COVALENT ASSOCIATION OF LIPOPOLYSACCHARIDE AT THE HEMOCYTE SURFACE OFINSECTS IS AN INITIAL STEP FOR ITS INTERNALIZATION - PROTEIN-TYROSINEPHOSPHORYLATION REQUIREMENT

It is well known that lipopolysaccharide (LPS) of Gram-negative bacter ia triggers antibacterial responses to mammalian macrophages [Weinstei n, S., Gold, M. R. & DeFranco, A. (1991) Pi oc. Natl Acad. Sci. USA 88 , 4148-4152] and insect hemocytes [Charalambidis, N. D., Zervas, C. G. , Lambropoulou, M., Katsoris, P. G. & Marmaras, V. J. (1995) Eur: J. C ell Biol. 67, 32-41], via protein-tyrosine phosphorylation. In this st udy we show that insect hemocytes in response to LPS facilitate intern alization of LPS (either cell-associated or cell-free). According to o ur data, the recognition and covalent association of LPS (either cell- associated or cell-free) to the hemocyte surface are essential initial steps for LPS internalization. LPS (Escherichia coli) recognizes memb rane effector 47-kDa protein (p47) and then crosslinks to membrane-ass ociated p47 (mp47) via the intermediacy of tyrosine derivatives genera ted by the action of phenol oxidase, as is the case for cuticular prot ein-chitin crosslinks during sclerotization [Shaefer, J., Kramer, K. J ., Garbow, J. R., Jacob, C. S., Stejskal, E. O., Hopkins, T. L. & Spei rs, R. D. (1987) Science 235, 1200-1204]. The covalent association of LPS to the hemocyte surface appears to be a prerequisite for LPS inter nalization as judged by the resistance of LPS binding to dissociation by proteinase K. In addition, our results show that the effector molec ules participating in LPS covalent association at the cell surface and LPS internalization are not involved in LPS-induced activation of hem ocytes. However, the fact that genistein, as well as the inhibitors of LPS-dependent secretion, block LPS covalent association at the cell s urface and LPS internalization provides a preliminary characterization of an LPS signal transduction-dependent process which is apparently i nvolved.