Preference toward a polylysine enantiomer in inhibiting prions
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- Jackson, K.S., Yeom, J., Han, Y. et al. Amino Acids (2013) 44: 993. doi:10.1007/s00726-012-1430-8
Differential anti-prion activity of polylysine enantiomers was studied. Based on our recent discovery that poly-l-lysine (PLK) is a potent anti-prion agent, we investigated suppression of prions in cultured cells using poly-d-lysine (PDK). The results showed that PDK was more efficacious than PLK to inhibit prions. Protein misfolding cyclic amplification assay demonstrated improved efficacy of PDK in inhibiting plasminogen-mediated prion propagation, corresponding to the enantio-preference of PDK observed in cultured cells. Furthermore, our study demonstrated that polylysines formed a complex with plasminogen. These results propose to hypothesize a plausible mechanism that elicits prion inhibition by polylysine enantiomers.
Prion diseases are fatal neurodegenerative disorders caused by infectious proteinaceous pathogens termed prions (Prusiner 1998; Ryou 2011). They are composed of scrapie prion protein (PrPSc), a misfolded isoform of cellular prion protein (PrPC) abundantly expressed in neuronal cells (Prusiner 1998; Ryou and Mays 2010). Prion diseases include scrapie in sheep and goats, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease in deer and elk, and Creutzfeldt–Jakob disease (CJD) in humans (Ryou 2007, 2010). While prion diseases pose a potential risk for human public health, either prevention or therapy is currently unavailable (Ryou 2011).
Polylysine is an artificial homo-polypeptide composed of several to hundreds of lysine residues which are linearly linked at the α-carbon groups by peptide bonds. Polylysine has been a useful model polypeptide to study formation and transition of the secondary structure of polypeptides (Greenfield and Fasman 1969). It was also frequently used as a biologically compatible medium to adhere cultured cells in vitro (Yavin and Yavin 1974) and as a non-viral vehicle to deliver nucleotide drugs (Luo and Saltzman 2000). Furthermore, biological activity of polylysine was reported to inhibit microbes (Shima et al. 1984), viruses (Langeland et al. 1988), and neoplastic cell growth (Arnold et al. 1979).
Recently, we demonstrated that plasminogen stimulates PrPSc propagation, playing a role as an auxiliary factor, while exclusion of plasminogen, destruction of plasminogen structure and interference of plasminogen–PrP interaction dampen stimulated PrPSc propagation (Mays and Ryou 2010). Our discovery on prion replication mechanism triggered development of anti-prion agents using polylysines that target the interaction of plasminogen with PrP. In our subsequent studies, polylysine as a form of poly-l-lysine (PLK) exhibited potent inhibitory effects against prion propagation in both cell-based and animal models of disease (Ryou et al. 2011). When ScN2a cells, a murine neuroblasotma cell line chronically infected with RML mouse-adapted scrapie prions (Butler et al. 1988), were incubated with PLK, the level of PrPSc was effectively decreased in a concentration responsive manner and the cells were completely cured with no reappearance of PrPSc even after the extended periods of cell culture. Most importantly, treatment with PLK significantly delayed the onset of disease in prion-infected mice and lowered the PrPSc level in their brains.
Polymerization of l-enantiomers of lysine leads to generation of PLK, whereas polymerization of d-enantiomers leads to generation of poly-d-lysine (PDK) (Fig. S1). PLK and PDK possess the same molecular formula and connectivity of the atoms, but only differ in spatial configuration of amino and carboxyl groups regarding the chiral α-carbon. Because the stereoisomers of compounds differ in their biological activities (David 1997), we hypothesized that the ability of polylysine stereoisomers is different to inhibit prion propagation.
In this study, we compared the anti-prion activity of the stereoisomers of polylysine by measuring the PrPSc levels in two independent cell lines chronically infected by different prion strains. We then investigated differential cytotoxicity of PLK and PDK to reveal whether the efficacious PLK and PDK concentrations to inhibit PrPSc propagation are below the cytotoxic concentrations. Furthermore, we ascertained the effect of polylysine with different physical states and investigated plausible scenarios by which polylysine stereoisomer-mediated anti-prion activity and enantio-preference were elicited.
Materials and methods
PLK and PDK
PLK30–70 and PDK30–70 were purchased from Sigma-Aldrich (St. Louis, MO). The molecular weight of these polylysines ranges from 30 to 70 kDa with the average at ~50 kDa. Their average polymerization degrees (the number of lysine monomers per polymer molecule) were ~240 ranging 144–336. PLK300 and PDK300 were also purchased from Sigma-Aldrich and their average molecular weights are 300 kDa. PLK52 and PDK52 were purchased from Alamanda Polymers Inc. (Huntsville, AL). The molecular weight of these polylysines ranges from 47 to 57 kDa with the average at ~52 kDa. Their deduced average polymerization degrees were ~250 ranging 225–275. The properties of PLK and PDK are summarized in Table S1.
Anti-prion efficacy assay in cells
Anti-prion activity of PLK and PDK was assayed as previously described using two different cell lines with chronic prion infection (Ryou et al. 2003, 2011). ScN2a and SMB cells were initially seeded at 2 % confluency in culture dishes (60-mm in diameter, Corning, Lowell, MA) and cultured with Dulbecco’s modified Eagle’s medium (DMEM) containing 10 % fetal bovine serum, 1 % penicillin–streptomycin, and 1 % Glutamax under 5 % CO2 and saturated humidity conditions. All cell culture reagents were purchased from Invitrogen (Carlsbad, CA). Treatment of cells with various concentrations of the PLK30–70/PDK30–70 and PLK52/PDK52 pairs began in 3 h as the cells anchored onto the plastic surface of culture containers by directly adding PLK and PDK to culture media. Incubation lasted for 6 days with a media replacement at the fourth day using fresh culture media containing PLK and PDK.
To measure anti-prion activity of immobilized PLK and PDK, culture dishes were coated with each polylysine stereoisomer using 0.1 mg/ml filter-sterilized stock solution prepared in water. After one half of a ml was aseptically transferred to a cell culture dish, it was gently rocked to evenly coat the surface for 5 min. Once the excess solution was removed, the surface of dish was thoroughly rinsed with a largely volume of sterile water and allowed to dry for several hours. The same numbers (5 × 105) of ScN2a cells were plated and cultured under the same condition described above for 5 days until cells became confluent.
Once the incubation period ended, cell lysate was prepared with Tris/NaCl buffer containing detergents (20 mM Tris, pH 8.0; 150 mM NaCl; 0.5 % Nonidet P-40; 0.5 % sodium deoxycholate). An aliquot (~30 μg) of cell lysate was analyzed for Western blotting for total PrP and β-actin. The rest (2 mg) of cell lysate was incubated with 20 μg/ml proteinase K (PK) for 1 h at 37 °C with vigorous shaking. PK-resistant PrPSc pelleted by centrifugation for 1 h at 16,000×g at 4 °C was detected by Western blotting. Detailed procedure for Western blotting is described elsewhere (Mays et al. 2008). Monoclonal anti-PrP antibodies, 6H4 (Prionics, Zurich, Switzerland) and 5C6 (gifted from G. Telling, Colorado State University), and anti-actin antibody, pan Ab-5 (Lab Vision Corp., Fremont, CA), were used for Western blotting. Densitometry analysis of Western blots was carried out using Doc-It Image Analysis software (UVP, Upland, CA).
MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay was used to measure cytotoxicity of PLK and PDK. The assay was carried out as previously described (Ryou et al. 2003, 2011). ScN2a cells were plated in a 12-well culture plate and incubated with PLK and PDK by the same method described for anti-prion activity assay. Following 6-day treatment, the cells were incubated for 2 h with DMEM containing 0.5 mg/ml MTT. Purple MTT formazan products converted by dehydrogenases and reductases of live cells were extracted in 0.05 N HCl–isopropanol and quantified by colorimetric readouts at 570 nm with background subtraction at 650 nm using DTX-880 Multimode Detector (Beckman-Coulter, Brea, CA).
In vitro prion propagation assay
Protein misfolding cyclic amplification (PMCA) (Saborio et al. 2001) was used to assay prion propagation in vitro. Plasminogen-mediated prion propagation assay was carried out as described previously (Mays and Ryou 2010). Briefly, purified human plasminogen (0.5 μM, Haematologic Technologies, Essex Junction, VT) was included in the PMCA reactions composed of the mixture of diseased and normal mouse brain extract (1:2,500 v/v). The effect of PLK and PDK was estimated in the PMCA reactions with plasminogen in which various concentrations of individual polylysine enantiomers were added. In this analysis, PLK300 and PDK300 were used in replacement of PLK30–70/PDK30–70 or PLK52/PDK52 to demonstrate that PLK and PDK with greater molecular weights are also capable of displaying anti-prion activity shown by PLK30–70/PDK30–70 or PLK52/PDK52. After 96 cycles of PMCA finished, the samples were analyzed by Western blotting as described previously (Mays et al. 2009).
Non-denaturing gel electrophoresis of plasminogen–polylysine complexes
Purified human plasminogen (0.5 μM, final concentration) was mixed with PLK or PDK (0–500 μg/ml, final concentration) in 20 μl of 10 mM Tris buffer (pH 7.5) containing 5 % glycerol (v/v). After incubation for 30 min at room temperature, the samples were subjected to 8 % non-denaturing polyacrylamide gel. The electrophoretic procedure was carried out using the modified method described elsewhere (Park and Raines 2004). The mobility of the plasminogen–polylysine complexes in the gel was visualized by staining with Silver Stain Kit (Pierce, Rockford, IL).
Measurement of particle size
The formation of particles composed of plasminogen and polylysine enantiomers was achieved by the similar manner described above. Briefly, purified human plasminogen (180 μg) and PLK/PDK (100 μg) were mixed in 10 mM Tris buffer (pH 7.5) (200 μl, final volume). The particle size of plasminogen–polylysine complexes was determined by dynamic light scattering (DLS) measurements using a Zetasizer Nano-ZS (Malvern, UK) (Bae et al. 2003). The instrument was equipped with a He–Ne laser (4 mW, 633 nm) and set up to collect 173° angle scattered light. Number distributions are presented as the mean particle size.
PrPSc destabilization assay
The ability of PLK and PDK to destabilize PrPSc was measured by the method described in our previous publication with modification (Lim et al. 2010). Brain homogenate (10 %, w/v) prepared in PBS from RML-infected CD-1 mice at the terminal stage was diluted 1:10 in 0.1 ml reaction buffer (50 mM sodium acetate buffer, 1 % NP-40) at acidic to neutral pH (pH 4, 5, 6, and 7). PLK52 and PDK52 were added to the reaction at a final concentration of 1 or 100 μg/ml. This PLK/PDK concentration was sufficient to exhibit anti-prion activity and enantio-preference in cell-based anti-prion efficacy assays. After incubation at 37 °C for 3 h with shaking at 300 rpm, 0.1 ml of PBS was added to the reaction. A 50 μl aliquot of samples were treated with PK under the same conditions described in the section above. The level of PrPSc was measured by Western blotting.
Results and discussion
The effect of PLK30–70 and PDK30–70 on expression of cellular proteins was negligible. The levels of β-actin and PrPC detected as a part of total prion protein (PrP) contents remained the same in cells incubated with or without PLK30–70 and PDK30–70 (Fig. 1a). Our results suggest that the observed anti-prion activity of PLK and PDK was not due to modulation of the PrPC abundance.
We then determined cytotoxicity of PLK and PDK. ScN2a cells showed no signs of cell death if they were incubated with up to 2 μg/ml of either PLK30–70 or PDK30–70 (Fig. 1c). PDK30–70 caused a drastic decrease of cell survival, leading to complete cell death at the higher concentrations, while PLK30–70 exhibited gradually increasing cytotoxicity at 4–10 μg/ml and caused complete cell death at >20 μg/ml. The dose responsiveness for PLK30–70 cytotoxicity in the current and previous (Ryou et al. 2011) studies was virtually identical. Taken together with the results of anti-prion efficacy assay in ScN2a cells, cytotoxicity study indicates that complete inhibition of PrPSc propagation by both PLK and PDK was achieved under non-toxic concentrations. Therefore, anti-prion activity of PLK and PDK appears to be involved in the processes of PrPSc propagation, but not to be facilitated by the mechanism that PLK and PDK inhibit survival of prion-infected cells.
Because the difference in the formation of the polylysine-plasminogen complex is not the only feasible mechanism to explain the enantio-preference, it remains to investigate other plausible scenarios. We envision that stability of the plasminogen–PDK complex is greater than that of the plasminogen–PLK complex, which will result in enantio-preference toward PDK in inhibiting plasminogen-mediated prion propagation. Previously, Hatton and Regoeczi (1975) showed that recovery of plasminogen [Type I fraction (Sodetz et al. 1972), which is deduced to include Glu-plasminogen, the plasminogen species used in this study] is greater from l-lysine-conjugated resin than from d-lysine-conjugated resin, suggesting that biochemical dissociation of plasminogen from PDK could be more difficult than that of plasminogen from PLK. Alternatively, preferential degradation of PLK may lead to enantio-specific anti-prion activity of PDK. Stability of free PDK in solution against proteolytic degradation by cellular proteases can be greater than that of PLK (Tsuyuki et al. 1956; Banecerraf and Levine 1964). This will result in a longer half-life of PDK, more complex formed between plasminogen and PDK, and differential anti-prion activity by polylysine enantiomers. Detection of these differences does not appear to be monitored by complex formation between polylysine and plasminogen. Xu et al. (2011) proposed differential degradation of polylysine enantiomers by cellular proteases, although experimental approaches were based on indirect determination of PLK degradation using flow cytometry that measured the level of PDK binding on the cells and a critical control experiment such as the binding of polylysine to the cells without PrP expression, which can guarantee to state the specific interaction of polylysines with PrP, is missing.
We demonstrate that PDK is more efficacious than PLK to inhibit multiple strains of scrapie prions. Furthermore, we found that prion propagation can be inhibited below the toxic levels of polylysine enantiomers. In vitro prion propagation assay with polylysine enantiomers suggests that a superior inhibitory activity of PDK in plasminogen-mediated prion propagation explains the enhanced anti-prion efficacy of PDK in cultured cells with chronic prion infection. Detailed mechanism for enantio-specific anti-prion activity of PDK remains open for further discussion. We emphasize that our study proposes an alternative suggestion that polylysines exert anti-prion effect through the interaction with plasminogen as compared to the interaction of PrP. Our study suggests an option to improve polylysine-based therapy for prion diseases, although additional preclinical and clinical investigations in the future remain to be performed. Because therapy for prion diseases is not available (Ryou 2011), application of the stereochemical principle proposes a unique strategy to advance development of potential anti-prion therapy.
The authors thank P. Thomason for editorial and H. Lee for technical assistance.
Conflict of interest
The authors declare that we have no conflict of interest.