Terumi Nakajima

Brevinin-1 and -2, unique antimicrobial peptides from the skin of the frog, Rana brevipoda porsa

Two unique antimicrobial peptides named brevinin-1 and -2 were isolated from the skin of the frog, Rana brevipoda porsa. Both of the peptides did not have any structural homology with bombinin nor magainin; the frog skin derived-antimicrobial peptides isolated from Bombina and Xenopus, nor even with other known antimicrobial peptides of non-amphibian origin. The minimum inhibitory concentration of brevinin-1 against the growth of St. aureus and E. coli was determined to be 8 micrograms/ml and 34 micrograms/ml while that of brevinin-2 was 8 micrograms/ml and 4 micrograms/ml, respectively, indicating the difference of the two peptides in the antimicrobial selectively on Gram-positive and Gram-negative bacteria.

Conformational change of mastoparan from wasp venom on binding with phospholipid membrane

The conformational change upon binding with phospholipid membrane has been studied of mastoparan from wasp venom, a tetradecapeptide causing the degranulation of mast cells. The 270‐MHz 1H‐NMR spectra and CD spectra indicate that the mastoparan molecule takes the α‐helical conformation in methanol solution, but a much less ordered form in aqueous solution. On binding with phospholipid membrane, the α‐helical conformation is formed even in aqueous medium. Such a conformational change is primarily due to the interaction between the aliphatic side chains of mastoparan and the hydrophobic interior of phospholipid membrane, in contrast to the case of melittin from bee venom.

Anoplin, a novel antimicrobial peptide from the venom of the solitary wasp Anoplius samariensis

A novel antimicrobial peptide, anoplin, was purified from the venom of the solitary wasp Anoplius samariensis. The sequence was mostly analyzed by mass spectrometry, which was corroborated by solid-phase synthesis. Anoplin, composed of 10 amino acid residues, Gly-Leu-Leu-Lys-Arg-Ile-Lys-Thr-Leu-Leu-NH2, has a high homology to crabrolin and mastoparan-X, the mast cell degranulating peptides from social wasp venoms, and, therefore, can be predicted to adopt an amphipathic alpha-helix secondary structure. In fact, the circular dichroism (CD) spectra of anoplin in the presence of trifluoroethanol or sodium dodecyl sulfate showed a high content, up to 55%, of the alpha-helical conformation. A modeling study of anoplin based on its homology to mastoparan-X supported the CD results. Biological evaluation using the synthetic peptide revealed that this peptide exhibited potent activity in stimulating degranulation from rat peritoneal mast cells and broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria. Therefore, this is the first antimicrobial component to be found in the solitary wasp venom and it may play a key role in preventing potential infection by microorganisms during prey consumption by their larvae. Moreover, this peptide is the smallest among the linear alpha-helical antimicrobial peptides hitherto found in nature, which is advantageous for chemical manipulation and medical application.

Syntheses of optically pure β-hydroxyaspartate derivatives as glutamate transporter blockers

DL-threo-beta-benzyloxyaspartate (DL-TBOA) is a non-transportable blocker of the glutamate transporters that serves as an indispensable tool for the investigation of the physiological roles of the transporters. To examine the precise interaction between a blocker and the transporters, we synthesized the optically pure isomers (L- and D-TBOA) and its erythro-isomers. L-TBOA is the most potent blocker for the human excitatory amino acid transporters (EAAT1-3), while D-TBOA revealed a difference in the pharmacophores between EAAT1 and EAAT3. We also synthesized the substituent variants (methyl or naphthylmethyl derivatives) of L-TBOA. The results obtained here suggest that bulky substituents are crucial for non-transportable blockers.

Distinct primary structures of the major peptide toxins from the venom of the spider Macrothele gigas that bind to sites 3 and 4 in the sodium channel1

Six peptide toxins (Magi 1–6) were isolated from the Hexathelidae spider Macrothele gigas. The amino acid sequences of Magi 1, 2, 5 and 6 have low similarities to the amino acid sequences of known spider toxins. The primary structure of Magi 3 is similar to the structure of the palmitoylated peptide named PlTx‐II from the North American spider Plectreurys tristis (Plectreuridae). Moreover, the amino acid sequence of Magi 4, which was revealed by cloning of its cDNA, displays similarities to the Na+ channel modifier δ‐atracotoxin from the Australian spider Atrax robustus (Hexathelidae). Competitive binding assays using several 125I‐labelled peptide toxins clearly demonstrated the specific binding affinity of Magi 1–5 to site 3 of the insect sodium channel and also that of Magi 5 to site 4 of the rat sodium channel. Only Magi 6 did not compete with the scorpion toxin LqhαIT in binding to site 3 despite high toxicity on lepidoptera larvae of 3.1 nmol/g. The K is of other toxins were between 50 pM for Magi 4 and 1747 nM for Magi 1. In addition, only Magi 5 binds to both site 3 in insects (K i=267 nM) and site 4 in rat brain synaptosomes (K i=1.2 nM), whereas it showed no affinities for either mammal binding site 3 or insect binding site 4. Magi 5 is the first spider toxin with binding affinity to site 4 of a mammalian sodium channel.

Effects of threo-β-hydroxyaspartate derivatives on excitatory amino acid transporters (EAAT4 and EAAT5)

d,lthreo‐β‐Benzyloxyaspartate (d,l‐TBOA), an analog of threo‐β‐hydroxyaspartate (THA) possessing a bulky substituent, is a potent non‐transportable blocker for the excitatory amino acid transporters, EAAT1, 2 and 3, while lthreo‐β‐methoxyaspartate (l‐TMOA) is a blocker for EAAT2, but a substrate for EAAT1 and EAAT3. To characterize the actions of these THA analogs and the function of EAAT4 and EAAT5, we performed electrophysiological analyses in EAAT4 or EAAT5 expressed on Xenopus oocytes. In EAAT4‐expressing oocytes, d,l‐TBOA acted as a non‐transportable blocker, while l‐TMOA like d,l‐THA was a competitive substrate. In contrast, d,l‐THA, d,l‐TBOA and l‐TMOA all strongly attenuated the glutamate‐induced currents generated by EAAT5. Among them, l‐TMOA showed the most potent inhibitory action. Moreover, d,l‐THA, d,l‐TBOA and l‐TMOA themselves elicited outward currents at negative potentials and remained inward at positive potentials suggesting that d,l‐TBOA and l‐TMOA, as well as d,l‐THA, not only act as non‐transportable blockers, but also block the EAAT5 leak currents. These results indicate that EAATs 4 and 5 show different sensitivities to THA analogs although they share properties of a glutamate‐gated chloride channel.

Purification, structure-function analysis, and molecular characterization of novel linear peptides from scorpion Opisthacanthus madagascariensis.

In the previous report [Biochem. Biophys. Res. Commun. 286 (2001) 820], we described a novel short linear peptide, IsCT, with cytolytic activity isolated from the venom of scorpion Opisthacanthus madagascariensis. From the same scorpion venom, we further purified and characterized three short linear peptides named IsCT2, IsCTf, and IsCT2f that shared high homology with IsCT, while with different C-terminal areas between IsCT/IsCT2 and IsCTf/IsCT2f. Structure-activity relationship was analyzed by performing vivo and vitro assays and circular dichroism spectroscopy. Like IsCT, IsCT2 showed broad activity spectra against microbes (Gram positive and negative bacteria as well as fungi) and relatively weak hemolytic activity against sheep red blood cells. It adopts an amphipathic alpha-helical structure in aqueous TFE and is able to disrupt the artificial membrane. However, the other two peptides IsCTf and IsCT2f showed no activity in antimicrobial or hemolytic assay. Furthermore, IsCTf and IsCT2f cannot form amphipathic alpha-helix while demonstrating random coil structure in aqueous TFE, which might result in their lost cytolytic activity. IsCTf and IsCT2f both exist in the crude venom and are proved to be enzymatic products from IsCT and IsCT2. Whether they have some other biological activity is still unclear. In addition, we got the cDNAs encoding the precursors of IsCT and IsCT2. Besides the signal peptide, they still contain an unusual acidic pro-peptide at the C-terminal that was quite different from other known precursors of scorpion venom peptides. The novel structure and biological activity of these peptides proposed them to be a new class in scorpion venom.

Secretion of endothelin and related peptides from renal epithelial cell lines

Using specific radioimmunoassays (RIAs) for endothelin (ET) and big ET, we have studied whether ET and related peptides are secreted from renal epithelial cell lines (LLCPK1 and MDCK) of non‐endothelial origin. Dilution curves of extracts of conditioned media from both LLCPK1 and MDCK cell lines were parallel to those of standard porcine (p) ET and big pET in each RIA. Both cell lines incubated in serum‐free medium secreted ET‐ and C‐terminal fragment (CTF)‐like immunoreactivity (LI) of big ET as a function of time. Reverse‐phase HPLC coupled with both RIAs of the extracted media from both cell lines revealed a single component with ET‐LI coeluting with pET(1–21) and several components with CTF‐LI, one corresponding to the elution position of big pET(1–39), one to its CTF(22–39), and the others eluting earlier than CTF. These data indicate that endothelin and related peptides are synthesized by and secreted from cells other than endothelial cells.

A Novel Protein Toxin from the Deadly Box Jellyfish (Sea Wasp, Habu-kurage) Chiropsalmus quadrigatus

The deadly box jellyfish (Sea Wasp, Habu-kurage in Japanese) Chiropsalmus quadrigatus Haeckel (Cubozoa) is distributed widely in the tropical Pacific region. In Japan, three fatal cases due to stings from this species have been reported officially. We successfully isolated C. quadrigatus toxin-A (CqTX-A, 44 kDa), a major proteinaceous toxin, for the first time, from the nematocysts of C. quadrigatus. CqTX-A showed lethal toxicity to crayfish when administered via intraperitoneal injection (LD50=80 μg/kg) and hemolytic activity toward 0.8% sheep red blood cells (ED50= 160 ng/ml). Furthermore, we sequenced the cDNA encoding CqTX-A. The deduced amino acid sequence of CqTX-A (462 amino acids) showed 25.2% and 21.6% sequence similarity to Carybdea rastoni toxins (CrTXs) and Carybdea alata toxin-A (CrTX-A), respectively, which are Cubozoan jellyfish toxins.

High‐performance liquid chromatography matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry peptide fingerprinting of tarantula venoms in the genus Brachypelma: chemotaxonomic and biochemical applications

Precise identification of arthropod species is fundamental in venom research, particularly in groups where taxonomy problems remain unsolved. High-performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis of crude venoms of six tarantula species in the genus Brachypelma showed that the characteristic chromatographic and peptide ion profiles obtained can be used to discriminate amongst closely related species. This method permits rapid mass fingerprinting of large numbers of samples in a reproducible manner, and offers a powerful systematic tool in combination with morphological methods for the classification of tarantula species. The sensitivity and precision of the method may offer a way to solve complex taxonomic relationships not easily resolved by morphological measurements, in a non-destructive manner. Additionally, peptide mapping of crude venoms by MALDI-TOFMS will speed up the discovery of novel ligands of neuronal receptors, since major venom components of related species share a high sequence homology and are likely to possess similar pharmacological properties.