Identification of a high-affinity binding protein for N-acetylchitooligosaccharide elicitor in the plasma membrane of suspension-cultured rice cells by affinity labeling

Abstract

A high-affinity binding protein for the N-acetylchito-oligosaccharide elicitor of phytoalexin biosynthesis was identified by photoaffinity labeling and affinity cross-linking in the plasma membrane of suspension-cultured rice cells. Both a [125I]-labeled photolabile 2-(4-azidophenyl)ethylamino conjugate ([125I]-GN8-AzPEA) and a [125I]-labeled 2-(4-aminophenyl)ethylamino conjugate ([125]-GN8-APEA) of N-acetylchito-octaose were synthesized. The two conjugates were separately incubated with the plasma membrane prepared by aqueous two-phase partitioning, and covalently cross-linked to the elicitor binding site by irradiation with UV light or treatment with the cross-linking agent glutaraldehyde, respectively. Autoradiography of the SDS-PAGE gel of the solubilized membrane proteins revealed the labeling of a single 75 kDa band in both cases. The incorporation of the radiolabeled ligands into the 75 kDa protein showed a saturable mode of binding, with half-maximal incorporation at 45 and 52 nM for photoaffinity labeling and affinity cross-linking, respectively. The labeling of the 75 kDa protein was inhibited by N-acetylchito-oligosaccharides in a size-dependent manner, and N-acetylchito-octaose (GlcNAc)8 showed a half-maximal inhibition at concentrations of the order of 10 nM. However, neither chito-octaose (GlcN)8, cellopentaose nor alpha-1,4 linked N-acetylgalactosamine octamer (GalNAc)8 at concentrations as high as 25 microM inhibited the labeling of the 75 kDa protein. These results are in good agreement with the sensitivity and the specificity of the 'high-affinity binding site' previously identified by binding assays, as well as with the activities of these oligosaccharides in the induction of phytoalexin biosynthesis and other cellular responses. These results suggest that the 75 kDa protein identified by the affinity labeling represents a functional receptor for this elicitor.