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.     

Cloning of gene cluster for sarcotoxin I, antibacterial proteins of Sarcophaga peregrina

A genomic clone of sarcotoxin I was isolated. This clone contained four genes of structurally related proteins belonging to the sarcotoxin I family present in tandem array. One of these genes was sequenced and found to be the sarcotoxin IB gene. This gene contained a single intron of 95 bases.     

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.     

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.     

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.     

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.     

Analysis of a gene cluster for sarcotoxin II, a group of antibacterial proteins of Sarcophaga peregrina.

Sarcotoxin II is a group of antibacterial proteins of Sarcophaga peregrina (flesh fly) with related primary structures. We have cloned three genes in this family. These genes formed a tandem array with about 2-kb intervals, and one of them was present in the opposite strand. The putative amino acid sequences of the proteins encoded by these genes were very similar except for a deletion in one of them. All of the genes were found to be activated transiently in the same way when the larval body wall was injured, suggesting that the encoded proteins are acute-phase-responsive proteins for protecting the insect from bacterial infection.     

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, 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.     

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.     

Purification of a 29-kDa hemocyte proteinase of Sarcophaga peregrina.

Previously, we suggested the participation of a hemocyte proteinase in the dissociation of fat body of Sarcophaga peregrina (flesh fly) at metamorphosis. We have now purified this proteinase to near homogeneity from pupal hemocytes. It is a cysteine proteinase with a molecular mass of 29 kDa and has a unique substrate specificity hydrolyzing both Suc-Leu-Leu-Val-Tyr-MCA and Z-Phe-Arg-MCA (Suc, succinyl; MCA, methylcoumaryl-7-amide; Z, carbobenzoxy), which are substrates for chymotrypsin and cathepsin B, respectively. Partial similarity was found between the amino-terminal sequence of this proteinase and that of cathepsin B, including Pro, Glu and Arg residues conserved in the papain superfamily of enzymes.     

Purification of three antibacterial proteins from the culture medium of NIH-Sape-4, an embryonic cell line of Sarcophaga peregrina

Three antibacterial proteins were purified from the culture medium of NIH-Sape-4, an embryonic cell line of Sarcophaga peregrina (flesh fly). Sequencing studies showed that two of these proteins belong to the sarcotoxin I family, potent antibacterial proteins purified from the hemolymph of Sarcophaga larvae, whereas the other protein, named sapecin, is a new protein consisting of 40 amino acid residues including 6 cysteine residues. Unlike sarcotoxin I, sapecin preferentially represses the growth of various Gram-positive bacteria. The proteins of the sarcotoxin I family produced by this cell line were found to have carboxyl-terminal glycine, whereas sarcotoxin I in the hemolymph has amidated amino acids. This suggests that the embryonic cells lack an enzyme that cleaves off carboxyl-terminal glycine to form a new amidated carboxyl terminus.     

免疫亲和层析法纯化棕尾别麻蝇(Sarcophagaperegrina)幼虫血淋巴凝集素

 报道了利用免疫亲和层析法纯化棕尾别麻蝇幼虫血淋巴凝集素的结果．哺乳动物红细胞能够特异地吸附凝集素．用兔红细胞与麻蝇幼虫血淋巴凝集素形成的复合体免疫供血家兔,得到麻蝇幼虫血淋巴凝集素的抗体．再利用抗体制备亲和吸附柱,通过免疫亲和层析一次性纯化了麻蝇幼虫血淋巴凝集素． Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ结果显示,该凝集素的分子量约为７３ ｋ Ｄ．这一结果,与用对麻蝇幼虫血淋巴凝集素有抑制作用的糖蛋白—胎球蛋白和甲状腺球蛋白为配基,亲和层析纯化的结果完全相同,表明用这种免疫亲和层析法纯化凝集素是可行的．为不清楚专一性识别糖或专一性识别糖不典型,难于用普通亲和层析纯化的凝集素,提供了一种有效的纯化方法．     

Purification of lectin induced in the hemolymph of Sarcophaga peregrina larvae on injury

A lectin was purified from the hemolymph of Sarcophaga peregrina larvae, obtained after injury of their body wall. This lectin agglutinated sheep red blood cells markedly and the hemagglutinating activity was inhibited by galactose and lactose. The active lectin was found to have a molecular weight of 190,000 and to consist of four alpha subunits and two beta subunits, with molecular weights of 32,000 and 30,000, respectively. During the early pupal stage, similar hemagglutinating activity in the hemolymph increased to several times than in larval hemolymph. This activity was completely inhibited by the antibody prepared against the lectin purified from the hemolymph of injured larvae. Thus, the same protein having lectin activity is apparently induced under two different physiological conditions: injury of the body wall of larvae and during pupation. The biological significance of this lectin is discussed.     

Purification of the 200 kDa hemocyte membrane protein of Sarcophaga peregrina and its specific interaction with fat body

A 200 kDa protein specifically expressed on the surface of pupal hemocytes of Sarcophaga peregrina was purified from the hemocyte membrane. This protein has been suggested to participate in dissociation of the fat body in the pupal stage of this insect. This protein was found to inhibit the dissociation of the fat body in vitro. Furthermore, it was shown to bind to the fat body and the binding could be saturated. These results suggested that pupal hemocytes expressing the 200 kDa protein interact directly with specific binding sites on the basement membrane of the fat body when they disintegrate this tissue.     

Structural analysis of a developmentally regulated 25-kDa protein gene of Sarcophaga peregrina

In the previous paper, we described the identification of two abundant mRNAs of Sarcophaga peregrina (flesh-fly) which are selectively expressed in the fat body of middle third instar larvae. One of these mRNAs was found to encode a protein with a molecular mass of about 25,000 (25-kDa protein) when translated in vitro (Tamura, H., et al. (1983) Dev. Biol. 99, 145-151). Present paper reports the nucleotide sequence of a 2.3 kb DNA containing the entire gene for the 25-kDa protein. This gene consisted of four exons and contained an open reading frame for 184 amino acids. A CAT box and a TATA box were found in the 5'-flanking sequence. A poly A addition signal of AATAAA was assigned to the non-coding region in the fourth exon. A sequence having 75% homology with SV40 enhancer core sequence was identified in the non-coding region of the first exon.     

Identification and characterization of an antibacterial peptide of the 26-kDa protease of Sarcophaga peregrina with antibacterial activity

Previously, we purified a serine protease with a molecular mass of 26 kDa that exhibits potent antibacterial activity from a pupal extract of Sarcophaga peregrina (flesh fly). We divided this protease into 12 peptides and examined their antibacterial activity. A peptide corresponding to residues 155 to 174 (peptide 9) was found to exhibit antibacterial activity comparable to that of the 26-kDa protease. When Escherichia coli was treated with peptide 9, the permeability of both the outer and inner membranes increased, and substrates for beta-lactamase and beta-galactosidase entered the cells, but beta-galactosidase did not leak out of the cells under these conditions. It was suggested that residues 6 to 18 of peptide 9 form an amphiphilic alpha-helix under hydrophobic conditions with an N-terminal basic loop and then interact with acidic phospholipids in the bacterial membranes.     

Identification and Purification of Sulfotransferases for 20-Hydroxysteroid from the Larval Fat Body of a Fleshfly,Sarcophaga peregrina

Sulfotransferase (ST) activity for 20-hydroxyecdysone (20E) was identified in a larval fat body lysate of the fleshfly, Sarcophaga peregrina, but not in the hemolymph. The activity was highly sensitive to 2,6-dichloro-4-nitrophenol (DCNP) (IC₅₀=0.61 μM), a specific inhibitor of phenol ST (P-ST), but insensitive to triethylamine, a hydroxysteroid ST inhibitor. These results suggest that 20E-specific ST enzymes belong to the P-ST family, despite the fact that 20E is a hydroxysteroid. In addition to 20E ST activity, a relatively high level of 2-naphthol ST activity was detected in the fat body lysate. The ST activity for both substrates transiently decreased to the 50% of maximal levels, 6 hrs after induction of pupation. The ST enzymes were separated on a DEAE-cellulose column. The 20E-ST enzymes were eluted around 50 mM KCl as two separate peaks of close proximity and the P-ST was eluted at 0.1 M KCl. The 20E ST enzymes were further purified using 3′-phosphoadenosine 5′-phosphate (PAP)-agarose affinity column chromatography. Both of the eluted active fractions demonstrated 43-kDa proteins on SDS-polyacrylamide gel. Photoaffinity labeling with [³⁵S]-3′-phosphoadenosine 5′-phosphosulfate (PAPS) showed 43-kDa bands in the fat body lysate, as well as in the purified fractions. These results suggest that the 43-kDa proteins catalyze 20E sulfation within the fat body of S. peregrina.     

Determination of the disulfide array in sapecin, an antibacterial peptide of Sarcophaga peregrina (flesh fly)

Sapecin is a 40-residue peptide containing 6 half-cystine residues. The disulfide structure of sapecin was determined by sequencing cystine-containing peptides obtained by digesting sapecin with thermolysin. Results showed that sapecin has a vortical structure fixed by 3 disulfide bonds between cysteine residues 3 and 30, 16 and 36, and 20 and 38, respectively, and that these disulfide bonds are essential for its antibacterial activity.