High-density lipoprotein mutant eye drops for the treatment of posterior eye diseases.

Age-related macular degeneration (AMD), in which choroidal neovascularization (CNV) affects the center of the retina (macula), leads to the irreversible visual loss. The intravitreal injection of anti-angiogenesis antibodies improved the prognosis of AMD, but relatively less invasive therapies should be explored. In the present study, we show that a high-density lipoprotein (HDL) mutant is a therapeutically active drug carrier capable of treating a posterior eye disease in mice via instillation. Various HDL mutants were prepared with apoA-I proteins fused with different cell-penetrating peptides (CPPs) and phospholipids with different alkyl chain lengths; their sizes were further controlled in the range of 10-25nm. They were screened based on the efficiency of fluorescent dye delivery to the inner retinal layer in mice. The best mutant was found to have penetratin (PEN) as a CPP, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and a size of 15nm. In preclinical studies on a laser-induced CNV murine model, 1week of instillation of the best mutant carrying the anti-angiogenesis drug pazopanib had dramatic therapeutic effects in reducing the CNV size. Importantly, the HDL mutant by itself contributed to the therapeutic effects. Future clinical trials for treating AMD with instillation of the HDL mutant are expected.

In Vitro Cellular Gene Delivery Employing a Novel Composite Material of Single-Walled Carbon Nanotubes Associated With Designed Peptides With Pegylation.

Single-walled carbon nanotubes (SWCNTs) attract great interest in biomedical fields including application for drug delivery system. In this study, we developed a novel gene delivery system employing SWCNTs associated with polycationic and amphiphilic H-(-Lys-Trp-Lys-Gly-)7-OH [(KWKG)7] peptides having pegylation. SWCNTs wrapped with (KWKG)7 formed a complex with plasmid DNA (pDNA) in aqueous solution based on polyionic interaction but later underwent aggregation. On the other hand, a complex of pDNA and SWCNT-(KWKG)7 modified with polyethylene glycol (PEG) chains of 12 units [SWCNT-(KWKG)7-(PEG)12] afforded good dispersion stability for 24 h even in a cell culture medium. The in vitro cellular uptake of SWCNT-(KWKG)7-(PEG)12/pDNA complex prepared with fluorescence-labeled pDNA was evaluated with fluorescent microscopic observation and flow cytometry. The uptake by A549 human lung adenocarcinoma epithelial cells increased along with the extent of pegylation, suggesting the importance of dispersion stability in addition to the cationic charge which facilitates ionic cellular interaction. The expression of pDNA encoding the monomeric Kusabira-Orange 2 fluorescent protein in the form of the SWCNT-(KWKG)7-(PEG)12/pDNA complex demonstrated remarkable enhancement of transfection depending also on the extent of pegylation and the N/P ratio. The potential of the SWCNT composite wrapped with polycationic and amphiphilic (KWKG)7 with pegylation as a carrier for gene delivery was demonstrated.

Sustained Release of Mitomycin C from Its Conjugate with Single-Walled Carbon Nanotubes Associated by Pegylated Peptide.

A novel sustained release formulation of mitomycin C (MMC) was developed by employing single-walled carbon nanotubes (SWCNTs) wrapped by designed peptide with polyethylene glycol (PEG) modification (pegylation) as a nano-scale molecular platform. The amino groups of polycationic and amphiphilic H-(-Cys-Trp-Lys-Gly-)(-Lys-Trp-Lys-Gly-)6-OH [CWKG(KWKG)6] peptide associated with SWCNTs were modified using PEG with 12 units (PEG12) to improve the dispersion stability of the composite. Then thiol groups of peptide were conjugated with MMC using N-ε-maleimidocaproic acid (EMCA) as a linker via transformation of aziridine group of MMC. The obtained SWCNTs-CWKG(KWKG)6-(PEG)12-C6-MMC composites particularly that with 13.6% PEG modification extent of amino groups, showed good dispersion stability both in water and in a cell culture medium for 24 h. The release of MMC from SWCNTs-CWKG(KWKG)6-(PEG)12-C6-MMC was confirmed to follow first-order kinetics being accelerated by the pH increase in good agreement with the results observed for MMC-dextran conjugate with the same conjugation structure. The SWCNTs-CWKG(KWKG)6-(PEG)12 composite exhibited a considerably low cytotoxicity against cultured human lung adenocarcinoma epithelial cell line (A549). In contrast, SWCNTs-CWKG(KWKG)6-(PEG)12-C6-MMC demonstrated delayed but relatively corresponding antitumor activity with free MMC at the same concentration. The results suggested the potential role of SWCNTs-CWKG(KWKG)6-(PEG)12 as a carrier for a controlled release drug delivery system (DDS).

Development of Novel Drug and Gene Delivery Carriers Composed of Single-Walled Carbon Nanotubes and Designed Peptides With PEGylation.

Single-walled carbon nanotubes (SWCNTs) attract great interest in biomedical applications including drug and gene delivery. In this study, we developed a novel delivery system using SWCNTs associated with designed polycationic and amphiphilic peptides. Wrapping of SWCNTs with H-(-Lys-Trp-Lys-Gly-)7-OH [(KWKG)7] resulted in stable dispersion in water, but the composite aggregated in the buffered solution. This dispersion instability was also evident in a cell culture medium with fetal bovine serum. To improve the aqueous dispersibility, the SWCNTs-(KWKG)7 composite was further modified with polyethylene glycol (PEG) at the lysine residues via amide bond formation and the highest modification extent of 13.3% of the amino groups which corresponded to 2 PEG chains in each peptide molecule was achieved with fluorescein isothiocyanate-labeled carboxyl-PEG12. The uptake of the SWCNTs composite by A549 human lung adenocarcinoma epithelial cells was evaluated by visual observation and fluorescence activated cell sorting analysis for SWCNTs wrapped with a mixture of (KWKG)7 with PEGylation and H-(-Cys-Trp-Lys-Gly-)-OH-(KWKG)6 [CWKG(KWKG)6] labeled with fluorescent boron-dipyrromethene tetramethylrhodamine and 7-fold higher uptake comparing with SWCNTs-peptide composite without PEGylation was obtained suggesting the importance of dispersibility in addition to a cationic charge. The superior potential of SWCNTs composites assisted by polycationic and amphiphilic peptides with PEGylation was thus demonstrated.

Asp3Gly polymorphism affects fatty acid-binding protein 3 intracellular stability and subcellular localization.

Fatty acid-binding proteins (FABP) play a crucial role in intracellular fatty acid transportation and metabolism. In this study, we investigate the effects of the FABP3 Asp3Gly (D3G) polymorphism on protein structure and function. Although the mutation did not alter protein secondary structure or the ability to bind 1-anilinonaphthalene-8-sulfonic acid and palmitate, the intracellular stability of the D3G mutant was significantly decreased. Immunocytochemical analysis reveals that the mutation alters FABP3 subcellular localization. Our results suggest that the D3G polymorphism may impact energy metabolism and physiological functions.

Preparation of immunostimulatory single-walled carbon nanotube/CpG DNA complexes and evaluation of their potential in cancer immunotherapy.

Carbon nanotubes (CNTs) have many interesting properties. In particular, their photohyperthermic effect by near-infrared (NIR) irradiation could be used to kill cancer cells, and could thus be applied in photohyperthermic therapy. However, the solubility of CNTs must be improved before they can be used in biological applications. As DNA is reported to disperse the CNTs in aqueous solution with π-π interactions, we hypothesis that immunostimulatory CpG DNA may also disperse the CNTs in aqueous solution. In this study, we used CpG DNA to disperse single-walled CNTs (SWCNTs) in aqueous solution, in order to combine photohyperthermic effect and immunoactivation together to achieve a more effective cancer therapy. As expected, CpG DNA effectively dispersed the SWCNTs in aqueous solution via the formation of SWCNT/CpG DNA complexes. Moreover, the immunoreactivity of the SWCNT/CpG DNA complexes was investigated. The results showed that intratumoral administration of the SWCNT/CpG DNA complexes in mice enhanced the production level of inflammatory cytokines in tumor tissues. Finally, we evaluated the antitumor effects of the SWCNT/CpG DNA complexes in tumor-bearing mice. The result indicated that intratumoral administration of the SWCNT/CpG DNA complexes combined with NIR irradiation was a more effective approach to prevent the proliferation of tumor growth.

Photothermal ablation of tumor cells using a single-walled carbon nanotube-peptide composite.

Single-walled carbon nanotubes (SWCNTs) are known to have great potential for biomedical applications such as photothermal ablation of tumor cells in combination with near-infrared (NIR) irradiation. In this study, the photothermal activity of a novel SWCNTs composite with a designed peptide having a repeated structure of H-(-Lys-Phe-Lys-Ala-)7-OH [(KFKA)7] against tumor cells was evaluated in vitro and in vivo. The SWCNT-(KFKA)7 composite demonstrated high aqueous dispersibility that enabled SWCNTs to be used in tumor ablation. The NIR irradiation of SWCNT-(KFKA)7 solution resulted in a rapid temperature increase dependent on the SWCNTs concentration up to 50µg/ml. Three minutes of NIR irradiation of a colon 26 or HepG2 cell culture incubated with SWCNT-(KFKA)7 resulted in remarkable cell damage, while that by single treatment with SWCNT-(KFKA)7 or NIR irradiation alone was moderate. The intratumoral injection of SWCNT-(KFKA)7 solution followed by NIR irradiation resulted in a rapid increase of the temperature to 43°C in the subcutaneously inoculated colon 26 tumor based on thermographic observation and remarkable suppression of tumor growth compared with treatment with only SWCNT-(KFKA)7 injection alone or NIR irradiation alone. These results suggest the a great potential of an SWCNT-peptide composite for use in photothermal cancer therapy.

Effects of conversion of the zinc-binding motif sequence of thermolysin, HEXXH, to that of dipeptidyl peptidase III, HEXXXH, on the activity and stability of thermolysin.

Most zinc metalloproteinases have the consensus zinc-binding motif sequence HEXXH, in which two histidine residues chelate a catalytic zinc ion. The zinc-binding motif sequence of thermolysin, H(142)ELTH(146), belongs to this motif sequence, while that of dipeptidyl peptidase III (DPP III), H(450)ELLGH(455), belongs to the motif sequence HEXXXH. In this study, we examined effects of conversion of HEXXH to HEXXXH in thermolysin on its activity and stability. Thermolysin variants bearing H(142)ELLGH(146) or H(142)ELTGH(146) (designated T145LG and T145TG respectively) were constructed by site-directed mutagenesis and were produced in Escherichia coli cells by co-expressing the mature and pro domains separately. They did not exhibit hydrolyzing activity for casein or N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide, but exhibited binding ability to a substrate analog glycyl-D-phenylalanine (Gly-D-Phe). The apparent denaturing temperatures based on the ellipticity at 222 nm of T145LG and T145TG were 85 ± 1 °C and 86 ± 1 °C respectively, almost the same as that of wild-type thermolysin (85 ± 1 °C). These results indicate that conversion of HEXXH to HEXXXH abolishes thermolysin activity, but does not affect its binding ability to Gly-D-Phe or its stability. Our results are in contrast to ones reported previously, that DPP III variants bearing H(450)ELGH(455) exhibit activity.

Development of a novel composite material with carbon nanotubes assisted by self-assembled peptides designed in conjunction with ß-sheet formation.

A novel composite material is developed with single-walled carbon nanotubes (SWCNTs) and artificially designed peptides, and its chemical and physicochemical characteristics are evaluated with an aim toward biomedical application. The peptides were designed to form a ß-sheet structure that would be suitable for wrapping SWCNTs. The complex of SWCNTs and peptide (SWCNT-peptide) showed good dispersibility in aqueous media and was considerably stable even in the absence of an excess amount of peptide in the media. The formation of SWCNT-peptide was confirmed by its performance in water, atomic force microscopy and transmission electron microscopy observation, and molecular modeling. The possibility of introducing various functions to SWCNT-peptide was also demonstrated by several methods, such as introduction of special amino acids, chemical modification, and additional complex formation based on electrostatic interaction. These results suggest the potential of the SWCNT-peptide complex as a molecular platform on which a desirable structure and/or function can be constructed for biomedical and industrial application.

Stabilization of bovine intestine alkaline phosphatase by sugars.

Bovine intestine alkaline phosphatase (BIALP) is widely used as a signaling enzyme in sensitive assays such as enzyme immunoassay. In this study, we evaluated the effects of sugars on the kinetic stability of BIALP in the hydrolysis of p-nitrophenylphosphate (pNPP). The temperatures reducing initial activity by 50% in a 30-min incubation, T(50), of BIALP with 1.0 M disaccharide (sucrose and trehalose) or 2.0 M monosaccharide (glucose and fructose) were 55.0-55.5 °C, 4.7-5.2 °C higher than without sugar (50.3±0.1 °C). The T(50) of BIALP increased to 58.4±0.3 °C when the trehalose concentration was from 1.0 to 1.5 M, but did not change when the glucose concentration was from 2.0 to 3.0 M. Thermodynamic analysis revealed that the stabilization of BIALP by sugars was driven by the increase in the enthalpy change of activation for thermal inactivation of BIALP. No sugars affected the k(cat) of BIALP in the hydrolysis of pNPP. These results suggest that not only trehalose, which is considered the most effective stabilizer of enzymes, but also sucrose, glucose, and fructose can be used as stabilizers of BIALP.

Effects of amines and aminoalcohols on bovine intestine alkaline phosphatase activity.

Bovine intestine alkaline phosphatase (BIALP) is widely used as a signaling enzyme in sensitive assays such as enzyme immunoassay (EIA). In this study, we evaluated the effects of various aminoalcohols and amines on the activity of BIALP in the hydrolysis of p-nitrophenyl phosphate (pNPP) at pH 9.8, at 20 °C. The k(cat) values at 0.05 M diethanolamine, 0.1 M triethanolamine, and 0.2 M N-methylethanolamine were 190±10, 840±30, and 500±10 s(-1), respectively. The k(cat) values increased with increasing concentrations of diethanolamine, triethanolamine, and N-methylethanolamine and reached 1240±60, 1450±30, and 2250±80 s(-1), respectively, at 1.0M. On the other hand, the k(cat) values at 0.05-1.0M ethanolamine, ethylamine, methylamine, and dimethylamine were in the range of 100-600 s(-1). These results indicate that diethanolamine, triethanolamine and N-methylethanolamine highly activate BIALP and might be suitable as a dilution buffer of BIALP in EIA. Interestingly, the K(m) values increased with increasing concentrations of diethanolamine and N-methylethanolamine, but not triethanolamine: the K(m) value at 1.0M diethanolamine (0.83±0.15 mM) was 12-fold higher than that at 0.05M (0.07±0.01 mM), and that at 1.0M N-methylethanolamine (2.53±0.20 mM) was 14-fold higher than that at 0.2M (0.18±0.02 mM), while that at 1.0M triethanolamine (0.31±0.01 mM) was similar as that at 0.2M (0.25±0.01 mM), suggesting that the mechanisms of BIALP activation are different between the aminoalcohols.

Engineering, expression, purification, and production of recombinant thermolysin.

Thermolysin [EC 3.4.24.27] is a thermostable neutral zinc metalloproteinase originally identified in the culture broth of Bacillus thermoproteolyticus Rokko. Since the discovery in 1962, the enzyme has been extensively studied regarding its structure and catalytic mechanism. Today, thermolysin is a representative of zinc metalloproteinase and an attractive target in protein engineering to understand the catalytic mechanism, thermostability, and halophilicity. Thermolysin is used in industry, especially for the enzymatic synthesis of N-carbobenzoxy L-Asp-L-Phe methyl ester (ZDFM), a precursor of an artificial sweetener, aspartame. Generation of genetically engineered thermolysin with higher activity in the synthesis of ZDFM has been highly desired. In accordance with the expansion of studies on thermolysin, various strategies for its expression and purification have been devised and successfully used. In this review, we aim to outline recombinant thermolysins associated with their engineering, expression, purification, and production.

Molecular mechanism of the inhibitory effect of cobalt ion on thermolysin activity and the suppressive effect of calcium ion on the cobalt ion-dependent inactivation of thermolysin.

Thermolysin activity in the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-l-leucine amide (FAGLA) and FA-l-leucyl-l-alanine amide (FALAA) was examined at various Co(2+) and Ca(2+) concentrations. It decreased to 28% with increasing [Co(2+)] up to 18 mM. The Co(2+)-dependent inactivation was in part suppressed by adding Ca(2+) ion up to 0.5 mM, but 33% of the activity remained to be inactivated even with a sufficient concentration of Ca(2+) (>0.5 mM). The Co(2+)-dependent inactivation was shown to be composed of Ca(2+)-sensitive and Ca(2+)-insensitive parts. In the latter part which is observed at [Ca(2+)] >0.5 mM, Co(2+) plays as a competitive inhibitor. On the other hand, the Co(2+)-dependent inactivation in the Ca(2+)-sensitive part observed at [Ca(2+)] <0.5 mM proceeds time-dependently following second-order kinetics, and the time-course is in good agreement with that of decrease in the thermolysin band due to autolysis in SDS-PAGE. This indicates that Co(2+) accelerates the autolysis. Here, we describe the co-regulation of thermolysin activity by Co(2+) and Ca(2+) ions and propose a molecular mechanism for the inhibition of thermolysin by Co(2+) and suppressive effect of Ca(2+) on the Co(2+)-dependent inhibition. Co(2+) ion inhibits thermolysin activity not only as a competitive inhibitor but also promoting the autolysis.

Kinetic analysis of the activation-and-inhibition dual effects of cobalt ion on thermolysin activity.

Thermolysin activity as well as its stability is remarkably enhanced by high concentration of neutral salts consisting of Na(+), K(+), Cl(-) and Br(-) in the synthesis and hydrolysis of N-carbobenzoxy-L-aspertyl-L-phenylalanine methyl ester and hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide (FAGLA) [Inouye, K. (1992) J. Biochem. 112, 335-340]. However, effect of divalent salts on thermolysin activity has not been investigated systematically. In this study, effect of Co(2+) ion on thermolysin activity in the hydrolysis of FAGLA was examined. Thermolysin activity increased 3-4 times with increasing the Co(2+) concentration to 2 mM, but the enhanced activity was considerably reduced with higher Co(2+) concentration (2-18 mM). The activation-and-inhibition dual effects of Co(2+) ion were analysed kinetically. Release of the catalytic Zn(2+) ion from thermolysin, concomitantly occurred with the Co(2+)-dependent activation, was measured with a Zn(2+)-specific fluorescent probe. This indicates that the activation is caused by substituting Co(2+) ion for the catalytic Zn(2+) ion. Meanwhile, the Co(2+)-dependent activation was inhibited competitively by Zn(2+) ion (0.1-1.0 muM) added, similarly to that it is inhibited by higher concentration of Co(2+) ion. These lines of evidence provide a strategy for regulating thermolysin activity with Co(2+) and Zn(2+) ions.

Effects of site-directed mutagenesis of the surface residues Gln128 and Gln225 of thermolysin on its catalytic activity.

Thermolysin is remarkably activated and stabilized by neutral salts with varying degrees depending on salt species, and particular surface residues are thought to be especially important in its activity and stability [Inouye, K. (1992) J. Biochem. 112, 335-340; Inouye, K. et al. (1998) Biochim. Biophys. Acta 1388, 209-214]. In this study, we examined the mutational effects of the surface residues of thermolysin. Gln128 and Gln225 were selected as the residues to be mutated because they are located on the surface loop and close to but not in the active site (23.5 and 15.8 A far from the active site zinc ion, respectively) and fully solvent accessible. Nine single mutants [Q128K (Gln128 is replaced with Lys), Q128E, Q128A, Q225K, Q225R, Q225E, Q225D, Q225A and Q225V] were constructed by site-directed mutagenesis. Mutational changes in catalytic activity were found only in the mutant thermolysins having a hydrophobic residue at the position 225 (Q225A and Q225V). In the hydrolysis of a neutral substrate N-[3-(2-furyl)acryloyl]-glycyl-l-leucine amide (FAGLA), the alkaline pK(a) value of Q225A is 8.48 +/- 0.04, being higher by 0.42 +/- 0.07 units than that of the wild-type thermolysin. The k(cat)/K(m) value of the wild-type enzyme is enhanced 14 times with 4 M NaCl, and those of Q225A and Q225V are enhanced 10 and 19 times, respectively. In the hydrolysis of a negatively charged substrate N-carbobenzoxy-l-aspartyl-l-phenylalanine methyl ester (ZDFM), unlike FAGLA, the initial velocities of Q225A and Q225V decreased to 30 and 50% of that of the wild-type enzyme, respectively. Their thermal stability is similar to that of the wild-type enzyme. These findings indicate that even a single mutation at the thermolysin surface induces changes in the electrostatic environment in the active site and affects the activity. Thus, site-directed mutagenesis of surface residues of thermolysin, including apparently thermodynamically unfavorable introduction of hydrophobic residues, should be explored to improve its activity and stability.

Engineering of the pH-dependence of thermolysin activity as examined by site-directed mutagenesis of Asn112 located at the active site of thermolysin.

Asn112 is located at the active site of thermolysin, 5-8 A from the catalytic Zn2+ and catalytic residues Glu143 and His231. When Asn112 was replaced with Ala, Asp, Glu, Lys, His, and Arg by site-directed mutagenesis, the mutant enzymes N112D and N112E, in which Asn112 is replaced with Asp and Glu, respectively, were secreted as an active form into Escherichia coli culture medium, while the other four were not. In the hydrolysis of a neutral substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide, the kcat/Km values of N112D and N112E exhibited bell-shaped pH-dependence, as did the wild-type thermolysin (WT). The acidic pKa of N112D was 5.7 +/- 0.1, higher by 0.4 +/- 0.2 units than that of WT, suggesting that the introduced negative charge suppressed the protonation of Glu143 or Zn2+-OH. In the hydrolysis of a negatively charged substrate, N-carbobenzoxy-l-Asp-l-Phe methyl ester (ZDFM), the pH-dependence of kcat/Km of the mutants decreased with increase in pH from 5.5 to 8.5, while that of WT was bell-shaped. This difference might be explained by the electrostatic repulsion between the introduced Asp/Glu and ZDFM, suggesting that introducing ionizing residues into the active site of thermolysin might be an effective means of modifying its pH-activity profile.

Extracellular production of recombinant thermolysin expressed in Escherichia coli, and its purification and enzymatic characterization.

Thermolysin is a representative zinc metalloproteinase derived from Bacillus thermoproteolyticus and a target in protein engineering to understand the catalytic mechanism and thermostability. Extracellular production of thermolysin has been achieved in Bacillus, but not in Escherichia coli, although it is the most widely used as a host for the production of recombinant proteins. In this study, we expressed thermolysin as a single polypeptide pre-proenzyme in E. coli under the original promoter sequences in the npr gene, the gene from B. thermoproteolyticus, which encodes thermolysin. Active mature thermolysin (34.6 kDa) was secreted into the culture medium. The recombinant thermolysin was purified to homogeneity by sequential column chromatography procedures of the supernatant with hydrophobic-interaction chromatography followed by affinity chromatography. The purified recombinant product is indistinguishable from natural thermolysin from B. thermoproteolyticus as assessed by hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide and N-carbobenzoxy-L-asparatyl-L-phenylalanine methyl ester. The results demonstrate that our expression system should be useful for structural and functional analysis of thermolysin.