Tomoyasu Aizawa

Heat-treatment method for producing fatty acid-bound alpha-lactalbumin that induces tumor cell death.

HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells), which was identified in human breast milk as an alpha-lactalbumin (LA)-oleic acid complex, kills tumor cells, selectively. Although it may have potential as a therapeutic agent against various tumor cells, only low-volume methods for its production exist. In this study, heat treatment was used to produce complexes from LAs and oleic acid using a simple method. In the case of human LA and oleic acid, heat-treated samples apparently showed much stronger activities than those treated at room temperature, with cytotoxicities equal to that of HAMLET. Furthermore, circular dichroism spectroscopy revealed that heat-treated samples lost their tertiary structure, suggesting a molten globule as oleic acid-bound LA. BLA samples also showed strong activities by heat treatment. Batch production with heat treatment can efficiently convert LAs into tumoricidal complexes.

Crystal Structure of Cel44A, a Glycoside Hydrolase Family 44 Endoglucanase from Clostridium thermocellum

The crystal structure of Cel44A, which is one of the enzymatic components of the cellulosome of Clostridium thermocellum, was solved at a resolution of 0.96Å. This enzyme belongs to glycoside hydrolase family (GH family) 44. The structure reveals that Cel44A consists of a TIM-like barrel domain and a β-sandwich domain. The wild-type and the E186Q mutant structures complexed with substrates suggest that two glutamic acid residues, Glu186 and Glu359, are the active residues of the enzyme. Biochemical experiments were performed to confirm this idea. The structural features indicate that GH family 44 belongs to clan GH-A and that the reaction catalyzed by Cel44A is retaining type hydrolysis. The stereochemical course of hydrolysis was confirmed by a 1H NMR experiment using the reduced cellooligosaccharide as a substrate.

Expression and purification of a small cytokine growth-blocking peptide from armyworm Pseudaletia separata by an optimized fermentation method using the methylotrophic yeast Pichia pastoris

A small multifunctional cytokine, growth-blocking peptide (GBP), from the armyworm Pseudaletia separata larvae was expressed as a soluble and active recombinant peptide in the methylotrophic yeast Pichia pastoris. An expression vector for GBP secretion was constructed using vector pPIC9, and GBP was expressed under the control of the alcohol oxidase (AOX1) promoter. Although we first tried to cultivate GBP in shake flask cultures, the yield was low, probably due to proteolysis of the recombinant protein. To overcome this problem, we utilized a high-density fermentation method. The pH of the medium in the fermenter was kept at 3.0, and the medium was collected within 48h post methanol shift to minimize exposure of the target peptide to proteases. Recombinant GBP was purified through three reverse-phase HPLC columns. We characterized the 25 amino acid GBP by molecular mass spectrometry and amino acid sequencing. Plasmatocyte spreading, one of the activities of GBP, was similar between chemically synthesized GBP and purified recombinant GBP. Up to 50mg GBP was recovered per 1L of yeast culture supernatant.

Interaction between tachyplesin I, an antimicrobial peptide derived from horseshoe crab, and lipopolysaccharide

Lipopolysaccharide (LPS) is a major constituent of the outer membrane of Gram-negative bacteria and is the very first site of interactions with antimicrobial peptides (AMPs). In order to gain better insight into the interaction between LPS and AMPs, we determined the structure of tachyplesin I (TP I), an antimicrobial peptide derived from horseshoe crab, in its bound state with LPS and proposed the complex structure of TP I and LPS using a docking program. CD and NMR measurements revealed that binding to LPS slightly extends the two β-strands of TP I and stabilizes the whole structure of TP I. The fluorescence wavelength of an intrinsic tryptophan of TP I and fluorescence quenching in the presence or absence of LPS indicated that a tryptophan residue is incorporated into the hydrophobic environment of LPS. Finally, we succeeded in proposing a structural model for the complex of TP I and LPS by using a docking program. The calculated model structure suggested that the cationic residues of TP I interact with phosphate groups and saccharides of LPS, whereas hydrophobic residues interact with the acyl chains of LPS.

Molecular mechanisms of the cytotoxicity of human α-lactalbumin made lethal to tumor cells (HAMLET) and other protein-oleic acid complexes

Background: The α-lactalbumin-oleic acid complex has a unique apoptotic activity for selectively killing tumor cells. Results: We identified the oleic acid-binding site in the human- and goat-α-lactalbumin complexes by two-dimensional NMR. Conclusion: Oleic acid is bound to a loosely organized hydrophobic core of the proteins in the molten globule state. Significance: The results are crucial for understanding the molecular mechanisms of the cytotoxicity of the protein-oleic acid complexes. Although HAMLET (human α-lactalbumin made lethal to tumor cells), a complex formed by human α-lactalbumin and oleic acid, has a unique apoptotic activity for the selective killing of tumor cells, the molecular mechanisms of expression of the HAMLET activity are not well understood. Therefore, we studied the molecular properties of HAMLET and its goat counterpart, GAMLET (goat α-lactalbumin made lethal to tumor cells), by pulse field gradient NMR and 920-MHz two-dimensional NMR techniques. We also examined the expression of HAMLET-like activities of complexes between oleic acid and other proteins that form a stable molten globule state. We observed that both HAMLET and GAMLET at pH 7.5 were heterogeneous, composed of the native protein, the monomeric molten globule-like state, and the oligomeric species. At pH 2.0 and 50 °C, HAMLET and GAMLET appeared in the monomeric state, and we identified the oleic acid-binding site in the complexes by two-dimensional NMR. Rather surprisingly, the binding site thus identified was markedly different between HAMLET and GAMLET. Furthermore, canine milk lysozyme, apo-myoglobin, and β2-microglobulin all formed the HAMLET-like complex with the anti-tumor activity, when the protein was treated with oleic acid under conditions in which their molten globule states were stable. From these results, we conclude that the protein portion of HAMLET, GAMLET, and the other HAMLET-like protein-oleic acid complexes is not the origin of their cytotoxicity to tumor cells and that the protein portion of these complexes plays a role in the delivery of cytotoxic oleic acid molecules into tumor cells across the cell membrane.

Adsorption of human lysozyme onto hydroxyapatite. Identification of its adsorbing site using site-directed mutagenesis.

To elucidate hydroxyapatite‐protein interaction, mutant human lysozymes in which the surface charge was modified by site‐directed mutagenesis were used. Five mutant human lysozymes (K1A, K13A, K33A, R10A, R14A) were expressed in yeast. The chromatographic behavior of these lysozymes was studied with a HPLC hydroxyapatite column. Elution molarities of K1A and R14A mutants were greatly lowered. While Lys‐13 and Arg‐10 are located around Lys‐1 and Arg‐14, K13A and R10A mutants bound onto hydroxyapatite stronger than K1A and R14A mutants. In combination with an X‐ray crystal structure of human lysozyme, it is concluded that the adsorbing site of human lysozyme is at the back of the active site and that Arg‐14, Lys‐1, Arg‐10 and Lys‐13 play important roles in binding.

A Novel Peptide Mediates Aggregation and Migration of Hemocytes from an Insect

Insect blood cells (hemocytes) comprise an essential arm of the immune system [1-7]. Several factors mediating recognition and phagocytosis of foreign intruders by hemocytes have been identified, but the mechanisms regulating hemocyte movement remain fragmentary. Embryonic hemocytes from Drosophila migrate along stereotypical routes in response to chemotactic signals from PVF ligands, members of the platelet-derived growth factor family [8-12]. Embryonic and larval hemocytes also accumulate at external wounds [11-13], but PVFs are not required for this response, suggesting involvement by other, unknown factors. Here we report the identification of hemocyte chemotactic peptide (HCP) from the moth Pseudaletia separata and present evidence that it stimulates aggregation and directed movement of phagocytic hemocytes. Spatiotemporal studies revealed that HCP is expressed in both epidermal cells and hemocytes, whereas structure-function studies identified post-translational modifications important for activity. HCP also shares similarities with another group of cytokines from moths called ENF peptides [14-17]. Taken together, our results identify HCP as a chemotactic cytokine that enhances clotting at wound sites in larvae.

In Vitro Antimicrobial Properties of Recombinant ASABF, an Antimicrobial Peptide Isolated from the Nematode Ascaris suum

ABSTRACT ASABF is a CSαβ-type antimicrobial peptide that contains four intramolecular disulfide bridges (Y. Kato and S. Komatsu, J. Biol. Chem. 271:30493–30498, 1996). In the present study, a recombinant ASABF was produced by using a yeast expression system, and its antimicrobial activity was characterized in detail. The recombinant ASABF was active against all gram-positive bacteria tested (7 of 7; minimum bactericidal concentration [MBC], 0.03 to 1 μg/ml) exceptLeuconostoc mesenteroides, some gram-negative bacteria (8 of 14; MBC, >0.5 μg/ml), and some yeasts (3 of 9; MBC >3 μg/ml). Slight hemolytic activity (4.2% at 100 μg/ml) against human erythrocytes was observed only under low-ionic-strength conditions. Less than 1 min of contact was enough to kill Staphylococcus aureus ATCC 6538P. The bactericidal activity against S. aureus was inhibited by salts.

Development of a novel multiplex lateral flow assay using an antimicrobial peptide for the detection of Shiga toxin-producing Escherichia coli

The binding capacity of peptides with broad antimicrobial activity, or antimicrobial peptides (AMPs), to microbes has recently been applied to the specific detection of bacteria and viruses. We established a novel lateral flow assay (LFA) that combines AMPs labeled with colloidal gold and a target-specific antibody immobilized on a nitrocellulose membrane. α-Helical AMPs, especially cecropin P1 (CP1), magainin 2 (MG2), and ceratotoxin A (CtxA), were shown to have optimal properties as probes in LFA. We also established a multiplex LFA for the simultaneous detection and identification of three serogroups of Shiga toxin-producing Escherichia coli (STEC) using the CP1 probe with polyclonal antibodies anti-O157, anti-O26, and anti-O111. Each serogroup of E. coli could easily and rapidly be detected by multiplex LFA using CP1 and each was clearly visualized in a different position on the LFA strip. The multiplex LFA could detect all tested E. coli strains from serogroups O157 (22/22), O26 (17/17), and O111 (7/7), and the detection limit was 10(4)CFU/mL. No other serogroups of E. coli, including STEC O45, O91, O103, O121, and O145, or non-E. coli strains, reacted. The multiplex LFA could detect E. coli O157, O26, and O111 in food samples at very low levels (6.3, 2.9, and 5.6 CFU per 25 g of ground beef, respectively) after 18-h enrichment, and these results were in accordance with the results of the culture method, immunochromatography (IC) strip, and PCR. Given the broad binding capacity, AMP probes in combination with specific antibodies in the novel multiplex LFA may have the potential to detect various microbes simultaneously with identification on a single strip.

Halorhodopsin from Natronomonas pharaonis Forms a Trimer Even in the Presence of a Detergent, Dodecyl‐β‐d‐maltoside

Halorhodopsin (HR) is a transmembrane seven‐helix retinal protein, and acts as an inward light‐driven Cl− pump. HR from Natronomonas pharaonis (NpHR) can be expressed in Escherichia coli inner membrane in large quantities. Here, we showed that NpHR forms the trimer structure even in the presence of 0.1% (2 mm) to 1% (20 mm) dodecyl‐β‐d‐maltoside (DDM), whose concentrations are much higher than the critical micelle concentration (0.17 mm). This conclusion was drawn from the following observations. (1) NpHR in the DDM solution showed an exciton‐coupling circular dichroism (CD) spectrum. (2) From the elution volume of gel filtration, the molecular mass of the NpHR–DDM complex was estimated. After evaluation of the mass of the bound DDM molecules, the mass of NpHR calculated was approximately equal to that of the trimer. (3) The cross‐linked NpHR by glutaraldehyde gave the SDS‐PAGE corresponding to the trimer. Mass spectra of these samples also support the notion of the trimer. Using the membrane fractions expressing NpHR (Escherichia coli and Halobacterium salinarum), CD spectra showed exciton‐coupling, which suggests strongly the trimer structure in the cell membrane.