Kiyoko Taniai

Lipopolysaccharide-lipophorin complex formation in insect hemolymph: a common pathway of lipopolysaccharide detoxification both in insects and in mammals

The formation of the lipophorin-lipopolysaccharide (LPS) complex in Bombyx mori hemolymph and its role in LPS detoxification were explored. LPS, an antibacterial protein inducer in insects, was injected into B. mori larvae. Analytical density gradient ultracentrifugation revealed that after injection the LPS peak shifts to a zone of lower density with time. The shifted peak was identified as the lipophorin-LPS complex. This complex formation was also achieved in an in vitro mixture of cell-free hemolymph and LPS at 25 degrees C but not at 1 degree C. The lipophorin-LPS complex had a significantly lower capacity to elicit the mRNA of cecropin B, an antibacterial protein. The biological activity of reextracted LPS from the complex was slightly reduced in the Limulus test and no structural modification was observed in sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). These results suggested that the formation of lipophorin-LPS strikingly reduces the cecropin inducibility of LPS without any structural change in LPS. Similar serum lipoprotein-LPS complex formation and reduction of biological activities of LPS were also observed in mammals. We, therefore, suggest that the formation of the serum lipoprotein-LPS complex is a common pathway to inactivate LPS both in insects and in mammals.

Expression and characterization of cDNAs for Cecropin B, and antibacterial protein of the silkworm, Bombyx mori

Abstract To analyze the induction mechanism of antibacterial protein gene expression, cDNAs coding for cecropin B have been cloned from the B. mori fat body cDNA library. Nucleotide sequences of two positive clones were determined and their amino acid sequences deduced. They revealed that these clones coded for the same cecropin, which is identical to purified cecropin B. However, the cDNAs contained different nucleotides at the third codon position and 5′ or 3′ non-coding regions. Results obtained by Northern blot analysis showed that the gene expression of B. mori cecropin B was rapidly induced by Escherichia coli and reached maximum levels 8 h after immunization. The expression of cecropin B gene occurred specifically in tissues, mainly in the fat body and hemocytes.

Isolation and nucleotide sequence of cecropin B cDNA clones from the silkworm, Bombyx mori

Two cDNA clones encoding cecropin B, an antibacterial protein, were isolated from a fat body cDNA library of the silkworm, Bombyx mori. Amino acid sequences of these clones, deduced from nucleotide sequences, were identical, including signal peptide regions. However, the nucleotide sequences were different at 30 positions. Deduced amino acid sequences of Bombyx mori cecropin B showed higher homology with cecropins from Lepidoptera than with those from Diptera.

Clearance of lipopolysaccharide in hemolymph of the silkworm Bombyx mori: Its role in the termination of cecropin mRNA induction

Abstract Clearance of lipopolysaccharide (LPS) in the hemolymph of silkworm, Bombyx mori, and its physiological role in the regulation of induction of an antibacterial protein, cecropin B, were investigated. In in vitro culture of fat body, cecropin B mRNA appeared 20 min after incubation with LPS and its accumulation persisted more than 72 h. No significant desensitization was observed for at least 24 h in vitro. On the contrary, cecropin B mRNA completely disappeared 24 h after injection of LPS in vivo. These results suggest that concentration and/or biological activity of LPS decreases in hemolymph with time. LPS concentration assay at various time intervals after injection revealed two phases of LPS clearance from hemolymph, an initial rapid ( T 1 2 = 1.0 h ) followed by a very slow phase ( T 1 2 > 18 h ). The threshold concentration of LPS to induce cecropin B mRNA in vivo was over 10-fold higher than that of in vitro. Although no cecropin mRNA accumulation was detected after 24 h of LPS injection, it appeared again by a reinjection of LPS at this time period. This suggests that the reduction of LPS concentration directly terminates cecropin induction in vivo. We, therefore, conclude that B. mori has a LPS clearance system from hemolymph which is sufficient to terminate LPS mediated antibacterial protein induction.

Isolation and α-amidation of the non-amidated form of cecropin D from larvae of Bombyx mori

Abstract Two forms of cecropin D, an antibacterial protein, were isolated from larvae of the silkworm, Bombyx mori, immunized with Escherichia coli. One was the mature form and the other seemed to contain a glycine residue at its C- terminus . The latter was converted into the mature form in vitro by peptidylglycine α-amidating enzymes from horse serum and the result clearly demonstrates that this protein is the precursor of cecropin D. The mature form had 4- to 5-fold higher antibacterial activity against E. coli or Acinetobacter sp., suggesting that the amidation is important for full expression of antibacterial activity.

Identification of a hemocyte membrane protein of the silkworm, Bombyx mori, which specifically binds to bacterial lipopolysaccharide

Abstract An in vitro system with isolated hemocytes of the silkworm, Bombyx mori (B. mori) was developed to examine the induction mechanism of insect antibacterial proteins by bacterial lipopolysaccharide (LPS). The gene expression of B. mori cecropin B, a representative antibacterial protein, was triggered by LPS in this in vitro system. To identify LPS-binding site(s) of the hemocytes, the [ 125 I]LPS binding assay to the hemocytes was performed in vitro . The amount of [ 125 I]LPS bound to hemocytes increased proportionately with the increase of incubation time and LPS dose. The binding was strongly inhibited by excess unlabeled LPS or lipid A, indicating that the binding of [ 125 I]LPS to hemocytes contains a highly specific reaction. Moreover, the specific binding could not be detected with Bm-N4 cells in which cecropin B gene expression was not induced by LPS, suggesting that the LPS binding is specific for LPS responsive cells. The LPS binding was fully sensitive to the proteinase K treatment of intact hemocytes, suggesting that a protein(s) located on the surface of hemocytes is involved in the LPS binding. Fluorescein isothiocyanate (FITC) conjugated-LPS binding assay demonstrated that this compound mainly binds to granular cells rather than other hemocytes under our assay conditions. Affinity-labeling with photoreactive-LPS allowed the identification of a 11 kDa LPS-binding protein in hemocytes, which might relate to the specific membrane receptor for LPS.

Distribution of Bm1, a SINE type transposable element, in cecropin B genes of the silkworm, Bombyx mori

Abstract In order to investigate the distribution of Bm1 , a SINE type transposable element, in cecropin B genes of the silkworm, Bombyx mori , a genomic library was first screened with cecropin B cDNA as a probe. Eighteen out of 275 positive clones were selected at random and Sa lI digestion patterns were compared. Ten clones which showed different patterns were further analyzed by Southern blotting using a cecropin B cDNA fragment encoding exon 1, exon 2 or the entire coding region as probes. The same Sa lI digested genomic clones were hybridized with a Bm1 probe. Comparison of positive patterns hybridized with the Bm1 probe to those hybridized with the cecropin B probes indicated that all cecropin B-fragments except one fragment had Bm1 . The exceptional fragment contained exon 2 only. The results indicate that Bm1 is widely distributed in cecropin B genes.