We recently isolated and characterized the lipopolysaccharide (LPS)-binding protein, BmLBP, from the larval hemolymph of the silkworm Bombyx mori. BmLBP is a pattern recognition molecule that recognizes the lipid A portion of LPS and participates in a cellular defense reaction. This paper describes the cDNA cloning of BmLBP. The deduced amino acid sequence of BmLBP revealed that BmLBP is a novel member of the C-type lectin superfamily with a unique structural feature that consists of two different carbohydrate-recognition domains in tandem, a short and a long form.z 1999 Federation of European Biochemical Societies.
Proteins having the ability to bind to Escherichia coli K12W3110 (rough (R) mutant) were isolated and purified by affinity precipitation from the larval hemolymph of the silkworm Bombyx mori. These proteins were found to consist of two components with molecular masses of 43 kDa and 40 kDa by SDS/ PAGE. They bound to all E. coli R mutants (Ra, Rb,, Rc, Rd, and Re) and Salmonella minnesota R mutants. However, they did not bind to smooth types of the above bacteria. They bound to both lipopolysaccharide(LPS)-coated and lipid-A-coated microtiter plates and have similar dissociation constants for LPS and lipid A. This indicates that the binding proteins recognize the lipid A portion of LPS and thus, we have named these proteins BmLBP (B. mori LPS-binding proteins). We also found that BmLBP participated in the clearance of E. coli cells injected into the body cavity of the silkworm.Keywords: pattern recognition; lipopolysaccharide; insect ; immunity.Insects have not acquired adaptive immunity which is based on clonal selection of individual specific lymphocytes, yet they have an efficient defense mechanism against invading microorganisms [ 1-31, This defense mechanism involves both cellular and humoral responses. The cellular response includes phagocytosis and/or nodule formation of bacteria and encapsulation of larger parasites by blood cells (hemocytes) [4]. The humoral response utilizes various antimicrobial peptides synthesized in the fat body (a functional homologue of the mammalian liver) and some hemocytes upon induction by septic injury and secreted into the hemolymph [S, 61. These responses are called innate immunity. Information is scanty on the initial step of these responses, that is, the recognition mechanism of invading microorgani sms .Microbes have highly conserved and widely distributed features, certain of which are not found on the cells of multicellular organisms. These features are called microbial pattern with a recognition system termed pattern recognition [7, 81. Pattern recognition is an important selfhon-self discrimination system in innate immunity in various animals including mammals, and the molecules that function in recognizing microbial pattern are called pattern-recognition molecules. It is known that gramnegative bacteria have a characteristic structure, lipopolysaccharide (LPS), on the outer membrane. In mammals, two effector molecules, LPS-binding protein (LBP) and CD14 are known as LPS-recognition proteins and play a role in pattern recognition [9]. In insects, the American cockroach Peripluneta americana has two LPS-recognition proteins, LBP [lo, 111 and Periplanteta lectin [12, 131. They recognize the outer core oligosaccharide of Escherichia coli K12 and 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), respectively, and function as an opsonin [13, 141. In the Mediterranean fruit fly Ceratitis capitata, a protein known as p47 participates in the binding of LPS to hemocytes and the internalization of the bound LPS [IS]. Recently, gramnegative-bacteria-binding protein (GNBP) which was...
CrylAa toxin-binding proteins from the midgut brush border membrane vesicles of Bombyx mori, a toxin-susceptible silkworm, were analyzed to find candidates for the toxin receptors. Ligand blotting showed that CrylAa toxin bound to a 120-kDa protein. A part of the 120-kDa protein was solubilized from the membrane vesicles with phosphatidylinositol-specific phospholipase C, resulting in a 110-kDa protein which therefore may be linked to a glycosyl-phosphatidylinositol anchor. The 120-kDa and 110-kDa Cryl Aa toxin-binding proteins were solubilized with detergent or pohosphatidylinositol-specific phospholipase C, respectively, and purified using anion-exchange chromatography. Scatchard plot analysis for the specific binding of purified 110-kDa protein to Cryl Aa toxin yielded a Kd value of 7.6 nM, which was similar to that for the binding of intact brush border membrane vesicles to the toxin. N-terminal and internal amino acid sequences of the 120-kDa and 110-kDa proteins showed high degrees of similarity to those of aminopeptidase N, a putative CrylAc toxin receptor, reported in Munducu sexta and Heliothis virescens. On this basis, the 120-kDa CrylAa toxin-binding protein from B. mori was identified as a member of the aminopeptidase family.Keywords: Bacillus thuringiensis; S-endotoxin ; Bombyx mori; receptor; aminopeptidase N.Bacillus thuringiensis, a gram-positive bacterium, produces various types of insecticidal proteinaceous crystal inclusions during spomlation [l-31. When this bacterium is ingested by susceptible insects, these crystal inclusions, composed of protoxins [4], are solubilized in the alkaline environment of the insect midgut and processed proteolytically to yield smaller active toxins [3, 51. The toxin is believed to bind specifically to receptor molecules on the midgut epithelial cells of host insects [7-101, disrupting the ion permeability of midgut cell membranes [ll]. This results in a net influx of ions and an accompanying influx of water, so that the cells swell and lyse [6, 121. The formation of cation-selective [13-151 or small non-specific [16] pores in the membrane has been proposed as a possible mechanism of the toxin action [17].Each toxin shows a specific insecticidal spectrum in vivo. In some cases, this activity spectrum correlates with the presence of specific toxin receptors in the brush border membrane vesicles (BBMV) [7][8][9][10] 181, while in other cases the binding of the toxin to the BBMV was unrelated to in vivo toxic activity. In the latter cases, toxin bound to BBMV, but very low-level in vivo toxicity resulted [18, 191. Although the binding of toxin to specific receptors on the midgut epithelial cells may be one of the requirements for the insecticidal activity in vivo, other factors, such as the ability of the toxin to interact with the epithelium membrane lipid layer, might also be involved. However, it is not clear what the normal function of the receptor molecules might be or how these molecules are involved in toxin action. Insects are able to acquire resistance to ...
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