specific cells that lack a key metabolic function. We have utilized the idea of targeting a protein, not to eliminate a specific cell as demonstrated previously by utilizing fusion proteins as targeted cytotoxic molecules (19), but rather to bestow a functional activity to the deficient cell.

In recent years new approaches for the treatment of PKU have been proposed. The first is based on gene therapy, suggesting the replacement of the mutant gene by the wild-type sequence encoding the normal PAH gene. This method, successfully tested on mice, can undoubtedly be used as the ultimate therapy (3, 4). Unfortunately, gene therapy is still considered not applicable to humans due to technical problems that have yet to be resolved. Liver replacement in PKU patients by the transplantation of a healthy organ will cure the disease but subject the patient to a wide variety of rejection complications (20).

Another promising potential cure has been suggested for PKU recently based on the PAL enzyme derived from the yeast Rhodosporidium toruloides. This enzyme is capable of degrading Phe by converting it to a harmless metabolite derivative that is cleared from the body through the kidneys. Oral administration of PAL has been shown to lower Phe levels in the blood of treated animals but is subject to proteolysis. Intravenous administration of PAL has been proven effective but will be recognized by the immune system as nonself (21). Current work on PAL involves PEGylation to reduce this effect (22).

Enzyme replacement therapy involves the administration of the wild-type PAH enzyme (23). Yet for the administration of this enzyme to the bloodstream to have an effect, it must be delivered to the liver, the site of action of PAH. Although all of these new approaches for treatment are considered promising, the low protein diet still remains the only clinical tool in PKU. Treatment of metabolic deficiencies by enzyme replacement therapy has already been proven effective in Gaucher disease type 1 (24). We, like others, believe that this could also be the most promising approach for the treatment of PKU. We believe that targeting the PAH enzyme to the liver by the use of a specific targeting moiety has the potential for the greatest benefits.

Using the approach we have presented here, human PAH enzyme can be delivered quickly and effectively into the preliminary site of action, the liver. After the PAH enzyme has internalized into the liver, it can act as a part of the normal metabolic pathway to lower Phe levels in the blood of PKU patients. This will also decrease the time of circulation of the enzyme in the blood, and thus can decrease the possible harmful immune effects. The idea of using PAH-based fusion proteins can be regarded as a potential alternative in the treatment of PKU.

Moreover, this approach, of targeting the wild-type enzyme to a specific tissue, could also be applied to treatment of any other metabolic deficiency for which the enzyme involved is known and cloned, and the target tissue identified. Thus, this new targeted enzyme replacement approach may open new paths in the treatment of metabolic diseases.


   FOOTNOTES
 
* This work was supported by the Israel Science Foundation (Grant 035358). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Tel.: 972-2-675-7465; Fax: 972-2-641-5848; E-mail: hayalg{at}md.huji.ac.il.

2 The abbreviations used are: PKU, phenylketonuria; PAH, phenylalanine hydroxylase; TAT, HIV transactivator of transcription; HGF, hepatocyte growth factor; NK1–3, N' terminus and first, second, and third kringle domains, respectively, of HGF; HIV, human immunodeficiency virus; cMet, Met oncogene; PBS, phosphate-buffered saline; HPLC, high-performance liquid chromatography. Back


   ACKNOWLEDGMENTS
 
We thank Ruth Belostotsky and Matan Rapoport for their valuable contribution to this work.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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