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.     

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.     

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.     

Channel-forming membrane permeabilization by an antibacterial protein, sapecin: determination of membrane-buried and oligomerization surfaces by NMR

The action mechanism of sapecin, an antibacterial peptide with membrane permeabilization activity, was investigated. The dose dependence of the membrane permeabilization caused by sapecin was sigmoidal, suggesting that sapecin oligomerization leads to the membrane permeabilization. Solution nuclear magnetic resonance analysis of the sapecin-phospholipid vesicle complex revealed the surface buried in the membrane and oligomerization surface on the sapecin molecule. The membrane-buried surface of sapecin was determined by observing the transferred cross-saturation phenomena from the alkyl chains of the phospholipid vesicle to the amide protons of sapecin. The membrane-buried surface contains basic and highly exposed hydrophobic residues, which are suitable for interacting with the acidic bacterial membrane. The oligomerization surface was also identified by comparisons between the results from hydrogen-deuterium exchange experiments and transferred cross-saturation experiments. On the basis of the results from the NMR experiments we built a putative model of sapecin oligomers, which provides insights into the membrane permeabilization caused by insect defensins.     

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.     

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.     

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.     

Molecular cloning and characterization of SRAM, a novel insect rel/ankyrin-family protein present in nuclei

Previously, we purified a 59-kDa protein that binds to the kappaB motif of the Sarcophaga lectin gene. Here we report its cDNA cloning and some of its characteristics as a novel member of the Rel/Ankyrin-family. This protein, named SRAM, contained a Rel homology domain, a nuclear localization signal and 4 ankyrin repeats, but lacked the Ser-rich domain and PEST sequence that Relish contained. We found that SRAM was localized in the nuclei of NIH-Sape-4 cells, which are an embryonic cell line of Sarcophaga. The Sarcophaga lectin gene promoter containing tandem repeats of the kappaB motifs was activated in NIH-Sape-4 cells. In Drosophila mbn-2 cells, Dif alone activated this reporter gene and a cooperative effect was detected when SRAM and Dif were co-transfected, although SRAM alone did not activate it. This is the first report of a Rel/Ankyrin molecule that exists in the nuclei.     

Cloning and sequencing of cDNA of Sarcophaga peregrina humoral lectin induced on injury of the body wall

A previous paper described the purification of a lectin induced in the hemolymph of larvae of Sarcophaga peregrina (flesh-fly) on injury of their body wall (Komano, H., Mizuno, D., and Natori, S. (1980) J. Biol. Chem. 255, 2919-2924). This paper describes cDNA cloning and the complete nucleotide sequence of the gene for Sarcophaga lectin. Although active lectin consists of alpha and beta subunits in a molar ratio of 2:1, the fat body of injured larvae was found to contain only mRNA for the alpha subunit, suggesting that these two subunits are derived from a common gene and that the alpha subunit is converted to the beta subunit post-translationally. The alpha subunit was found to consist of 260 amino acid residues with an additional signal sequence of 19 or 23 amino acid residues.     

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.     

Mapping the peptide and protein immune response in the larvae of the fleshfly Sarcophaga bullata.

We chose the larvae of fleshfly Sarcophaga bullata to map the peptide and protein immune response. The hemolymph of the third-instar larvae of S. bullata was used for isolation. The larvae were injected with bacterial suspension to induce an antimicrobial response. The hemolymph was separated into crude fractions, which were subdivided by RP-HPLC, gel electrophoresis, and free-flow electrophoresis. In several fractions, we determined significant antimicrobial activities against the pathogenic bacteria Escherichia coli, Staphylococcus aureus, or Pseudomonas aeruginosa. Among antimicrobially active compounds we identified dipeptide beta-alanyl-L-tyrosine, protein transferrin, and two variants of peptide sapecin. We also partially characterized two novel antimicrobially active polypeptides; odorant-binding protein 99b, and a peptide which remains unidentified.     

Purification and Heterogeneous Localization of Sarcophaga Lectin Receptor on the Surface of Imaginal Discs of Sarcophaga peregrina (Flesh Fly)

Previously, we reported autocrine involvement of Sarcophaga lectin in the development of Sarcophaga imaginal discs (Kawaguchi et al. , Dev. Biol. 144 , 86–93 (1991)). In this study, we purified Sarcophaga lectin binding protein from the membrane fraction of cultured embryonic cells of Sarcophaga to near homogeneity and raised a monoclonal antibody against it. Histochemical analysis using the monoclonal antibody revealed that this binding protein is distributed heterogeneously on the surface of leg imaginal discs. This binding protein was especially clearly localized in the central region of the basal side of leg discs which forms the junction between the leg and body, suggesting the participation of Sarcophaga lectin in morphogenesis of the basal region of the developing leg.     

Structure and expression of a Manduca sexta larval cuticle gene homologous to Drosophila cuticle genes

A genomic clone was isolated from the tobacco hornworm, Manduca sexta, by virtue of its similarity to a Drosophila larval cuticle gene. RNA analysis shows that this clone, B311, is expressed at times appropriate for a larval cuticle gene. Hybrid-selection experiments using B311 DNA show that it encodes a 14 x 10(3) Mr protein, LCP-14, which is precipitated by an antiserum to Manduca larval cuticle. We have sequenced both genomic and cDNA clones for the LCP-14 gene. A conceptual translation of the cDNA sequence shows that the LCP-14 protein is similar not only to another Manduca cuticle protein, but also to Drosophila, Sarcophaga and Hyalophora cecropia cuticle proteins. Since these proteins are found in flexible cuticle and have similar sequences, we conclude they are encoded by homologous genes.     

A ribosomal protein is encoded in the chloroplast DNA in a lower plant but in the nucleus in angiosperms. Isolation of the spinach L21 protein and cDNA clone with transit and an unusual repeat sequence

The distribution of chloroplast ribosomal protein genes between the organelle DNA and the nuclear DNA is highly conserved in land plants, but a notable exception is rpl21. This gene has been found in the completely sequenced chloroplast genome of a lower plant but not in that of two higher plants. We describe the purification and characterization of the spinach chloroplast ribosomal protein L21 and the isolation and nucleotide sequence of a cDNA clone that encodes its cytoplasmic precursor. The mature protein, identified by NH2-terminal sequencing, has 201 residues (Mr 22,766) and is thus substantially larger than either its Escherichia coli (103 residues) or the lower plant homologue (116 residues). The extra length is in peptide extensions at both amino and carboxyl termini. The COOH-terminal extension is unusual in that it comprises seven Ala-Glu repeats, a feature not found in any other ribosomal proteins described so far. The cDNA clone also encodes a 55-residue long transit peptide (with a high proportion of the polar residues, threonine and serine), to target the L21 protein into chloroplasts. The identification of rpl21 as a nuclear gene in a higher plant (spinach) and chloroplast gene in a lower plant (liverwort) suggests an organelle-to-nucleus gene relocation during the evolution of the former.     

Anti-tumor factor in insects' hemolymph

We have purified a lectin from the hemolymph of Sarcophaga peregrina larvae, obtained after injury of their body wall. This lectin recognizes galactose residues and suggested to be involved in the defense mechanism of this insect. We also demonstrated that this lectin induced cytotoxic effects on tumor cells in the presence of murine macrophages. It was found that the murine macrophages had Sarcophaga lectin binding proteins. Using pupal hemocytes and fatbody of this insect, we established an in vitro system that mimics dissociation of the fatbody in vivo. New membrane protein, induced on the surface of the hemocytes at pupation, suggested to participate in the recognition of the fatbody.