Purification of a beta-1,3-glucan recognition protein in the prophenoloxidase activating system from hemolymph of the silkworm, Bombyx mori

Abstract

The plasma fraction (referred to as plasma-CPB) of silkworm hemolymph, from which a protein with affinity to beta-1,3-glucan was specifically removed according to Yoshida et al. (Yoshida, H., Ochiai, M., and Ashida, M. (1986), Biochem. Biophys. Res. Commun. 141, 1177-1184), was used to develop a method for quantitating the beta-1,3-glucan recognition protein of the prophenoloxidase activating system. In principle, a sample was judged to contain beta-1,3-glucan recognition protein when that sample could restore the ability of the system in plasma-CPB to be triggered by beta-1,3-glucan. Purification procedures for the recognition protein from silkworm hemolymph consisted of fractionation with ammonium sulfate, chromatography on DEAE-Toyopearl, Affi-Gel-heparin, and Mono Q and Superose 12 on the fast protein liquid chromatography system of Pharmacia LKB Biotechnology Inc. About 2.03 mg of beta-1,3-glucan recognition protein was obtained from 300 ml of hemolymph. The purified beta-1,3-glucan recognition protein was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing-polyacrylamide gel electrophoresis. beta-1,3-Glucan recognition protein had a molecular mass of 62 kDa composed of a single polypeptide and an isoelectric point of pH 4.3. It bound to curdlan beads (composed of beta-1,3-glucan with average particle size of 80 micron) in the absence of divalent cation, whereas its binding to glucans with beta(1----4)- or alpha(1----6)-glycosidic linkages was not detected under the experimental conditions. Elution of the beta-1,3-glucan recognition protein bound to curdlan beads could be achieved under strongly denaturing conditions (after incubation of the beads with sodium dodecyl sulfate and beta-mercaptoethanol in boiling water for 5 min), but elution at room temperature was poor. Since beta-1,3-glucan recognition protein is the only protein in silkworm plasma with strong affinity to beta-1,3-glucan and endows the prophenoloxidase activating system in plasma-CPB with the ability to be triggered by beta-1,3-glucan, it was concluded that binding of the purified beta-1,3-glucan recognition protein with beta-1,3-glucan causes the triggering of the prophenol-oxidase activating system in silkworm plasma. However, the nature of the activity that is generated as the result of binding is not yet known. The purified beta-1,3-glucan recognition protein bound to beta-1,3-glucan did not hydrolyze appreciably any of the 26 commercially available peptidyl-7-amino-4-methylcoumarins, substrates for various proteases.