Covalent Association of Lipopolysaccharide at the Hemocyte Surface of Insects is an Initial Step for its Internalization

Abstract

It is well known that lipopolysaccharide (LPS) of Gram-negative bacteria triggers antibacterial responses to mammalian macrophages [Weinstein, S., Gold, M. R. & DeFranco, A. (1991) Proc. 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. Cell Biol. 67, 32-41], via protein-tyrosine phosphorylation. In this study we show that insect hemocytes in response to LPS facilitate internalization of LPS (either cell-associated or cell-free). According to our 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 membrane effector 47-kDa protein (p47) and then crosslinks to membrane-associated p47 (mp47) via the intermediacy of tyrosine derivatives generated by the action of phenol oxidase, as is the case for cuticular protein-chitin crosslinks during sclerotization [Shaefer, J., Kramer, K.J., Garbow, J.R., Jacob, G.S., Stejskal, E.O., Hopkins, T.L. & Speirs, R.D. (1987) Science 235, 1200-1204]. The covalent association of LPS to the hemocyte surface appears to be a prerequisite for LPS internalization as judged by the resistance of LPS binding to dissociation by proteinase K. In addition, our results show that the effector molecules participating in LPS covalent association at the cell surface and LPS internalization are not involved in LPS-induced activation of hemocytes. However, the fact that genistein, as well as the inhibitors of LPS-dependent secretion, block LPS covalent association at the cell surface and LPS internalization provides a preliminary characterization of an LPS signal-transduction-dependent process which is apparently involved.

Phagocytosis of Escherichia coli by Insect Hemocytes Requires Both Activation of the Ras/Mitogen-activated Protein Kinase Signal Transduction Pathway for Attachment and β3 Integrin for Internalization

“…Hemocytes Possess Putative Tyrosine-phosphorylated Proteins-Insect hemocytes require protein tyrosine phosphorylation in order to attach and internalize cell-free or cell-associated LPS (3)(4)(5).…”

“…Anti-p47 and anti-phenol oxidase are rabbit polyclonal antisera specific for C. capitata p47 and phenol oxidase, respectively, raised at our laboratory, as described previously (5,15). Rabbit polyclonal antiserum against the Drosophila Drk protein, was kindly provided by Dr. T. Pawson (University of Toronto).…”

“…and the assays of LPS-Y binding and internalization into the hemocytes were performed as described previously (5). For inhibition experiments, hemocyte monolayers were first incubated in 100 l/well of 1 g/ml anti-␤ 3 , or 2 mM RGDS peptide (Sigma), or 10 M cytochalasin D (Sigma) diluted in Grace's insect medium for 30 min at 25°C before the addition of the labeled LPS-Y complex.…”

“…Upon LPS triggering, a protein, p47, is released from hemocytes, in vitro, which together with tyrosine and phenol oxidase cause aggregation of E. coli cells (3,4). The aggregation is based on the cross-linking of LPS to p47 via the intermediacy of tyrosine derivatives generated by the action of phenol oxidase (5). This protein is also weakly associated with the hemocyte surface (4) and covalently binds to LPS with the same mechanism.…”

“…This protein is also weakly associated with the hemocyte surface (4) and covalently binds to LPS with the same mechanism. The LPS⅐catechol⅐p47 complex is then internalized into hemocytes, as judged by proteinase K treatment (5). The key regulatory component for the complex formation is pro (phenol oxidase)-activators (serine proteases), which are released by hemocytes upon LPS triggering and activate the hemocyte surface phenol oxidase (6).…”