IDENTIFICATION OF A HEMOCYTE MEMBRANE-PROTEIN OF THE SILKWORM, BOMBYX-MORI, WHICH SPECIFICALLY BINDS TO BACTERIAL LIPOPOLYSACCHARIDE

An in vitro system with isolated hemocytes of the silkworm, Bombyx mor i (B, mori) was developed to examine the induction mechanism of insect antibacterial proteins by bacterial lipopolysaccharide (LPS). The gen e expression of B, mori cecropin B, a representative antibacterial pro tein, was triggered by LPS in this in vitro system, To identify LPS-bi nding site(s) of the hemocytes, the [I-125]LPS binding assay to the he mocytes was performed in vitro. The amount of [I-125]LPS bound to hemo cytes increased proportionately with the increase of incubation time a nd LPS dose, The binding was strongly inhibited by excess unlabeled LP S or lipid A, indicating that the binding of [I-125]LPS to hemocytes c ontains a highly specific reaction, Moreover, the specific binding cou ld not be detected with Bm-N4 cells in which cecropin B gene expressio n was not induced by LPS, suggesting that the LPS binding is specific for LPS responsive cells, The LPS binding was fully sensitive to the p roteinase 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 demonst rated that this compound mainly binds to granular cells rather than ot her hemocytes under our assay conditions, Affinity-labeling with photo reactive-LPS allowed the identification of a 11 kDa LPS-binding protei n in hemocytes, which might relate to the specific membrane receptor f or LPS.