Detection of
petidoglycan and β-glucan with |
Abstract A method to detect peptidoglycan and (1→3)-β-D-glucan with silkworm larvae plasma (SLP) derived from the the hemolymph of the silkworm, Bombyx mori was developed. SLP contains all of the factors of the pro-phenol oxidase cascade, an important self-defense mechanism of insects. Peptidoglycan or (1→3)-β-D-glucan initiates the cascade, in which pro-phenol oxidase is finally activated to phenol oxidase. Thephenol oxidase activity was was colorimetrically or visually detected with 3,4-dihydroxyphenylan as a substrate. SLP displayed high reactivity with peptidoglycan and polysaccharides containing 1,3-β-glucosidic linkages, but not with endotoxins. SLP is useful for the detection of microbial contamination because peptidoglycan and (1→3)-β-D-glucan are cell wall components of bacteria and fungi, respectively. Keywords : Peptidoglycan; β-glucan; Silkworm larvae plasma; Pro-phenol oxidase cascade
Introduction The pro-phenol oxidase(proPO) cascade, which is present in insect hemolymph and cuticle, plays an important role in the defense mechanism of insects [1,2]. Local melanization around microbial invaders in insects results from the activation of the proPO in this cascade. This cascade is composed of a (1→3)-β-D-glucan recognition protein, a peptidoglycan recognition protein, zymogens of serine proteases, proPO, and as yet unidentified factors. Ashida reported a method to collect intact plasma from silkworm larvae, Bombyx mori[3]. The proPo cascade of the plasma was shown to be activated by peptidoglycan or (1→3)-β-D-glucan[3,4]. Peptidoglycan, a cell wall component of bacteria, has many kinds of biological activities such as immunoadjuvant activity and stimulation of macrophages[5]. Yeast and fungi have (1→3)-β-D-glucan in their cell wall. Detection of these cell wall components is important in clinical and pharmaceutical fields. Methods to detect (1→3)-β-D-glucan with Limulus amebocyte lysate, which is commonly used for the detection of bacterial endotoxins, have been reported[6,7]. Since no method has been reported to detect intact peptidoglycan, the purpose of this study was to develop a method for detecting peptidoglycan and (1→3)-β-D-glucan using the proPO cascade of silkworm hemolymph. |