1 Institute of Medical Biochemistry and Medical Molecular Biology, University of Graz, Harrachgasse 21/III 2 Institute of Organic Chemistry, University of Graz, Heinrichstrasse 28, 8010 Graz, Austria
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Abstract |
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Keywords: apo(a)/1H-NMR/kringle/Lys-Sepharose/refolding
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Introduction |
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Apo(a) is highly glycosylated protein sharing a high degree of sequence homology with plasminogen (McLean et al., 1987). This includes the sequences of the KIV domain of plasminogen, which are found in type 1 to type 10 (T1T10) apo(a) kringles, KV and the protease domain, the latter lacking an apparent proteolytic function in Lp(a). The Lp(a) assembly takes place extracellularly and a two-step model has been proposed (Chiesa et al., 1992
; Koschinsky et al., 1993
; White and Lanford, 1994
; Trieu and McConathy, 1995
; Frank and Kostner, 1997
). In the first step, unique kringles bind non-covalently to Lys and possibly other residues of apoB100 from LDL yielding an unstable complex which is dissociable with Lys analogues, proline and other charged molecules (Frank et al., 1995
). In the second step, the complex is stabilized by disulfide bridge formation between Cys4057 of apo(a) and Cys4326 of apoB100. Previous studies (Frank et al., 1994
, 1995
; Trieu and McConathy, 1995
) ascribed kringle IV-T6 the leading role in the first step of Lp(a) assembly. To study the mechanisms involved in Lp(a) assembly further we cloned, overexpressed and purified apo(a) KIV-T6 using an Escherichia coli expression system.
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Materials and methods |
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Restriction enzymes were obtained from New England Biolabs, unless specified otherwise. Human apo(a) KIV-T6 was PCR amplified together with its interkringle sequences (amino acids 11151227) using human cDNA (McLean et al., 1987) as a template. PCR amplification was performed using the following two synthetic oligonucleotides:
To permit the cloning of KIV-T6 sequence into the expression vector, an EcoRI restriction site was included in the N-terminal primer and a HindIII and two STOP codons were included in the C-terminal primer. The His6-tag sequence in the N-terminal primer allows single-step purification of recombinant protein by use of TALON metal affinity chromatography (Clontech); the His-tag may be removed by factor Xa cleavage. The conditions for 50 µl polymerase chain reaction (PCR) volume were as follows: 10 pM of oligonucleotides A and B each, 250 ng dNTP, 0.6 units of Taq-polymerase (Finnzymes) and 50 ng of human apo(a) cDNA as a template. The reaction conditions were as follows: denaturing temperature, 94°C, 60 s; annealing, 60°C, 60 s; extension, 72°C, 90 s; 25 cycles. PCR amplification of the 375 bp product was followed by gel purification, digestion with EcoRI and HindIII and ligation into EcoRI/HindIII cleaved pT7-7 vector. This construct (pT7-7/KIV-T6) was used to transform E.coli DH5- competent cells in order to amplify recombinant plasmid. Positive clones, confirmed by restriction analysis and DNA sequencing, were finally transformed into E.coli strain BL-21 (DE3) (Novagen).
Expression of apo(a) KIV-T6
The recombinant protein was expressed in E.coli strain BL-21 (DE3) under the following conditions: 1 l of LB medium [10 g of tryptone (Difco), 5 g of yeast extract (Difco), 5 g of NaCl/l distilled H2O, pH 7.4] containing 50 µg/ml ampicillin (Sigma) was inoculated with 10 ml of overnight culture of BL-21 (DE3) containing the pT7-7/KIV-T6 plasmid and incubated at 37°C with agitation (250 r.p.m.) until the culture achieved an A600 nm of 0.5. At this point the expression of recombinant apo(a) KIV-T6 was induced by addition of 0.05 mM isopropyl-ß-D-thiogalactoside (IPTG) (Sigma) and the culture was incubated for an additional 4 h. During this time, A600 nm reached ~1.0 and ~1.9 for induced and non-induced cultures, respectively. Cells were harvested by centrifugation at 6000 g for 10 min at 4°C and the cell pellets were frozen at 70°C until use. Approximately 2 g of cells were obtained per liter of culture medium. Aliquots from induced and non-induced cells were taken, frozen at 20°C and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE), followed by Coomassie Brilliant Blue staining in order to visualize protein bands.
Cell fractionation
After thawing, the cell pellet from 1 l of culture was resuspended in 50 ml of lysis buffer [(20 mM TrisHCl, 100 mM NaCl, 8 M urea (Sigma), 200 µl of 5 mg/ml DNase I (Sigma), final pH 8.0] and this suspension was gently stirred for 30 min on the turn-wheel (100 r.p.m.) at room temperature. To reduce viscosity, the suspension was additionally passed through A 21-gauge syringe needle and finally centrifuged at 12 000 g for 15 min at 4°C. The supernatant was used for further purification of apo(a) KIV-T6.
Purification of recombinant apo(a) KIV-T6
The purification of KIV-T6 was a combination of batch/gravity flow column. TALON metal affinity chromatography is based on the affinity of TALON for His-tagged recombinant protein (Clontech). TALON resin was thoroughly resuspended, an aliquot (2 ml of resin suspension per 5 mg of anticipated His-tagged protein) was briefly centrifuged at 700 g for 2 min at room temperature to pellet the resin, the supernatant was discarded and the resin was additionally washed with three volumes of lysis buffer as described in the original protocol (Clontech). Cell supernatant after cell fractionation was added to the resin and the suspension was gently stirred for 30 min on the turn-wheel (60 r.p.m.) at room temperature. The sample was centrifuged at 700 g for 5 min at room temperature and the supernatant was discarded. The resinprotein complex was washed twice with 30 ml of lysis buffer, followed by incubation for 10 min at room temperature and centrifugation at 700 g for 5 min each time. The resinprotein complex was transferred into the column (5x50 mm) and washed three more times using 20 ml of wash buffer lacking urea (20 mM TrisHCl, 100 mM NaCl, 10 mM imidazole, final pH 8.0) in order to remove traces of urea and non-specific proteins. Finally, apo(a) KIV-T6 was eluted with elution buffer (20 mM TrisHCl, 100 mM NaCl, 50 mM imidazole, 1 mM CaCl2, final pH 7.5) at a flow-rate of 0.5 ml/min. Protein was monitored at 280 nm and 0.5 ml fractions were collected. Proteolytic cleavage of purified His-tagged KIV-T6 was performed immediately after purification (see Results).
To confirm the proper kringle structure after refolding, the KIV-T6 was applied on a Lys-Sepharose 4B column (Pharmacia Biotech) followed by a wash with wash buffer A (50 mM TrisHCl, pH 8.0) and twice with wash buffer B (50 mM TrisHCl, 0.5 M NaCl, pH 8.0). Protein was eluted with elution buffer [50 mM TrisHCl, 0.2 M -aminocaproic acid (
-ACA), pH 8.0] at a flow-rate of 1 ml/min and 1.0 ml fractions were collected.
Proteolytic cleavage
For removal of the His-tag, recombinant His-tagged KIV-T6 was treated with factor Xa. Cleavage was performed immediately after purification (see Results) in modified cleavage buffer (20 mM TrisHCl, 100 mM NaCl, 50 mM imidazole, 1 mM CaCl2, final pH 8.0). A ratio of 5 units of factor Xa per milligram of recombinant protein was used and the mixture was gently stirred at 25°C for 16 h. To monitor the cleavage efficiency, the mobility shift of KIV-T6 was followed by SDSPAGE and immunoblotting.
SDSPAGE and immunoblot analysis
Aliquots of cell lysates and fractions containing KIV-T6 were mixed with sample buffer [20% (w/v) glycerol, 5% (w/v) SDS, 0.15% (w/v) bromophenol blue, 63 mmol/l TrisHCl, pH 6.8] and incubated for 10 min at 95°C. Samples were analyzed by SDSPAGE (15%) for 1 h at 150 V (running buffer: 25 mM Tris, 0.2 M glycine, 3.5 mM SDS) and protein bands were visualized by Coomassie Brilliant Blue staining for 30 min. In some cases samples were transferred to nitrocellulose membranes and incubated with a specific antibody against KIV-T6 (see below). Bands were visualized by enhanced chemiluminescence assay (ECL, Amersham) following the original protocol.
The specific polyclonal antibody against KIV-T6 was prepared by immunizing rabbits with three 500 µg portions of KIV-T6 for 3 weeks. A 50 ml volume of blood was then collected and centrifuged at 1500 g for 15 min at room temperature and plasma was treated with 600 mg/l of CaCl2 to eliminate fibrin. After 1 h of incubation at room temperature the sample was centrifuged at 45 000 g for 10 min; the supernatant was collected, supplemented with 1 mg/ml EDTA and 1 mg/ml NaN3 and stored at 20°C in small aliquots. An antiserum dilution of 1:500 was found to be optimal for immunoblotting.
Refolding of KIV-T6
This was performed as described (Buchner and Rudolph, 1991; LoGrasso et al., 1994
). After exchange of elution buffer by ultrafiltration (Amicon membrane, YM3 = 3000 MW), recombinant protein from 1 l of E.coli culture was resuspended in 3 ml of buffer A [100 mM TrisHCl, 6 M guanidineHCl (Sigma), 1 mM EDTA, 0.1 M DTT (Sigma), final pH 8.0] and stirred (40 r.p.m.) at room temperature for 2 h. This solution was diluted with buffer B [100 mM TrisHCl, 0.4 M arginine (Sigma), 1 mM EDTA, 4 mM oxidized glutathione (Sigma), final pH 8.0] to 100 ml and incubated at 10°C for 5 days. Samples were stored at 20°C until further use.
Reversed-phase high-performance liquid chromatographic (RP-HPLC) analysis
RP-HPLC was carried out on a Hewlett-Packard Series 1050 liquid chromatograph equipped with a 125x4 mm Vydac RP-4 column. A wateracetonitrile gradient, with acetonitrile concentration increasing from 10 to 90% in 60 min, was applied at a flow-rate of 1 ml/min. Both gradient compounds were supplemented with 0.1% trifluoroacetic acid (TFA). The UV absorbance was measured at 215 nm.
1H-NMR spectroscopy
One-dimensional 1H-NMR spectra were recorded at 27°C, using a Varian 600 MHz Inova Unity spectrometer. The NMR sample contained 0.1 mM of human apo(a) KIV-T6 in 15 mM sodium phosphate buffer, pH 7.4, supplemented with 5% D2O. Chemical shifts are reported in p.p.m. relative to the HOD resonance.
125I labeling of KIV-T6
After purification and refolding, 2 mg of KIV-T6 were dialyzed against PBS (phosphate-buffered saline, pH 7.4) and finally incubated with 1.0 mCi of 125I for 5 min at room temperature. In order to separate labeled protein from free 125I, the sample was chromatographed on a Sephadex G25M column followed by dialysis against 15 l of PBS for 14 h at 4°C. The final protein concentration as determined by the Lowry method for the particular batch was 1.82 mg/ml. The specific activity measured in a -counter (Cobra QuantumPackard) was 238 c.p.m./ng protein. The amount of free 125I in the sample was 5.4%.
Lys binding properties of refolded KIV-T6
125I-labeled KIV-T6 was incubated for 1 h with 2 ml of Lys-Sepharose at room temperature. After filling into a small column, the Lys-Sepharose was washed once with 4 ml of PBS, twice with 4 ml of 40 mM NaCl and additionally twice with 4 ml of PBS in order to remove NaCl. Elution was performed with increasing concentrations (0.2, 0.4, 0.6, 0.8, 1.0, 1.4, 1.8 and 2.2 mM) of -ACA. Fractions of 4 ml were collected and the protein concentrations were determined by measuring the radioactivity.
In order to study the affinity for LDL, refolded KIV-T6 was subjected to competition experiments for Lp(a) assembly. LDL was purified from healthy volunteers as described previously (Frank and Kostner, 1997). Amounts of 2 µg of human LDL were incubated with the recombinant apo(a) `XL' (Frank et al., 1994
) expressed in transiently transfected COS-7 with the admixture of various ratios of refolded KIV-T6 in DMEM medium, for 16 h at 37°C. The total reaction volume was 300 µl. The amount of apo(a) XL complexed to LDL (assembly rate) was determined by a DELFIA immunoassay, as described previously (Frank et al., 1999
).
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Results |
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The cDNA for recombinant KIV-T6 was engineered by PCR in such a way as to contain two specific restriction sites (EcoRI/HindIII) for insertion into the E.coli expression vector. The steps involved in this procedure are summarized in Figure 1. Additionally, a His6-tag and an Xa-cleavage site were added to the N-terminus to simplify purification of recombinant protein and the possibility of proteolytic removal of the His6-tag. The remaining three amino acids which precede the first histidine encoded by pT7-7 bacterial vector are MetAlaArg. The calculated molecular weight of the whole recombinant protein was 15.9 kDa.
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We examined various fractions of E.coli cells for the presence of KIV-T6 in order to determine the subcellular distribution of recombinant protein (data not shown). As reported previously for plasminogen and for other apo(a) kringles (Cleary et al., 1989; LoGrasso et al., 1994
; Chung et al., 1998
), most of the expressed KIV-T6 was present as insoluble protein in inclusion bodies. To obtain pure KIV-T6, inclusion bodies were solubilized with lysis buffer containing 8 M urea (see Materials and methods) and recombinant apo(a) KIV-T6 was isolated using TALON metal affinity chromatography. All proteins lacking His6-tag were eluted in the few wash steps, whereas apo(a) KIV-T6 remained bound to the column until elution with imidazole-containing elution buffer (Figure 3A
). Figure 3B
shows that the scale-up isolation in a one-step purification is very efficient, resulting in high purity (>95%) of human apo(a) KIV-T6. The solubility of pure KIV-T6 in TrisHCl, pH 8.0, was >10 mg/ml. In 10 mM HEPES buffer, pH 7.0, the solubility decreased to ~8 mg/ml.
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In order to remove N-terminal His6-tag from KIV-T6, proteolytic cleavage was performed with factor Xa. This protease recognizes the IleGluGlyArg tetrapeptide that was cloned between the His6-tag and the KIV-T6 sequences. We noticed that in our case the proteolytic cleavage was inefficient using cleavage buffer recommended by Pharmacia Biotech (50 mM TrisHCl, 100 mM NaCl, 1 mM CaCl2, pH 7.5). This was the case especially for the fractions which were stored at 20°C for more than 48 h and we presumed that this is caused by masking of the Xa cleavage site due to formation of intra- or inter-kringle accidental disulfide bonds during the protein storage. In order to avoid this, we prepared cleavage immediately after purification through the TALON column. After loading, the column was washed with buffer containing 8 M urea in order to remove non-specific proteins. Three additional wash steps followed using wash buffer without urea in order to remove traces of urea from the column. Finally, the cleavage was performed immediately after elution in the elution buffer with addition of 1 mM CaCl2 and factor Xa in the absence of urea. These modifications of cleavage procedure were necessary to increase the cleavage efficiency and they enabled us to obtain His-tag free KIV-T6 with a yield of ~70%. The cleavage efficiency was determined by SDSPAGE and immunoblotting, yielding a shift in electrophoretic mobility which corresponds to a molecular size shift of ~1.8 kDa (Figure 4).
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As the purification of recombinant apo(a) KIV-T6 from E.coli requires denaturing conditions, refolding was necessary to obtain a native kringle structure. As measures of proper kringle folding, four different methods were applied: binding to Lys-Sepharose (Figure 5A), SDSPAGE, RP-HPLC and 1H-NMR spectroscopy. Figure 5B
shows the SDSPAGE pattern of the identical sample once under reducing and once under non-reducing conditions; the non-reduced fraction migrated faster than the reduced fraction owing to its compact kringle structure after refolding.
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Purified refolded human apo(a) KIV-T6 was investigated by 1H-NMR spectroscopy to verify the correct folding. Several investigators have demonstrated in the past the appearance of proton resonances at ~0.2 and 1.0 p.p.m., which were considered as fingerprints for a proper kringle folding (DeMarco et al., 1982; Trexler et al., 1983
; Ramesh et al., 1987
; Thewes et al., 1987
). These resonances arise from the
,
1-CH3 protons of the conserved Leu46 residue by interaction with several ring currents caused by aromatic amino acid residues (DeMarco et al., 1985
). This fact has also been used previously to provide evidence for a proper refolding of recombinant human plasminogen kringle I (Menhart et al., 1991
) and human apolipoprotein(a) kringle 37 (LoGrasso et al., 1994
). The high-field region of a one-dimensional 1H-NMR spectrum is shown in Figure 7
. Resonances observed at 0.20, 0.15 and 1.05 p.p.m., respectively, in our case also demonstrated a proper structure of the refolded apo(a) KIV-T6.
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In order to study the binding affinity of KIV-T6 to surface-bound Lys, 125I-labeled protein in PBS, pH 7.4, was incubated with Lys-Sepharose and specifically bound material was eluted with increasing concentrations of -ACA ranging from 0.2 to 2.2 mM. By semilogarithmically plotting the fraction of bound KIV-T6 versus the
-ACA concentration, an EC50 value of 0.47 mM was obtained (Figure 8
).
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In order to investigate the extent to which refolded KIV-T6 might be able to interfere with the Lp(a) assembly using LDL and an intact recombinant apo(a) containing 18 KIV repeats, KV and the protease domain, apo(a)-XL was incubated with LDL in the absence and presence of increasing amounts of KIV-T6. As shown in Figure 9, a 100-fold molar excess of KIV-T6 inhibited Lp(a) assembly by only ~20%. At a 1000-fold molar excess the inhibition was not more than 60% compared with the assembly in the absence of KIV-T6.
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Discussion |
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The homogeneity of refolded and purified apo(a) KIV-T6 was assessed by RP-HPLC, showing a single peak and >99% purity. Furthermore, our purified and refolded KIV-T6 was studied by 1H-NMR spectroscopy and exhibited methyl doublet signals at 0.2 and 1.05 p.p.m., an important measure of proper kringle folding (DeMarco et al., 1982, 1985
; Ramesh et al., 1987
; Thewes et al., 1987
). These signals arise from the protons of the
,
1-CH3 groups of Leu45 and are a strong argument for the integrity of the folding of KIV-T6. This was further confirmed by the demonstration that purified and refolded apo(a) KIV-T6 bind to Lys-Sepharose. However, Lys-Sepharose binding of KIV-T6 was only approximately four times weaker than that of the previously described recombinant apo(a) showing the weakest lysine binding affinity (Frank and Kostner, 1997
). In that study we observed EC50 values for Lys-Sepharose binding in the range 211 mM
-ACA under conditions where KIV-T6 had an EC50 value of 0.47 mM. This is in line with previous observations that the binding affinity of recombinant apo(a) correlates positively with the number of kringles with lysine binding affinity (Frank and Kostner, 1997
). Our original intention was to investigate the interaction of KIV-T6 with LDL and to map the binding to surface structures of apoB100 in order to obtain further information on the mode of Lp(a) assembly. However, the binding affinity of this single kringle to LDL under physiological conditions was very low (Kd > 103 M) (data not shown). In competition experiments using LDL and intact recombinant apo(a) the relatively low affinity of the single kringle KIV-T6 was further underlined: At a 1000-fold molar excess this kringle inhibited only about 60% of the Lp(a) assembly. These findings suggest that the first step of Lp(a) assembly is more complex than simple interaction of apo(a) with Lys residues on the LDL surface. In line with this assumption is the fact that Lp(a) assembly is inhibited not only with Lys analogues but also with Pro and hydroxy-Pro (McConathy and Trieu, 1991
; Frank and Kostner, 1997
).
The availability of large amounts of pure native apo(a) KIV-T6 should enable us to perform further functional assembly studies. In addition, KIV-T6 is currently crystallized and its structure will be studied by X-ray crystallography.
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Notes |
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Acknowledgments |
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References |
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Received February 11, 2000; revised July 24, 2000; accepted August 10, 2000.