A new drug delivery system for intravenous coronary thrombolysis with thrombus targeting and stealth activity recoverable by ultrasound.

OBJECTIVES: The purpose of this study was to develop a new intelligent drug delivery system for intracoronary thrombolysis with a strong thrombolytic effect without increasing bleeding risk. BACKGROUND: Rapid recanalization of an occluded coronary artery is essential for better outcomes in acute myocardial infarction. Catheter-based recanalization is widely accepted, but it takes time to transport patients. Although the current fibrinolytic therapy can be started quickly, it cannot achieve a high reperfusion rate. Recently, we generated nanoparticles comprising tissue-type plasminogen activator (tPA), basic gelatin, and zinc ions, which suppress tPA activity by 50% with 100% recovery by ultrasound (US) in vitro. METHODS: The thrombus-targeting property of nanoparticles was examined by an in vitro binding assay with von Wilbrand factor and with a mouse arterial thrombosis model in vivo. The thrombolytic efficacy of nanoparticles was evaluated with a swine acute myocardial infarction model. RESULTS: Nanoparticles bound to von Wilbrand factor in vitro and preferentially accumulated at the site of thrombus in a mouse model. In a swine acute myocardial infarction model, plasma tPA activity after intravenous injection of nanoparticles was approximately 25% of tPA alone and was recovered completely by transthoracic US (1.0 MHz, 1.0 W/cm(2)). During US application, plasma tPA activity near the affected coronary artery was recovered and was higher than that near the femoral artery. Although treatment with tPA alone (55,000 IU/kg) recanalized the occluded coronary artery in only 1 of 10 swine, nanoparticles containing the same dose of tPA with US achieved recanalization in 9 of 10 swine within 30 min. CONCLUSIONS: We developed an intelligent drug delivery system with promising potential for better intravenous coronary thrombolysis.

Ultrasound-responsive thrombus treatment with zinc-stabilized gelatin nano-complexes of tissue-type plasminogen activator.

This study is undertaken to design zinc-stabilized gelatin nano-complexes of tissue-type plasminogen activator (t-PA) for thrombolytic therapy where the t-PA activity can be recovered in the blood circulation upon ultrasound irradiation. Various molecular weights of gelatin were complexed with t-PA by their simply mixing in aqueous solution. Then, zinc acetate, calcium acetate or magnesium acetate was added to form nano-sized gelatin-t-PA complexes. The complexes had the apparent molecular size of about 100 nm. When zinc ions were added to the gelatin-t-PA complexes, the t-PA activity was suppressed most strongly to 57% of the original, free t-PA activity. Upon ultrasound exposure in vitro, the t-PA activity was fully recovered. A cell culture experiment with L929 fibroblasts demonstrated no cytotoxicity of complexes at the concentration used for the in vivo experiment. The half-life of t-PA in the blood circulation prolonged by the complexation with gelatin and zinc ions. The zinc-stabilized t-PA-gelatin complex is a promising t-PA delivery system which can manipulate the thrombolytic activity by the local ultrasound irradiation.

Highly potent fibrinolytic serine protease from Streptomyces.

We introduce a highly potent fibrinolytic serine protease from Streptomyces omiyaensis (SOT), which belongs to the trypsin family. The fibrinolytic activity of SOT was examined using in vitro assays and was compared with those of known fibrinolytic enzymes such as plasmin, tissue-type plasminogen activator (t-PA), urokinase, and nattokinase. Compared to other enzymes, SOT showed remarkably higher hydrolytic activity toward mimic peptides of fibrin and plasminogen. The fibrinolytic activity of SOT is about 18-fold higher than that of plasmin, and is comparable to that of t-PA by fibrin plate assays. Furthermore, SOT had some plasminogen activator-like activity. Results show that SOT and nattokinase have very different fibrinolytic and fibrinogenolytic modes, engendering significant synergetic effects of SOT and nattokinase on fibrinolysis. These results suggest that SOT presents important possibilities for application in the therapy of thrombosis.

Molecular dissection of Streptomyces trypsin on substrate recognition.

We recently identified residue 71 of two homologous serine proteases from Streptomyces omiyaensis (SOT) and Streptomyces griseus (SGT) as a crucial residue for differences in their topological specificities, i.e. recognition of a distinct three-dimensional structure. To study the role of this key residue in substrate recognition, we used surface plasmon resonance analysis to evaluate the affinities of inactive mutants, in which residues 71 of SOT and SGT were substituted respectively with Leu and Tyr, toward different types of collagens. We identified another amino acid residue involved in the interaction with collagens from analyses of inactive chimeras between SOT and SGT using an in vivo DNA shuffling system. Results showed that residue 72 contributes to collagen binding. By substituting Leu71 and Gln72 with Tyr and Arg, respectively, SGT mutant showed a change in topological specificity and high hydrolytic activity toward type IV collagen comparable to SOT. We demonstrated that the neighboring residues 71 and 72 in the N-terminal ß-barrel domain of the enzyme synergistically play an important role in substrate recognition.

Extracellular production and characterization of two streptomyces L-asparaginases.

L-Asparaginase (ASNase) has proved its use in medical and food industries. Sequence-based screening showed the thermophilic Streptomyces strain Streptomyces thermoluteus subsp. fuscus NBRC 14270 (14270 ASNase) to positive against predicted ASNase primary sequences. The 14270 ASNase gene and four L-asparaginase genes from Streptomyces coelicolor, Streptomyces avermitilis, and Streptomyces griseus (SGR ASNase) were expressed in Streptomyces lividans using a hyperexpression vector: pTONA5a. Among those genes, only 14270 ASNase and SGR ASNase were successful for overexpression and detected in culture supernatants without an artificial signal peptide. Comparison of the two Streptomyces enzymes described above demonstrated that 14270 ASNase was superior to SGR ASNase in terms of optimum temperature, thermal stability, and pH stability.

An ultrasound-responsive nano delivery system of tissue-type plasminogen activator for thrombolytic therapy.

This study is undertaken to design a novel nano-sized delivery system of tissue-type plasminogen activator (t-PA) which has a suppressed thrombolytic activity of t-PA, but recovered the activity only when exposed to ultrasound. Various amounts of ethylenediamine were chemically introduced into gelatin (cationized gelatins) to complex with t-PA. To modify the surface of complexes with polyethylene glycol (PEG), PEG was chemically grafted to the anionic gelatin (PEG-gelatin). The simple mixing with the PEG-gelatin enabled the t-PA-cationized gelatin complex to form a nano-sized delivery complex with PEG chains on the surface. The t-PA activity of PEG-modified complexes was significantly suppressed to be 45% of original t-PA. However, when exposed to ultrasound in vitro, the t-PA activity was fully recovered. A cell culture experiment demonstrated no cytotoxicity of PEG-modified complexes. The body distribution study indicated that the half-life of t-PA in the blood circulation was prolonged about 3 times. In a rabbit thrombosis model, the intravenous administration of PEG-modified complexes followed by ultrasound irradiation resulted in complete recanalization, in remarked contrast to the complex administration alone. It is concluded that the PEG-modified complex is a promising t-PA delivery system to enhance the biological activity at the site necessary only by a local ultrasound irradiation.

Engineered transaminopeptidase, aminolysin-S for catalysis of peptide bond formation to give linear and cyclic dipeptides by one-pot reaction.

Aminopeptidase from Streptomyces thermocyaneoviolaceus NBRC14271 was engineered into transaminopeptidase and used to catalyze an aminolysis reaction to give linear and cyclic dipeptides from cost-effective substrates such as the ester derivatives of amino acids.

The role of Tyr71 in Streptomyces trypsin on the recognition mechanism of structural protein substrates.

Studies of substrate recognition by serine proteases have focused on specificities at the primary S1-Sn sites, but topological specificities (i.e. recognition at distinct three-dimensional structural motifs) have not been established. This is the first report to identify the key amino acid residue conferring topological specificity. A serine protease from Streptomyces omiyaensis (SOT), which is a trypsin-like enzyme, was chosen as a model enzyme to clarify the recognition mechanism of structural protein substrates in serine proteases. We have found previously that the topological specificities of SOT and S. griseus trypsin (SGT) for high molecular mass substrates differ greatly, even though the enzymes have similar primary structures. In this study, we constructed chimeras between SOT and SGT using an in vivo DNA shuffling system and several mutants to identify the key residues involved in topological specificities. By comparing the substrate specificities of chimeras and mutants, we found that residue 71 of SOT, which is separate from the catalytic triad, contributes to the topological specificity. Using site-directed mutagenesis, residue 71 of SOT was also found to be crucial for catalytic efficiency and enzyme conformation.

Activation of oligopeptidase B from Streptomyces griseus by thiol-reacting reagents is independent of the single reactive cysteine residue.

Oligopeptidase B from Streptomyces griseus was cloned and characterized to clarify the substrate recognition mechanism and the role of a reactive cysteine residue in family S9 prolyl oligopeptidases (POPs). The cloned enzyme, SGR-OpdB, was annotated as a putative family S9 prolyl oligopeptidase based on its deduced amino acid sequence, in which a sole cysteine residue Cys(544) is present close to the catalytic Asp residue in the C-terminal region. The protein was identified as oligopeptidase B, a member of the subfamily S9a of the family S9 POPs, as judged by its substrate specificity and enzymatic characteristics. Its enzymatic activity was markedly enhanced by high NaCl concentration and the reducing reagents dithiothreitol (DTT) and reduced glutathione (GSH). It is particularly interesting that oxidized glutathione (GSSG) also enhanced SGR-OpdB activity. The SGR-OpdB C544A mutant was constructed and characterized to clarify the role of the putative reactive Cys residue, Cys(544). Surprisingly, the enzymatic activity of the Cys-free mutant was also markedly activated by the general thiol-reacting reagent DTT, GSH, and GSSG. To our knowledge, this is the first report of activity-enhancing effects of thiol-reacting reagents toward Cys-free enzymes. Results clarified the role of additives in inducing conformational change of SGR-OpdB into active peptidase.

Phospholipase D mechanism using Streptomyces PLD.

Phospholipase D (PLD) plays various roles in important biological processes and physiological functions, including cell signaling. Streptomyces PLDs show significant sequence similarity and belong to the PLD superfamily containing two catalytic HKD motifs. These PLDs have conserved catalytic regions and are among the smallest PLD enzymes. Therefore, Streptomyces PLDs are thought to be suitable models for studying the reaction mechanism among PLDs from other sources. Furthermore, Streptomyces PLDs present advantages related to their broad substrate specificity and ease of enzyme preparation. Moreover, the tertiary structure of PLD has been elucidated only for PLD from Streptomyces sp. PMF. This article presents a review of recently reported studies of the mechanism of the catalytic reaction, substrate recognition, substrate specificity and stability of Streptomyces PLD using various protein engineering methods and surface plasmon resonance analysis.

Bacillus D-stereospecific metallo-amidohydrolase: active-site metal-ion substitution changes substrate specificity.

From investigation of 2000 soil isolates, we identified a D-stereospecific metallo-amidohydrolase that can hydrolyze D-aminoacyl derivatives from the culture supernatant of Bacillus sp. 62E11: 62E11DppA. The enzyme binds two equivalents of zinc, exhibits 70% identity with that of D-aminopeptidases from Bacillus subtilis (DppA). In fact, 62E11DppA has strict specificity toward D-aminoacyl derivatives, i.e., the enzyme shows high activity toward D-aminoacyl benzyl esters and little activity toward D-amino acid containing peptides. Moreover, 62E11DppA exhibits a dramatic change in its activity and substrate specificity by substitution of metal ions in its active site. Based on results of kinetic studies using apo-62E11DppA with various metal ion and substrate concentrations, we propose a possible mechanism for the change in its activity and specificity by substitution of metal ions: the substitution of metal ions in 62E11DppA dramatically changes its activity by altering the substrate specificity.

Putative "acylaminoacyl" peptidases from Streptomyces griseus and S. coelicolor display "aminopeptidase" activities with distinct substrate specificities and sensitivities to reducing reagent.

Aminopeptidases from Streptomyces griseus (SGRAP) and S. coelicolor (SCOAP) were cloned and characterized to clarify their biochemical characteristics. Although both enzymes had been annotated as putative oligopeptidases of family S9 enzymes, they showed "aminopeptidase" activities, not "oligopeptidase" activities. Although their deduced amino acid sequences showed high similarity (69% overall sequence homology), they showed distinct substrate specificities and sensitivities to the reducing reagent dithiothreitol (DTT). The reaction pH and addition of DTT dramatically affected the substrate preference of SGRAP. Furthermore, SCOAP selectively hydrolyzed phenyalanine p-nitroanilide (Phe-pNA) in the presence or absence of DTT. The chimera protein between SGRAP and SCOAP was constructed to identify the region responsible for the properties described above. Furthermore, Cys(409) of SCOAP was identified as a functional residue responsible for activation by reducing reagent DTT.

pTONA5: a hyperexpression vector in Streptomycetes.

We constructed the Streptomyces hyperexpression vector pTONA5 based on pIJ702 vector; it includes a metalloendopeptidase (SSMP) promoter isolated from Streptomyces cinnamoneus TH-2 and a metalloendopeptidase terminator isolated from Streptomyces aureofaciens TH-3. The vector contains recognition sites for restriction enzymes NdeI and EcoRI/XbaI/HindIII between the promoter and terminator to facilitate heterologous gene cloning. The plasmids were transferred from Escherichia coli to streptomycetes via conjugation from oriT; the transformants were able to be selected using kanamycin and/or thiostrepton. The SSMP promoter functions constitutively in the presence of a rich inorganic phosphate source and glucose. We constructed expression plasmids including three Streptomyces aminopeptidases-leucine aminopeptidase, proline aminopeptidase (PAP), and aminopeptidase P (APP)-using the pTONA5 vector and Streptomyces lividans. Although they lack signal peptides for secretion, PAP and APP were secreted at high levels in the culture broth.

Two bacterial collagenolytic serine proteases have different topological specificities.

From among 2000 soil isolates, we purified a secreted serine protease from Streptomyces omiyaensis (SOT), which has extremely high gelatinolytic activity. Using sequence analysis, the primary structure of SOT showed 77% identity with that of S. griseus trypsin (SGT). We constructed recombinants SOT and SGT using S. lividans. They indicated similar properties on optimum pH and temperature, thermostability, and substrate preference using fluorescence energy transfer combinatorial libraries. SOT greatly hydrolyzed both type I and type IV collagens, but SGT has poor ability to hydrolyze type IV collagen. Furthermore, SOT exhibits higher hydrolytic activities toward other protein substrate such as gelatin and casein than SGT. These results suggest that these two enzymes have different topological specificities in spite of their similar primary structures. We also constructed chimeras between SOT and SGT to investigate which domain is associated with differences in their substrate specificity. In comparison to substrate specificities of chimeras, we found that the N-terminal domain contributes to the determination of topological specificity.

Streptomyces aminopeptidase P: biochemical characterization and insight into the roles of its N-terminal domain.

We purified and characterized the aminopeptidase P from Streptomyces costaricanus TH-4 (thAPP). This enzyme has a tetramer structure, a metal-ion preference toward Zn, broad substrate specificity and a narrow pH dependency for activity. The primary structure of thAPP, respectively, exhibits 91% and 65% identity with those of two other APPs-APP I and APP II-from Streptomyces lividans (slAPP I and slAPP II). We next overexpressed the genes encoding thAPP and slAPP II in Escherichia coli and characterized them. Two differences were apparent in their properties: slAPP II formed a dimer, whereas thAPP formed a tetramer; also, the alkaline side pKa for the catalytic action of slAPP II is higher than that of thAPP. Investigation using chimeras of both enzymes revealed that the N-terminal domain is associated with the determination of pKa values for catalytic action and quaternary structure.

Effect of salt on the activity of Streptomyces prolyl aminopeptidase.

A salt-tolerant prolyl aminopeptidase from Streptomyces aureofaciens TH-3 (TH-3PAP) was purified from a culture supernatant. The gene encoding TH-3PAP was cloned and sequenced. The primary structure of TH-3PAP showed 65% identity with that of PAP from Streptomyces lividans (SLPAP) and possessed a conserved catalytic motif, GxSxGG, which is conserved in the alpha/beta hydrolase fold family. The characterization of the recombinants TH-3PAP and SLPAP indicated a difference: in 4.0 M NaCl, TH-3PAP showed enzyme activity, whereas SLPAP was inactive. Next, we constructed chimeras between TH-3PAP and SLPAP using an in vivo DNA shuffling system and a sandwich chimera (sc-PAP), whose region from 63 to 78 amino acids of TH-3PAP was substituted with that of SLPAP. Comparison of the biochemical properties between TH-3PAP and the salt-sensitive sc-PAP suggested that the fine tuning of the N-terminal conformation of TH-3PAP by hydrophobic interaction is important for the salt tolerance mechanism of the enzyme.

Sensor of phospholipids in Streptomyces phospholipase D.

Recently, we identified Ala426 and Lys438 of phospholipase D from Streptomyces septatus TH-2 (TH-2PLD) as important residues for activity, stability and selectivity in transphosphatidylation. These residues are located in a C-terminal flexible loop separate from two catalytic HxKxxxxD motifs. To study the role of these residues in substrate recognition, we evaluated the affinities of inactive mutants, in which these residues were substituted with Phe and His, toward several phospholipids by SPR analysis. By substituting Ala426 and Lys438 with Phe and His, respectively, the inactive mutant showed a much stronger interaction with phosphatidylcholine and a weaker interaction with phosphatidylglycerol than the inactive TH-2PLD mutant. We demonstrated that Ala426 and Lys438 of TH-2PLD play a role in sensing the head group of phospholipids.

Characterization, cloning, sequencing, and expression of an aminopeptidase N from Streptomyces sp. TH-4.

The aminopeptidase N (TH-4AP) of Streptomyces sp. TH-4 was purified from a culture supernatant. The purified enzyme had a molecular mass of 95 kDa. The gene encoding TH-4AP was cloned and sequenced. The primary structure of the protein possessed the PepN-conserved motif GxMEN and the zinc-binding motif HExxHx18E, and showed 88% identity with that of PepN from Streptomyces lividans strain 66. We succeeded in overproducing a His-tagged recombinant enzyme using Escherichia coli. The enzyme had a 1.5-fold higher activity in the presence of cobalt ions than in their absence. To evaluate the possible application of TH-4AP to decrease the content of bitter peptides, we investigated the ability of Streptomyces aminopeptidases to hydrolyze synthetic peptides by a coupling method using L-amino acid oxidase and peroxidase. The substrate specificity of TH-4AP toward synthetic peptides was significantly different from that toward aminoacyl-p-nitroanilide derivatives.

C-terminal loop of Streptomyces phospholipase D has multiple functional roles.

We have recently shown that two flexible loops of Streptomyces phospholipase D (PLD) affect the catalytic reaction of the enzyme by a comparative study of chimeric PLDs. Gly188 and Asp191 of PLD from Streptomyces septatus TH-2 (TH-2PLD) were identified as the key amino acid residues involved in the recognition of phospholipids. In the present study, we further investigated the relationship between a C-terminal loop of TH-2PLD and PLD activities to elucidate the reaction mechanism and the recognition of the substrate. By analyzing chimeras and mutants in terms of hydrolytic and transphosphatidylation activities, Ala426 and Lys438 of TH-2PLD were identified as the residues associated with the activities. We found that Gly188 and Asp191 recognized substrate forms, whereas residues Ala426 and Lys438 enhanced transphosphatidylation and hydrolysis activities regardless of the substrate form. By substituting Ala426 and Lys438 with Phe and His, respectively, the mutant showed not only higher activities but also higher thermostability and tolerance against organic solvents. Furthermore, the mutant also improved the selectivity of the transphosphatidylation activity. The residues Ala426 and Lys438 were located in the C-terminal flexible loop of Streptomyces PLD separate from the highly conserved catalytic HxKxxxxD motifs. We demonstrated that this C-terminal loop, which formed the entrance of the active well, has multiple functional roles in Streptomyces PLD.

Change in substrate preference of Streptomyces aminopeptidase through modification of the environment around the substrate binding site.

We attempted to alter the substrate preference of aminopeptidase from Streptomyces septatus TH-2 (SSAP). Because Asp198 and Phe221 of SSAP are located in the substrate binding site, we screened 2,000 mutant enzymes with D198X/F221X mutations. By carrying out this examination, we obtained two enzymes; one specifically hydrolyzed an arginyl derivative, and the other specifically hydrolyzed a cystinyl derivative (65- and 12.5-fold higher k(cat) values for hydrolysis of p-nitroanilide derivatives than those of the wild type, respectively).