Molecular cloning of cDNA for sapecin and unique expression of the sapecin gene during the development of Sarcophaga peregrina

A cDNA clone for sapecin, an antibacterial protein produced by an embryonic cell line of Sarcophaga peregrina, was isolated and characterized. This clone was found to encode a precursor of sapecin consisting of 94 residues, with sapecin (40 residues) constituting its carboxyl-terminal half. RNA blot hybridization revealed that the gene for the sapecin precursor is activated in the hemocytes of the third instar larvae of Sarcophaga in response to body injury. Thus, sapecin is probably a defense protein synthesized by Sarcophaga to prevent bacterial infection through the damaged body wall. This gene was also found to be activated in the embryonic and early pupal stages, suggesting that sapecin also plays a role in the ontogenetic processes of Sarcophaga.     

Purification of sarcotoxin III, a new antibacterial protein of Sarcophaga peregrina

A glycine-rich antibacterial protein with a molecular mass of 7,000 termed sarcotoxin III, was purified to homogeneity from the hemolymph of third instar larvae of Sarcophaga peregrina. When the hemolymph was fractionated, this protein was recovered in the same fraction as sarcotoxin I, a group of potent antibacterial proteins that have been purified. But, it was clearly different from sarcotoxin I in amino acid composition and molecular mass. Sarcotoxin III was shown to be induced in the hemolymph in response to injury of the larval body wall.     

Primary structure of sarcotoxin I, an antibacterial protein induced in the hemolymph of Sarcophaga peregrina (flesh fly) larvae

The primary structure of sarcotoxin I, a potent bactericidal protein induced in the hemolymph of larvae of Sarcophaga peregrina (flesh fly), was investigated. Sarcotoxin I was a mixture of three proteins (sarcotoxins IA, IB, and IC) with almost identical primary structures. These proteins were found to consist of 39 amino acid residues and to differ in only 2-3 amino acid residues. The amino-terminal half of the molecules was rich in charged amino acids and was hydrophilic, whereas the carboxyl-terminal half was hydrophobic. It is suggested that the carboxyl-terminal half of sarcotoxin I penetrates into the bacterial membrane and that its amino-terminal half rich in basic amino acid residues interacts with acidic phospholipids in the bacterial membrane, resulting in perturbation of the membrane and loss of viability of the bacteria.     

Molecular cloning of a cDNA and assignment of the C-terminal of sarcotoxin IA, a potent antibacterial protein of Sarcophaga peregrina.

A previous paper described the complete amino acid sequences of sarcotoxins IA, IB and IC, which are a group of potent antibacterial proteins with almost identical primary structures produced by Sarcophaga peregrina (fleshfly) larvae [Okada & Natori (1985) J. Biol. Chem. 260, 7174-7177]. The present paper describes the cDNA cloning and complete nucleotide sequencing of a cDNA clone for sarcotoxin IA. The C-terminal amino acid residue of sarcotoxin IA deduced from the nucleotide sequence was glycine, whereas it was found to be arginine by amino acid sequencing of purified sarcotoxin IA. Analysis of the elution profiles on h.p.l.c. of the synthetic derivatives of sarcotoxin IA showed that the C-terminal amino acid residue of authentic sarcotoxin IA is amidated arginine, which is probably produced by enzymic cleavage of terminal glycine.     

Production of recombinant sarcotoxin IA in Bombyx mori cells.

A cDNA for sarcotoxin IA, an antibacterial protein of Sarcophaga peregrina (fleshfly), was inserted into a silkworm baculovirus vector and expressed in Bm-N cells, a line of Bombyx mori cells. When a cysteine proteinase inhibitor, p-chloromercuribenzenesulphonic acid, was present in the culture medium, a significant amount of recombinant sarcotoxin IA accumulated, but without this reagent the product seemed to be degraded in this system. The C-terminus of the recombinant sarcotoxin IA seemed to be glycine, not amidated arginine as found in authentic sarcotoxin IA. Probably, Bm-N cells lack the C-terminal alpha-amidation enzyme.     

Purification of sarcotoxin II, antibacterial proteins of Sarcophaga peregrina (flesh fly) larvae

Three antibacterial proteins with almost identical primary structures termed sarcotoxin IIA, IIB, and IIC were purified to homogeneity from the hemolymph of third instar larvae of Sarcophaga peregrina. The molecular masses of these proteins were about 24,000. These proteins were found to have common antigenicity, and antibody against sarcotoxin IIA cross-reacted with sarcotoxin IIB and IIC. Radioimmunoassay using this antibody showed that these proteins are induced in the hemolymph in response to injury of the larval body wall.     

Involvement of sapecin in embryonic cell proliferation of Sarcophaga peregrina (flesh fly)

Addition of antibodies against sapecin to the culture medium of NIH-Sape-4 cells derived from a Sarcophaga embryo greatly inhibited cell proliferation, whereas addition of sapecin stimulated cell proliferation. These results suggest that sapecin is involved in the proliferation of embryonic cells of Sarcophaga. Sapecin is known to have potent antibacterial activity, so it seems to have two different biological functions: i.e. protection against bacterial infection and stimulation of embryonic cell proliferation.     

A potent antibacterial protein in royal jelly. Purification and determination of the primary structure of royalisin

A new potent antibacterial protein, for which we propose the name royalisin, was found in royal jelly of the honeybee Apis mellifera L. and purified to homogeneity for the first time by acid extraction, gel filtration, and reverse-phase high pressure liquid chromatography. The primary structure of royalisin was determined to consist of 51 residues, with three intramolecular disulfide linkages, having a calculated molecular mass of 5523 Da. Royalisin is an amphipathic protein, with the C-terminal half of the molecule being rich in charged amino acids; and it showed extensive sequence homology to two other antibacterial proteins, sapecin from embryonic Sarcophaga peregrina cells and phormicins from Phormia terranovae larvae. Royalisin was found to have potent antibacterial activity against Gram-positive bacteria at low concentrations, but not against Gram-negative bacteria. Royalisin may be involved in a defense system active against bacterial invasion of the honeybee.     

Interaction between liposomes and sarcotoxin IA, a potent antibacterial protein of Sarcophaga peregrina (flesh fly)

The direct interaction between phospholipids and sarcotoxin IA, a potent bactericidal protein of Sarcophaga peregrina, was studied using authentic sarcotoxin IA, its synthetic derivatives, and various liposomes. Results showed that sarcotoxin IA interacted with liposomes constituted from acidic phospholipids, resulting in the release of glucose trapped in these liposomes. The amidated carboxyl-terminal of this protein was found to be important for this interaction. Liposomes constituted from total phospholipids of Escherichia coli became less susceptible to sarcotoxin IA with an increase in their cholesterol content. Since bacterial membranes do not contain cholesterol, this finding may partly explain the selective toxicity of sarcotoxin I to bacteria.     

Purification of Sarcophaga (fleshfly) lectin and detection of sarcotoxins in the culture medium of NIH-Sape-4, an embryonic cell line of Sarcophaga peregrina.

An established cell line originating from a Sarcophaga peregrina (fleshfly) embryo, NIH-Sape-4, was found to synthesize mRNAs for Sarcophaga lectin and sarcotoxin IA, but not those for storage protein or 25 kDa protein. These four proteins are known to be synthesized in the fat-body of third-instar larvae, and the two former in particular are known to participate in the defence mechanism of this insect and to be induced in response to injury of the body wall. Thus the embryonic cell line NIH-Sape-4 synthesizes certain defence proteins constitutively. This cell line will be useful for large-scale purification of Sarcophaga lectin, since 50 micrograms of purified Sarcophaga lectin could be obtained from about 400 ml of culture medium.     

Expression of the recombinant antibacterial peptide sarcotoxin IA in Escherichia coli cells

Sarcotoxin IA is an antibacterial peptide that is secreted by a meat-fly Sarcophaga peregrina larva in response to a hypodermic injury or bacterial infection. This peptide is highly toxic against a broad spectrum of both Gram-positive and Gram-negative bacteria and lethal to microbes even at nanomolar concentrations. However, research needs as well as its potential use in medicine require substantial amounts of highly purified sarcotoxin. Because heterologous expression systems proved to be inefficient due to sarcotoxin sensitivity to intracellular proteases, here we propose the biosynthesis of sarcotoxin precursors in Escherichia coli cells that are highly sensitive to the mature peptide. To optimize its biosynthesis, sarcotoxin was translationally fused with proteins highly expressed in E. coli. A fusion partner and the position of sarcotoxin in the chimeric polypeptide were crucial for protecting the sarcotoxin portion of the fusion protein from proteolysis. Released after chemical cleavage of the fusion protein and purified to homogeneity, sarcotoxin displayed antibacterial activity comparable to that previously reported for the natural peptide.     

Saccharomyces cerevisiae cells harboring the gene encoding sarcotoxin IA secrete a peptide that is toxic to plant pathogenic bacteria.

Sarcotoxin IA is a cecropin-type antibacterial protein produced by the flesh fly, Sarcophaga peregrina. Similar to other bactericidal small proteins produced by insects, sarcotoxin IA is released into the hemolymph of larvae and nymphs upon mechanical injury or bacterial infection. The gene (sarco) that encodes this toxin was introduced into Saccharomyces cerevisiae yeast cells and was expressed under a constitutive yeast promoter. The transformed yeast cells were grown in a liquid medium, and a peptide with a similar molecular size to that of the mature sarcotoxin IA was detected in the medium by Western blot analysis. The secreted sarcotoxin-like peptide (SLP) had a potent cytotoxic effect against several bacteria, including plant pathogenic bacteria, similar to the toxic effects of the authentic sarcotoxin IA. Erwinia carotovora was more susceptible to the toxic medium than Pseudomonas solanacearum and Pseudomonas syringae pv. lachrymans. Thus, yeast may be used in the production of such proteins for employment against various bacterial pathogens.     

Molecular cloning, sequencing, and characterization of cDNA for sarcotoxin IIA, an inducible antibacterial protein of Sarcophaga peregrina (flesh fly)

A cDNA clone for sarcotoxin IIA, an antibacterial protein of Sarcophaga peregrina (flesh fly) larvae [Ando, K., Okada, M., & Natori, S. (1987) Biochemistry 26, 226-230], was isolated and characterized. Sarcotoxin IIA was found to consist of 270 amino acid residues. Northern blot analysis showed that the sarcotoxin IIA gene was activated in response to injury of the body wall of the larvae. The gene was activated for much longer after injection of Escherichia coli into the abdominal cavity of larvae than after injection of saline alone. A common nucleotide sequence for mammalian inflammatory mediator protein cDNAs, TTATTTAT, was found in the 3'-untranslated region of sarcotoxin IIA cDNA, suggesting that this protein plays a role in the inflammatory response of this insect.     

Insect immunity. Purification and characterization of a family of novel inducible antibacterial proteins from immunized larvae of the dipteran Phormia terranovae and complete amino-acid sequence of the predominant member, diptericin A

Injury or injection of live bacteria into third instar larvae of the dipteran insect Phormia terranovae results in the appearance in the haemolymph of at least five groups of heat-stable, more or less basic peptides with antibacterial activity against Escherichia coli. Three of these peptides have been purified. The amino acid sequence has been completely established for one of these and partially (first 40 residues from the N-terminus) for the two others. The sequences show marked homologies indicating that the three peptides belong to a common family. They are not related to other known antibacterial peptides from insects [lysozymes, cecropins (including sarcotoxin I) and attacins]. We propose the name of diptericins for this new family of antibiotic molecules.     

1H nuclear magnetic resonance study of the solution conformation of an antibacterial protein, sapecin

The solution conformation of an antibacterial protein sapecin has been determined by 1H nuclear magnetic resonance (NMR) and dynamical simulated annealing calculations. It has been shown that the polypeptide fold consists of one flexible loop (residues 4-12), one helix (residues 15-23), and two extended strands (residues 24-31 and 34-40). It was found that the tertiary structure of sapecin is completely different from that of rabbit neutrophil defensin NP-5, which is homologous to sapecin in the amino acid sequences and also has the antibacterial activity. The three-dimensional structure determination has revealed that a basic-residue rich region and the hydrophobic surface face each other on the surface of sapecin.     

Molecular cloning of cDNA for sarcocystatin A and analysis of the expression of the sarcocystatin A gene during development of Sarcophaga peregrina

Sarcocystatin A is a cysteine proteinase inhibitor purified from the hemolymph of Sarcophaga peregrina larvae [Suzuki, T., & Natori, S. (1985) J. Biol. Chem. 260, 5115-5120]. We isolated a cDNA clone for sarcocystatin A and analyzed the structure and expression of the sarcocystatin A gene. Sarcocystatin A consists of 102 amino acid residues. Significant homology was found between amino acid sequences of sarcocystatin A and other mammalian cystatins, and highly conserved sequences among mammalian cystatins were also found in sarcocystatin A. Using cloned cDNA as a probe, we investigated expression of the sarcocystatin A gene during the development of Sarcophaga. Results showed that this gene was transiently activated in the very early embryonic stage and in the pupal stage, suggesting that sarcocystatin A participates in morphogenesis of larval and adult structures of Sarcophaga.     

Inhibitory effect of sarcotoxin IIA, an antibacterial protein of Sarcophaga peregrina, on growth of Escherichia coli

The effect of sarcotoxin IIA, an antibacterial protein of Sarcophaga peregrina (flesh fly), on Escherichia coli was investigated. Sarcotoxin IIA was found to have a bacterial effect on growing bacteria, but little on non-growing bacteria. At a concentration of 25 micrograms/ml, it induced significant morphological change of growing E. coli cells. In its presence, growing cells became greatly elongated, and spheroplast-like bulges and projections appeared on their surface. A rough mutant strain of E. coli with a defect in the structure of lipopolysaccharide was more sensitive than the parent strain to sarcotoxin IIA. These results suggest that the main effect of sarcotoxin IIA is to inhibit cell wall synthesis, including septum formation.     

Purification and characterization of an antibacterial protein from haemolymph of Sarcophaga peregrina (flesh-fly) larvae.

Three antibacterial proteins were induced when the body wall of Sarcophaga peregrina (flesh-fly) larvae was injured with a hypodermic needle. These proteins were separated and one was purified to homogeneity. The molecular weight of the purified protein was 5000 and its amino acid composition was similar to that of cecropins, which are antibacterial proteins in Hyalophora cecropia (cecropia moth) pupae. This protein was found to have bactericidal activity and to be effective at a concentration of 0.1 micrograms/ml against certain Gram-negative and Gram-positive bacteria.     

Ionophore activity of sarcotoxin I, a bactericidal protein of Sarcophaga peregrina.

When Escherichia coli was treated with sarcotoxin I, a potent bactericidal protein of Sarcophaga peregrina (fleshfly), K+ inside of the cells leaked out rapidly and the ATP pool of the cells rapidly decreased. These results suggested that the bactericidal effect of sarcotoxin I was due to its ionophore activity, and that it blocked the generation of ATP by inhibiting formation of the proton gradient essential for oxidative phosphorylation. This was confirmed by use of an uncA mutant, which was much less susceptible than the wild-type strain to sarcotoxin I under fixed ionic conditions.