Streptolysin S induces pronounced calcium-ion influx-dependent expression of immediate early genes encoding transcription factors.

Anginosus group streptococci (AGS) are opportunistic human pathogens of the oral cavity. The ß-hemolytic subgroup of Streptococcus anginosus subsp. anginosus secretes streptolysin S (SLS) and exhibits not only hemolytic activity but also cytotoxicity toward cultured human cell lines. However, the detailed mechanism of action of SLS and the cellular responses of host cells have not yet been fully clarified. To determine the pathogenic potential of SLS-producing ß-hemolytic S. anginosus subsp. anginosus, the SLS-dependent response induced in the human oral squamous cell carcinoma HSC-2 cells was investigated to determine the pathogenic potential of SLS-producing ß-hemolytic S. anginosus subsp. anginosus. This study revealed that the Ca2+ influx and the expression of immediate early genes (IEGs) encoding transcription factors such as early growth responses (EGRs) and activator protein-1 (AP-1) were greatly increased in HSC-2 cells incubated with the culture supernatant of SLS-producing ß-hemolytic S. anginosus subsp. anginosus. Moreover, this SLS-dependent increase in expression was significantly suppressed by Ca2+ chelation, except for jun. These results suggest that SLS caused Ca2+ influx into the cells following greatly enhanced expression of IEG-encoding transcription factors. The results of this study may help in understanding the pathogenicity of SLS-producing AGS.

Molecular Aspects of C-glycosides: Interactive Analysis of C-linked Compounds With the SGLT2 Molecular Model.

BACKGROUND/AIM: Interaction analysis between modeled human sodium/glucose cotransporter 2 (hSGLT2) and antidiabetic C-glycoside drugs, such as canagliflozin, dapagliflozin, ipragliflozin, empagliflozin, tofogliflozin, and luseogliflozin was performed. MATERIALS AND METHODS: The hSGLT2 was modeled using the X-ray data of Vibrio parahaemolyticus SGLT2 (protein data bank ID=2XQ2) as a template. Conformational analyses of C-glycosides were performed using CAChe-Conflex. Interactive analyses between hSGLT2 and C-glycosides were performed using Molegro Virtual Docker. RESULTS: Canagliflozin interacted with hSGLT2 via Asn75, Ser287, Lys321 and Gln457 Dapagliflozin interacted with six amino acids (Arg46, Arg49, Ile76, Ser78, Met216 and Ser393). Ipragliflozin (Ala69, Met596 and Gln600), empagliflozin (Ser78, Gly79, Lys154, Asp158 and Ser393), tofogliflozin (Arg49, Met216, Ala389, Ser392 and Ser393), and luseogliflozin (Arg49, Ser74, Ser78, Gly79, His80, Lys154, Asp158 and Ser393) interacted with hSGLT2 via the amino acids described in the parentheses. CONCLUSION: The binding mode of each C-glycoside drug to hSGLT2 was different, and structural features of each compound were revealed. The reactive base points of C-glycosides were the sugar moiety, with the sugar structure being important for hSGLT2 inhibitory action.

Human serum albumin stabilizes streptolysin S activity secreted in the extracellular milieu by streptolysin S-producing streptococci.

Anginosus group streptococci (AGS) are opportunistic pathogens of the human oral cavity; however, their pathogenicity has not been discussed in detail. Oral streptococci live in the gingival sulcus, from where they can easily translocate into the bloodstream due to periodontal diseases and dental treatment and cause hazardous effects on the host through their virulence factors. Streptolysin S (SLS), a pathogenic factor produced by ß-hemolytic species/strains belonging to AGS, plays an important role in damaging host cells. Therefore, we investigated the SLS-dependent cytotoxicity of ß-hemolytic Streptococcus anginosus subsp. anginosus (SAA), focusing on different growth conditions such as in the bloodstream. Consequently, SLS-dependent hemolytic activity/cytotoxicity in the culture supernatant of ß-hemolytic SAA was stabilized by blood components, particularly human serum albumin (HSA). The present study suggests that the secreted SLS, not only from ß-hemolytic SAA, but also from other SLS-producing streptococci, is stabilized by HSA. As HSA is the most abundant protein in human plasma, the results of this study provide new insights into the risk of SLS-producing streptococci which can translocate into the bloodstream.

Dual functions of discoidinolysin, a cholesterol-dependent cytolysin with N-terminal discoidin domain produced from Streptococcus mitis strain Nm-76.

Background: Some strains of Streptococcus mitis exhibit ß-hemolysis due to the ß-hemolytic activity of cholesterol-dependent cytolysin (CDC). Recently, a gene encoding an atypical lectinolysin-related CDC was found in S. mitis strain Nm-76. However, the product of this gene remains uncharacterized. We aimed to characterize this atypical CDC and its molecular functions and contribution to the pathogenicity of S. mitis strain Nm-76. Methods: Phylogenetic analysis of the CDC gene was conducted based on the web-deposited information. The molecular characteristics of CDC were investigated using a gene-deletion mutant strain and recombinant proteins expressed in Escherichia coli. Results: The gene encoding CDC found in Nm-76 and its homolog are distributed among many S. mitis strains. This CDC is phylogenetically different from other previously characterized CDCs, such as S. mitis-derived human platelet aggregation factor (Sm-hPAF)/lectinolysin and mitilysin. Because this CDC possesses an additional N-terminal domain, including a discoidin motif, it was termed discoidinolysin (DLY). In addition to the preferential lysis of human cells, DLY displayed N-terminal domain-dependent facilitation of human erythrocyte aggregation and intercellular associations between human cells. Conclusion: DLY functions as a hemolysin/cytolysin and erythrocyte aggregation/intercellular association molecule. This dual-function DLY could be an additional virulence factor in S. mitis.

Molecular Interaction Between Boron Tracedrug UTX-51 Derivatives and Bovine Serum Albumin: Application to an Analytical Model of AGEs Destruction by Thermal Neutron Irradiation.

BACKGROUND/AIM: Boron tracedrugs possess global molecular tracking abilities and localized destructive power. We investigated the molecular properties of synthesized boron tracedrugs, including UTX-51, and their interactions with the advanced glycation end-product (AGE)-related protein bovine serum albumin (BSA). MATERIALS AND METHODS: A conformational analysis of the compounds used in the present study was performed using CAChe (Fujitsu Inc., Tokyo, Japan) and the degree of stereo-hydrophobicity of the conformers obtained was verified using Mopac (Fujitsu Inc.). The interactive properties of global minimum conformers of the derivatives tested with BSA were assessed using Molegro Virtual Docker (CLC bio., Aarhus, Denmark). RESULTS: Among the compounds investigated, UTX-51 was confirmed to interact with BSA based on the formation of hydrogen bonds between BSA and UTX-51. CONCLUSION: UTX-51 is a promising boron tracedrug and can be used as the lead structure for developing a therapeutic agent for AGE-related diseases, including cancer.

Construction of a Drug Release Evaluation System: Application of Mitochondrial Respiration to Monitor Drug Release.

BACKGROUND/AIM: Efficient drug encapsulation and regulation of drug release are important factors for sustained drug release and application for release-controlled anti-cancer and anti-inflammatory drug delivery. In the present study, a direct evaluation system for drug-release from model carrier (e.g., alginate-gel beads) was examined using the mitochondrial oxygen consumption rate as an index. MATERIALS AND METHODS: Alginate-gel beads were coated with the uncoupler SF6847 (SF beads) and used as a model microparticle-type drug. The real-time monitoring of SF6847 release from prepared alginate-gel beads was performed using the mitochondrial oxygen consumption rate. Release profiles of nonsteroidal anti-inflammatory drugs [NSAIDs, mefenamic acid (MEF) and diclofenac (DIC)] from alginate-gel beads as well as SF beads were investigated using the real time monitoring system. RESULTS: SF6847 release from alginate-gel beads was clearly detected using the rat liver mitochondrial oxygen consumption rate. The release features of MEF and DIC from alginate-gel beads were defined by the present trial monitoring system, and these NSAIDs exhibited different release profiles. CONCLUSION: The present drug monitoring system detected released drugs, and the release profile reflected the molecular properties of the test drugs. This system may be applied to the design and development of precise sustained drug release systems (e.g., anti-cancer and anti-inflammatory drugs).

Molecular characteristics of an adhesion molecule containing cholesterol-dependent cytolysin-motif produced by mitis group streptococci.

Streptococcus pseudopneumoniae (SPpn) is a relatively new species closely related to S. pneumoniae (SPn) and S. mitis (SM) belonging to the Mitis group of the genus Streptococcus (MGS). Although genes encoding various pneumococcal virulence factors have been observed in the SPpn genome, the pathogenicity of SPpn against human, including the roles of virulence factor candidates, is still unclear. The present study focused on and characterized a candidate virulence factor previously reported in SPpn with deduced multiple functional domains, such as lipase domain, two lectin domains, and cholesterol-dependent cytolysin-related domain using various recombinant proteins. The gene was found not only in SPpn but also in the strains of SM and SPn. Moreover, the gene product was expressed in the gene-positive strains as secreted and cell-bound forms. The recombinant of gene product showed lipase activity and human cell-binding activity depending on the function of lectin domain(s), but no hemolytic activity. Thus, based on the distribution of the gene within the MGS and its molecular function, the gene product was named mitilectin (MLC) and its contribution to the potential pathogenicity of the MLC-producing strains was investigated. Consequently, the treatment with anti-MLC antibody and the mlc gene-knockout significantly reduced the human cell-binding activity of MLC-producing strains. Therefore, the multifunctional MLC was suggested to be important as an adhesion molecule in considering the potential pathogenicity of the MLC-producing strains belonging to MGS, such as SPpn and SM.

Effect of Isomerization of TX-2036 Derivatives on the Interaction With Tyrosine Kinase Domain of EGF Receptor.

BACKGROUND: From the design and synthesis of enantiomers, we can expect to obtain two compounds with different pharmacokinetics and pharmacological activities at the same time, which is thought to lead to the development of efficient anticancer agents. Chiral-2-nitroimidazole TX-2036 derivatives exhibit stereo-configuration (R- and S-configuration)-dependent tyrosine kinase inhibitory activity, and the activity of the tyrosine kinase domain of EGF receptor (EGFR-tyk) is suppressed. In order to clarify the reason why the effects on EGFR-tyk activity differ depending on stereoisomers, we tried to analyze the interaction between each TX-2036 derivative and EGFR-tyk. MATERIALS AND METHODS: The 2-nitroimidazole-based radiosensitizer TX-2036 series were synthesized and their molecular features were examined using protein kinase inhibition assay and molecular structural analysis. RESULTS: R-configured TXs (TX-2043, -2030, and -2036) exhibited more potent protein kinase inhibitory activity than S-configured TXs (TX-2044, - 2031, and -2037), and the IC50 value of TX-2036 was 1.8 µM. CONCLUSION: R-configured TXs interacted with Lys721 and Thr766 of EGFR-tyk. The combinations of amino acid residues targeted by the S-configured TXs were different from each other (Ile765 and Thr766 (TX-2044), Ser696, Thr766, and Thr830 (TX-2031), Gly772, Cys773, and Thr830 (TX-2037)). Preparing a series of isomers with different target sites was considered beneficial when the target was mutated.

Correlation Between Radiosensitizing Activity and the Stereo-structure of the TX-2036 Series of Molecules.

BACKGROUND/AIM: The stereo-configuration (R-, S-configuration) of chiral-2-nitroimidazole derivatives alters their radiosensitizing activity. This study aimed at examining the molecular features of these enantiomers by molecular simulation techniques. MATERIALS AND METHODS: A series of 2-nitroimidazole-based radiosensitizer TX-2036 molecules were synthesized, and their profiles were examined using molecular structural analysis such as conformation analysis, molecular orbital analysis, and electrostatic potential analysis. RESULTS: R-configured TXs (TX-2043, -2030, -2036) had a weaker radiosensitizing activity than S-configured TXs (TX-2044, -2031, -2037), and R-compounds had a small minus electrostatic potential (ESP) field in the cyclopentene-1,3-dione region. S-configured TX-2046 had weaker radiosensitizing activity than R-configured TX-2045, and TX-2046 had a small minus ESP field as well as R-configured TX-2043, -2030, - 2036. CONCLUSION: The cyclopentene-1,3-dione involved in the small minus ESP field affected the radiosensitizing activity of the TX-2036 series of molecules.

ß-Hemolytic Streptococcus anginosus subsp. anginosus causes streptolysin S-dependent cytotoxicity to human cell culture lines in vitro.

Background: Streptococcus anginosus subsp. anginosus (SAA) is one of the opportunistic pathogens in humans that inhabits the oral cavity. The type strain of SAA, NCTC10713T, showed clear ß-hemolysis on blood agar plates, and the sole ß-hemolytic factor revealed two streptolysin S (SLS) molecules. SLS is well known as the peptide hemolysin produced from the human pathogen S. pyogenes and shows not only hemolytic activity on erythrocytes but also cytotoxic activity in cell culture lines in vitro and in vivo, such as in a mouse infection model. However, no cytotoxic activity of SLS produced from ß-hemolytic SAA (ß-SAA) has been reported so far. Objective and Design: In this study, the SLS-dependent cytotoxicity of the ß-SAA strains including the genetically modified strains was investigated in vitro. Results: The SLS-producing ß-SAA showed cytotoxicity in human cell culture lines under the co-cultivation condition and it was found that this cytotoxicity was caused by the SLS secreted into the extracellular milieu. Conclusion: The results from this study suggest that the SLS produced from ß-SAA might indicate the cytotoxic potential similar to that of the SLS from S. pyogenes and the SLS-producing ß-SAA would be recognized as "a wolf in sheep's clothing" More attention will be paid to the pathogenicity of ß-hemolytic Anginosus group streptococci.

Structure-associated Functional Control of TX-1877 Series by Glyco-conjugation.

BACKGROUND/AIM: Sugar molecules are often used as a tool to structurally modify chemical compounds. The features of synthesized sugar-conjugated TX-1877 derivatives were herein examined. MATERIALS AND METHODS: The molecular stabilities (reactivity) and hydrophobicities of sugar (e.g., monosaccharide and tetra-O-acetylated monosaccharide)-conjugated TXs were analyzed using a molecular simulation (e.g. molecular mechanics (MM) and molecular orbital (MO) analysis). RESULTS: The hydrophobicities of TX-1877 derivatives were increased by tetra-O-acetylation, and TX-2244 exhibited the most potent radiosensitizing activity (enhancement ratio: ER=2.30). CONCLUSION: The conformations and hydrophobicities of chemical compounds may be controlled by adding monosaccharide- and tetra-O-acetyl-conjugated sugars to TX-1877.

Effects of the Nonsteroidal Anti-inflammatory Drug Celecoxib on Mitochondrial Function.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used to treat inflammation and pain. In the present study, we examined the effects of celecoxib, a cyclooxygenase-2 (COX-2)-selective NSAID, on rat liver mitochondrial function. Celecoxib dose-dependently induced mitochondria swelling, which was not suppressed by cyclosporine A (CsA). The oxygen consumption rate in mitochondria-suspended solution was facilitated by the addition of celecoxib, and its uncoupling activity was observed. Celecoxib also suppressed SF6847-induced uncoupling, and appeared to exert inhibitory effects on the electron transport chain. Celecoxib suppressed the state 3 oxygen consumption rate in the presence of ADP. Protein release from the mitochondrial matrix was detected following the addition of celecoxib, and aldehyde dehydrogenase 2 (ALDH2) and hydroxymethylglutaryl-CoA (HMG-CoA) synthase 2 (HMGCS2) bands were confirmed in a Western blot analysis. On the other hand, protein release of cytochrome C (CytC), which is an inducer of apoptosis, from the intermembrane space was not observed. Celecoxib enhanced the membrane permeability of human erythrocytes and synthesized liposomes dose-dependently. It then induced the membrane-involving mitochondrial swelling and suppressed mitochondrial function.

Interactive Analysis of TX-1123 with Cyclo-oxygenase: Design of COX2 Selective TX Analogs.

BACKGROUND: To date, two cyclo-oxygenase (COX) isoforms, COX1 and COX2, have been identified. In the present study, the COX-inhibitory activities of TX-1123 derivatives with the 2-hydroxyarylidene-4-cyclopentene-1,3-dione structure were examined, and the binding profiles of TX-1123 to COXs were analyzed using docking simulations. MATERIALS AND METHODS: X-Ray data on COX1 [protein data bank (PDB) ID=1PGG] and COX2 (PDB ID=3LN1) were used for molecular interactive simulations. The interactive profiles of TX-1123 derivatives with COXs were examined using a molecular simulation technique with Molegro Virtual Docker (CLC bio, Aarhus, Denmark). RESULTS: TX-1123 exhibited COX1-inhibitory activity [half-maximal-inhibitory concentration (IC50)=1.57×10-5 M]. The COX2 inhibitory activity of TX-1123 was potent (IC50=1.16×10-6 M), and the ratio of COX1/COX2 inhibition was 13.5. TX-1123 bound to the COX2 molecule, and the oxygen atom of the 4-cyclopentene-1,3-dione region of TX-1123 interacted with Cys26 and Gln447 of COX2. CONCLUSION: The TX-1123-binding pocket of COX2 differs from that of the COX2-selective celecoxib-binding pocket. TX-1123 exhibited a different COX2-interactive mechanism from that of celecoxib.

An Antitumor 2-Hydroxyarylidene-4-cyclopentene-1,3-Dione as a Protein Tyrosine Kinase Inhibitor: Interaction Between TX-1123 Derivatives and Src Kinase.

BACKGROUND: Protein tyrosine kinases (PTKs) play major roles in signal transduction during cell proliferation and apoptosis. Tyrphostin AG17 was previously shown to be a potent tumor growth inhibitor, while AG17 induced apoptosis and inhibited activity of cyclin-dependent kinase 2. We herein describe the binding features of tyrphostin AG17 analogs, such as TX-1123, with Src kinase (Src-K). MATERIALS AND METHODS: Structural data for Src-K were obtained from a protein data bank (ID=2SRC), and the molecular interactions between Src-K and TX-1123 derivatives were examined. RESULTS: TX-1123 exihibited potent Src-K inhibitory activity (half maximal-inhibitory concentration=2.2 µM), and fit into the pocket of the Src-K molecule as well as c-AMP did. CONCLUSION: The binding profiles of TX-1123 derivatives differed from each other, while their Src-K inhibitory activities were affected by their fit in the Src-K molecule.

Effect of N-Phenylanthranilic Acid Scaffold Nonsteroidal Anti-inflammatory Drugs on the Mitochondrial Permeability Transition.

Hepatotoxicity is a known side effect of nonsteroidal anti-inflammatory drugs (NSAIDs). In the present study, the effects of N-phenylanthranilic acid (NPA) scaffold NSAIDs on rat liver mitochondria were examined. Mefenamic acid (MEF, 200 µM) induced mitochondrial swelling, which was inorganic phosphate (Pi)-dependent and suppressed by cyclosporin A (CsA, 2.5 µM), similar to calcium-induced swelling. Mitochondrial swelling was also observed following the addition of 200 µM flufenamic acid (FLU), meclofenamic acid (MCL), and tolfenamic acid (TOL). Less swelling was observed with the addition of 200 µM diclofenac (DIC) or NPA. Diphenylamine (DPA)-induced swelling occurred in a Pi-independent manner and was not sensitive to CsA. The mechanism by which DPA interacted with the mitochondrial inner membrane differed from those of the other NPA scaffold NSAIDs. The addition of 50 µM MEF, MCL, TOL, and FLU had uncoupling effects in mitochondrial inner membrane. These NSAIDs dose-dependently obstructed electron transport in the respiratory chain. NSAIDs are known to have various dynamic structures, and the solvation free energies (dGWs: an index of stereo-hydrophobicity) of the conformers obtained were determined using a molecular orbital analysis. The relationship between the dynamic structures and swelling induced by NPA scaffold NSAIDs was also examined.

Immunoblotting with Peptide Antibodies: Differential Immunoreactivities Caused by Certain Amino Acid Substitutions in a Short Peptide and Possible Effects of Differential Refolding of the Peptide on a Nitrocellulose or PVDF Membrane.

Immunodetection using antibodies, e.g., Western blotting, is generally utilized to measure the amount of a certain protein in a protein mixture. For valid interpretation of results observed by immunodetection, strict attention must be paid to the factors affecting the immunoreactivities of the antibodies. We here describe the step-by-step procedures to demonstrate that substitution of certain amino acids in a peptide can cause remarkable differences in its immunoreactivity with antibodies against epitope tags in the immobilized peptide. Refolding of the peptide on the membrane in a way that masks the epitope to different degrees was the possible reason for their distinct immunoreactivities with the antibodies. The results in this chapter suggest that we need to interpret carefully the experimental results involving immunodetection.

Development of a Sortase A-mediated Peptide-labeled Liposome Applicable to Drug-delivery Systems.

BACKGROUND/AIM: In order to develop an efficient drug-delivery system (DDS), a lipopeptide-loaded liposome that functions as a platform for the transpeptidase reaction mediated by sortase A (SrtA) was constructed and its stability, as well as cell-specific targeting were evaluated in the present study. MATERIALS AND METHODS: Several lipopeptides possessing an acceptor peptide sequence (oligoglycine ≥ three residues) or donor peptide sequence (LPETG) for the SrtA-mediated reaction were chemically synthesized and then inserted into the liposome membrane composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol (DPPC-Chol-lipo) to obtain the lipopeptide-loaded liposomes. The transpeptidase reaction mediated by recombinant SrtA (His-ΔN59SrtA) was employed to modify the peptide moiety on the liposomal surface using a fluorescently-labeled substrate peptide corresponding to the species of each loaded lipopeptide. Furthermore, lung tumor-binding peptide (LTBP)-labeled liposomes, prepared by this transpeptidase reaction, were investigated for selective targeting to lung cancer cells in vitro. RESULTS AND DISCUSSION: The His-ΔN59SrtA-mediated transpeptidation of fluorescently-labeled peptide on the lipopeptide-loaded DPPC-Chol-lipo was confirmed. The selective targeting of LTBP-labeled liposomes to the lung cancer cell line A549 was also observed in vitro. These results suggest that the labeling of acceptor or donor lipopeptide-loaded liposomes with the transpeptidase SrtA could be a useful method for developing a platform applicable to a cancer-targeting DDS.

Cytolytic Activity and Molecular Feature of Cardiotoxin and Cardiotoxin-like Basic Protein: The Electrostatic Potential Field Is an Important Factor for Cell Lytic Activity.

BACKGROUND/AIM: Cardiotoxin (CT) is a well-known cell lytic protein and has been purified from cobra venom. Cardiotoxin-like basic protein (CLBP) has two amino acid insertions and does not exhibit cell lytic activity. The molecular features of these CT family proteins were examined in the present study using molecular modeling and molecular simulation techniques. MATERIALS AND METHODS: Molecular models of CT and CLBP were constructed based on the X-ray data of Naja mossambica mossambica CT VII4 (Protein Data Bank ID: 1CDT). The structural features of these models were examined using molecular orbital and electrostatic potential parameters. RESULTS: The stereo-hydrophobicities and molecular torsions of CT and CLBP, which are indexes of structural features, were similar. Electrostatic potential fields (ESP) differed between CT and CLBP and this was considered one of the critical factors in molecular titer. CONCLUSION: The distribution of ESP fields may affect the cytolytic activity of the CT family.

Bongkrekic acid analogue, lacking one of the carboxylic groups of its parent compound, shows moderate but pH-insensitive inhibitory effects on the mitochondrial ADP/ATP carrier.

Bongkrekic acid, isolated from Burkholderia cocovenenans, is known to specifically inhibit the mitochondrial ADP/ATP carrier. However, the manner of its interaction with the carrier remains elusive. In this study, we tested the inhibitory effects of 17 bongkrekic acid analogues, derived from the intermediates obtained during its total synthesis, on the mitochondrial ATP/ATP carrier. Rough screening of these chemicals, performed by measuring their inhibitory effects on the mitochondrial ATP synthesis, revealed that 4 of them, KH-1, KH-7, KH-16, and KH-17, had moderate inhibitory effects. Further characterization of the actions of these 4 analogues on mitochondrial function showed that KH-16 had moderate; KH-1 and KH-17, weak; and KH-7, negligible side effects of both permeabilization of the mitochondrial inner membrane and inhibition of the electron transport, indicating that only KH-7 had a specific inhibitory effect on the mitochondrial ADP/ATP carrier. Although the parental bongkrekic acid showed a strong pH dependency of its action, the inhibitory effect of KH-7 was almost insensitive to the pH of the reaction medium, indicating the importance of the 3 carboxyl groups of bongkrekic acid for its pH-dependent action. A direct inhibitory effect of KH-7 on the mitochondrial ADP/ATP carrier was also clearly demonstrated.

Molecular analysis of Streptococcus anginosus-derived SagA peptides.

BACKGROUND: SagA1 and SagA2 molecules produced from beta-hemolytic Streptococcus anginosus subsp. anginosus are composed of a leader peptide and a propeptide, and their mature form has hemolytic activity as a well-known Streptococcal peptide toxin, streptolysin. The function of these SagA molecules is thought to be dependent on intra-molecular heterocycle formation. In this study, we examined the heterocycle-involved molecular features of SagA1, SagA2, and S. pyogenes SagA (SPySagA), focusing on their heterocycle formation. MATERIALS AND METHODS: Molecular models of SagA1, SagA2, and SPySagA were constructed using a molecular modeling technique. Molecular dynamics and molecular mechanic analyses of the modeled SagA molecules were performed to obtain their energy profiles. RESULTS: Total energy of the modeled SagA1, SagA2, and SPySagA decreased with heterocycle formation, and the border between the leader peptide and propeptide was clearly observed after heterocycle formation. CONCLUSION: The flexibility of SagA molecules was changed by intramolecular heterocycle formation, and their function (e.g. hemolytic activity) seems to be regulated by structural transition with heterocycle formation.