Akihito Ochiai

Antimicrobial activity and mechanism of action of a novel cationic α-helical octadecapeptide derived from α-amylase of rice.

AmyI‐1–18, an octadecapeptide derived from α‐amylase (AmyI‐1) of rice (Oryza sativa L. japonica), is a novel cationic α‐helical antimicrobial peptide (AMP) that contains two lysine and two arginine residues. The antimicrobial activity of AmyI‐1–18 against human pathogens was quantitatively evaluated using a chemiluminescence method that measures ATP derived from viable cells. Of the ten kinds of human pathogens, AmyI‐1–18 exhibited antimicrobial activity against nine. Its 50% growth‐inhibitory concentrations (ICs50) against Porphyromonas gingivalis, Propionibacterium acnes, Pseudomonas aeruginosa, Candida albicans, and Streptococcus mutans were 13, 19, 50, 64, and 77 μM, respectively. AmyI‐1–18 had little or no hemolytic activity even at 500 μM, and showed negligible cytotoxicity up to 1200 μM. The degree of 3,3′‐dipropylthiadicarbocyanine iodide release from P. gingivalis cells induced by the addition of AmyI‐1–18 was significantly lower than that induced by the addition of melittin. Flow cytometric analysis showed that the percentages of P. aeruginosa, S. mutans, and C. albicans cells stained with propidium iodide (PI), a DNA‐intercalating dye, were 89%, 43%, and 3%, respectively, when AmyI‐1–18 was added at a concentration equal to its 4×IC50. Therefore, the antimicrobial activity of AmyI‐1–18 against P. aeruginosa and S. mutans appears to be mainly attributable to its membrane‐disrupting activity. In contrast, its antimicrobial activity against P. gingivalis and C. albicans most likely depends upon interactions with intracellular targets other than their cell membranes. Collectively, these results indicate that AmyI‐1–18 is useful as a safe and potent AMP against the pathogens described above in many fields of healthcare.

α‐Amylase is a potential growth inhibitor of Porphyromonas gingivalis, a periodontal pathogenic bacterium

BACKGROUND AND OBJECTIVE Porphyromonas gingivalis is a major etiological agent in the development and progression of periodontal diseases. In this study, we isolated a cell growth inhibitor against P. gingivalis species from rice protein extract. MATERIAL AND METHODS The cell growth inhibitor active against P. gingivalis was purified from polished rice extract using a six-step column chromatography process. Its antimicrobial properties were investigated through microscope analysis, spectrum of activity and general structure. RESULTS The inhibitor was identified as AmyI-1, an α-amylase, and showed significant cell growth inhibitory activity against P. gingivalis species. Scanning electron microscopy micrograph analysis and bactericidal assay indicated an intriguing possibility that the inhibitor compromises the cell membrane structure of the bacterial cells and leads to cell death. Moreover, α-amylases from human saliva and porcine pancreas showed inhibitory activity similar to that of AmyI-1. CONCLUSIONS This is the first study to report that α-amylases cause cell death of periodontal pathogenic bacteria. This finding highlights the potential importance and therapeutic potential of α-amylases in treating periodontal diseases.

Effect of substituting arginine and lysine with alanine on antimicrobial activity and the mechanism of action of a cationic dodecapeptide (CL(14‐25)), a partial sequence of cyanate lyase from rice

The antimicrobial activity of analogs obtained by substituting arginine and lysine in CL(14‐25), a cationic α‐helical dodecapeptide, with alanine against Porphyromonas gingivalis, a periodontal pathogen, varied significantly depending on the number and position of cationic amino acids. The alanine‐substituted analogs had no hemolytic activity, even at a concentration of 1 mM. The antimicrobial activities of CL(K20A) and CL(K20A, K25A) were 3.8‐fold and 9.1‐fold higher, respectively, than that of CL(14‐25). The antimicrobial activity of CL(R15A) was slightly lower than that of CL(14‐25), suggesting that arginine at position 15 is not essential but is important for the antimicrobial activity. The experiments in which the alanine‐substituted analogs bearing the replacement of arginine at position 24 and/or lysine at position 25 were used showed that arginine at position 24 was crucial for the antimicrobial activity whenever lysine at position 25 was substituted with alanine. Helical wheel projections of the alanine‐substituted analogs indicate that the hydrophobicity in the vicinity of leucine at position 16 and alanines at positions 18 and/or 21 increased by substituting lysine at positions 20 and 25 with alanine, respectively. The degrees of diSC3‐5 release from P. gingivalis cells and disruption of GUVs induced by the alanine‐substituted analogs with different positive charges were not closely related to their antimicrobial activities. The enhanced antimicrobial activities of the alanine‐substituted analogs appear to be mainly attributable to the changes in properties such as hydrophobicity and amphipathic propensity due to alanine substitution and not to their extents of positive charge (cationicity).

Crystal structure of α-amylase from Oryza sativa: molecular insights into enzyme activity and thermostability

AmyI-1 is an α-amylase from Oryza sativa (rice) and plays a crucial role in degrading starch in various tissues and at various growth stages. This enzyme is a glycoprotein with an N-glycosylated carbohydrate chain, a unique characteristic among plant α-amylases. In this study, we report the first crystal structure of AmyI-1 at 2.2-Å resolution. The structure consists of a typical (β/α)8-barrel, which is well-conserved among most α-amylases in the glycoside hydrolase family-13. Structural superimposition indicated small variations in the catalytic domain and carbohydrate-binding sites between AmyI-1 and barley α-amylases. By contrast, regions around the N-linked glycosylation sites displayed lower conservation of amino acid residues, including Asn-263, Asn-265, Thr-307, Asn-342, Pro-373, and Ala-374 in AmyI-1, which are not conserved in barley α-amylases, suggesting that these residues may contribute to the construction of the structure of glycosylated AmyI-1. These results increase the depths of our understanding of the biological functions of AmyI-1. Graphical Abstract Crystal structure of α-amylase AmyI-1 from rice.

Contribution of cationic amino acids toward the inhibition of Arg‐specific cysteine proteinase (Arg‐gingipain) by the antimicrobial dodecapeptide, CL(14–25), from rice protein

CL(14–25), a dodecapeptide, exhibits antimicrobial activity against Porphyromonas gingivalis with the 50% growth‐inhibitory concentration (IC50) value of 145 µM, and arginine‐specific gingipain (Rgp)‐inhibitory activity. Kinetic analysis revealed that CL(14–25) is a mixed‐type inhibitor, with inhibition constants (Ki and Ki′ values) of 1.4 × 10−6 M and 4.3 × 10−6 M, respectively. To elucidate the contributions of four cationic amino acid residues at the N‐ and C‐termini of CL(14–25) toward Rgp‐inhibitory activity, we investigated the Rgp‐inhibitory activities of truncated and alanine‐substituted analogs of CL(14–25). Rgp‐inhibitory activities significantly decreased by truncated analogs, CL(15–25) and CL(16–25), whereas those of CL(14–24) and CL(14–23) were almost as high as that of CL(14–25). Rgp‐inhibitory activities of alanine‐substituted analogs, CL(R14A) and CL(R14A, R15A) also significantly decreased, whereas those of CL(K25A) and CL(R24A, K25A) were higher than that of CL(14–25). These results suggest that the arginine residue at position 15 substantially contributes to the Rgp‐inhibitory activity and that the arginine residue at position 14 plays important roles in exerting Rgp‐inhibitory activity. In this study, we demonstrated that CL(K25A) was a potent, dual function, peptide inhibitor candidate, exhibiting Rgp‐inhibitory activity with Ki and Ki′ of 9.6 × 10−7 M and 1.9 × 10−6 M, respectively, and antimicrobial activity against P. gingivalis with an IC50 value of 51 µM.

Pyrrhocoricin, a proline-rich antimicrobial peptide derived from insect, inhibits the translation process in the cell-free Escherichia coli protein synthesis system

Previous studies have shown that pyrrhocoricin, a proline-rich antimicrobial peptide (PrAMP), killed sensitive species in a dose-dependent manner by specifically binding to DnaK. Here, on the basis of the finding that DnaK-deficient Escherichia coli strains are susceptible to PrAMPs, we used pyrrhocoricin to investigate internal targets other than DnaK. Using conventional antibiotics (bleomycin, streptomycin, and fosfomycin) that have known modes of action, first, we validated the availability of an assay using a cell-free rapid translation system (RTS), which is an in vitro protein synthesis system based on E. coli lysate, for evaluating inhibition of protein synthesis. We found that, similarly to bleomycin and streptomycin, pyrrhocoricin inhibited GFP synthesis in RTS in a concentration-dependent manner. In addition, blockage of transcription and translation steps in RTS was individually estimated using RT-PCR after gene expression to determine mRNA products and using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine the amounts of GFP expressed from purified mRNA, respectively. The results demonstrated that this inhibition of GFP synthesis by pyrrhocoricin did not occur at the transcription step but rather at the translation step, in a manner similar to that of GFP synthesis by streptomycin, an inhibitor of the translation step by causing misreading of tRNA. These results suggest that RTS is a powerful assay system for determining if antimicrobial peptides inhibit protein synthesis and its transcription and/or translation steps. This is the first study to have shown that pyrrhocoricin inhibited protein synthesis by specifically repressing the translation step.

Endotoxin-neutralizing activity and mechanism of action of a cationic α-helical antimicrobial octadecapeptide derived from α-amylase of rice

We have previously reported that AmyI-1-18, an octadecapeptide derived from α-amylase (AmyI-1) of rice, is a novel cationic α-helical peptide that exhibited antimicrobial activity against human pathogens, including Porphyromonas gingivalis, Pseudomonas aeruginosa, Propionibacterium acnes, Streptococcus mutans, and Candida albicans. In this study, to further investigate the potential functions of AmyI-1-18, we examined its inhibitory ability against the endotoxic activities of lipopolysaccharides (LPSs, smooth and Rc types) and lipid A from Escherichia coli. AmyI-1-18 inhibited the production of endotoxin-induced nitric oxide (NO), an inflammatory mediator, in mouse macrophages (RAW264) in a concentration-dependent manner. The results of a chromogenic Limulus amebocyte lysate assay illustrated that the ability [50% effective concentration (EC50): 0.17 μM] of AmyI-1-18 to neutralize lipid A was similar to its ability (EC50: 0.26 μM) to neutralize LPS, suggesting that AmyI-1-18 specifically binds to the lipid A moiety of LPS. Surface plasmon resonance analysis of the interaction between AmyI-1-18 and LPS or lipid A also suggested that AmyI-1-18 directly binds to the lipid A moiety of LPS because the dissociation constant (KD) of AmyI-1-18 with lipid A is 5.6×10(-10) M, which is similar to that (4.3×10(-10) M) of AmyI-1-18 with LPS. In addition, AmyI-1-18 could block the binding of LPS-binding protein to LPS, although its ability was less than that of polymyxin B. These results suggest that AmyI-1-18 expressing antimicrobial and endotoxin-neutralizing activities is useful as a safe and potent host defense peptide against pathogenic Gram-negative bacteria in many fields of healthcare.

Effect of alanine, leucine, and arginine substitution on antimicrobial activity against candida albicans and action mechanism of a cationic octadecapeptide derived from α‐amylase of rice

AmyI‐1‐18, an antimicrobial peptide, is a cationic α‐helical octadecapeptide derived from α‐amylase of rice (Oryza sativa L. japonica) that contains four cationic amino acid residues (two arginines and two lysines). To enhance the antifungal activity of AmyI‐1‐18 against Candida albicans, 11 analogs bearing substitutions with alanine, leucine, and/or arginine, which were designed on the basis of the helical wheel projection of AmyI‐1‐18, were synthesized, and their antifungal activity was investigated. The antifungal activities of four analogs obtained by replacing arginine or lysine with alanine were significantly reduced. The results suggested that the cationic arginine and lysine residues in AmyI‐1‐18 are important for its antifungal activity. The antifungal activities of two single leucine‐substituted analogs were not improved, but among three single arginine‐substituted analogs, AmyI‐1‐18(D15R) had approximately a twofold higher antifungal activity [50% growth‐inhibitory concentration (IC50): 31 μM] than AmyI‐1‐18 (IC50: 64 μM) and exhibited low hemolytic activity (4% at 100 μM). Flow cytometric analysis using propidium iodide revealed that the antifungal activity of AmyI‐1‐18(D15R) was dependent on its membrane‐disrupting activity in a manner different from that of AmyI‐1‐18. Further enhancement of the cationicity and hydrophobicity of AmyI‐1‐18(D15R) resulted in no improvement in antifungal activity and a significant increase in hemolytic activity. In this study, the results demonstrated that the antifungal activity of AmyI‐1‐18 against C. albicans was enhanced through increasing its membrane‐disrupting activity by replacing aspartic acid at position 15 with arginine without a significant increase in hemolytic activity.

Cationic peptides from peptic hydrolysates of rice endosperm protein exhibit antimicrobial, LPS-neutralizing, and angiogenic activities

HIGHLIGHTSPeptides were fractionated from hydrolysates of rice endosperm protein by autofocusing.Cationic peptides were purified by RP‐HPLC and then identified by MALDI‐TOF MS.Of identified peptides, five cationic peptides exhibited dual or multiple functions.Five peptides exhibited angiogenic and LPS‐neutralizing activities.Fractions obtained from hydrolysates of rice endosperm protein contain non‐toxic cationic peptides with multiple functions. ABSTRACT In this study, we hydrolyzed rice endosperm protein (REP) with pepsin and generated 20 fractions containing multifunctional cationic peptides with varying isoelectric point (pI) values using ampholyte‐free isoelectric focusing (autofocusing). Subsequently, we determined antimicrobial activities of each fraction against the pathogens Prophyromonas gingivalis, Propionibacterium acnes, Streptocossus mutans, and Candida albicans. Fractions 18, 19, and 20 had pI values greater than 12 and exhibited antimicrobial activity against P. gingivalis, P. acnes, and C. albicans, but not against S. mutans. In further experiments, we purified and identified cationic peptides from fractions 18, 19, and 20 using reversed‐phase high‐performance liquid chromatography and matrix‐assisted laser/desorption ionization‐time‐of‐flight mass spectroscopy. We also chemically synthesized five identified peptides (RSVSKSR, RRVIEPR, ERFQPMFRRPG, RVRQNIDNPNRADTYNPRAG, and VVRRVIEPRGLL) with pI values greater than 10.5 and evaluated antimicrobial, lipopolysaccharide (LPS)‐neutralizing, and angiogenic activities. Among these synthetic peptides, only VVRRVIEPRGLL exhibited antimicrobial activity against P. gingivalis, with an IC50 value of 87 &mgr;M. However, all five cationic peptides exhibited LPS‐neutralizing and angiogenic activities with little or no hemolytic activity against mammalian red blood cells at functional concentrations. These present data show dual or multiple functions of the five identified cationic peptides with little or no hemolytic activity. Therefore, fractions containing cationic peptides from REP hydrolysates have the potential to be used as dietary supplements and functional ingredients in food products.

Effects of arginine and leucine substitutions on anti-endotoxic activities and mechanisms of action of cationic and amphipathic antimicrobial octadecapeptide from rice α-amylase

Previously, we showed that the antimicrobial cationic and amphipathic octadecapeptide AmyI‐1‐18 from rice α‐amylase (AmyI‐1) inhibited the endotoxic activity of lipopolysaccharide (LPS) from Escherichia coli. In addition, we demonstrated that several AmyI‐1‐18 analogs containing arginine or leucine substitutions, which were designed on the basis of the helical wheel projection of AmyI‐1‐18, exhibited higher antimicrobial activity against human pathogenic microorganisms than AmyI‐1‐18. In the present study, anti‐inflammatory (anti‐endotoxic) activities of five AmyI‐1‐18 analogs containing arginine or leucine substitutions were investigated. Two single arginine‐substituted and two single leucine‐substituted AmyI‐1‐18 analogs inhibited the production of LPS‐induced nitric oxide in mouse macrophages (RAW264) more effectively than AmyI‐1‐18. These data indicate that enhanced cationic and hydrophobic properties of AmyI‐1‐18 are associated with improved anti‐endotoxic activity. In subsequent chromogenic Limulus amebocyte lysate assays, 50% inhibitory concentrations (IC50) of the three AmyI‐1‐18 analogs (G12R, D15R, and E9L) were 0.11–0.13 μm, indicating higher anti‐endotoxic activity than that of AmyI‐1‐18 (IC50, 0.22 μm), and specific LPS binding activity. In agreement, surface plasmon resonance analyses confirmed direct LPS binding of three AmyI‐1‐18 analogs. In addition, AmyI‐1‐18 analogs exhibited little or no cytotoxic activity against RAW264 cells, indicating that enhancements of anti‐inflammatory and LPS‐neutralizing activities following replacement of arginine or leucine did not result in significant increases in cytotoxicity. This study shows that the arginine‐substituted and leucine‐substituted AmyI‐1‐18 analogs with improved anti‐endotoxic and antimicrobial activities have clinical potential as dual‐function host defense agents. Copyright