Adhesion forces in the self-recognition of oligosaccharide epitopes of the proteoglycan aggregation factor of the marine sponge Microciona prolifera

Abstract

Cell aggregation in the marine sponge Microciona prolifera is mediated by a multimillion molecular-mass aggregation factor, termed MAF. Earlier investigations revealed that the cell aggregation activity of MAF depends on two functional domains: (i) a Ca(2+)-independent cell-binding domain and (ii) a Ca(2+)-dependent proteoglycan self-interaction domain. Structural analysis of involved carbohydrate fragments of the proteoglycan in the self-association established a sulfated disaccharide beta-D: -GlcpNAc3S-(1-->3)-alpha-L: -Fucp and a pyruvated trisaccharide beta-D: -Galp4,6(R)Pyr-(1-->4)-beta-D: -GlcpNAc-(1-->3)-alpha-L: -Fucp. Recent UV, SPR, and TEM studies, using BSA conjugates and gold nanoparticles of the synthetic sulfated disaccharide, clearly demonstrated self-recognition on the disaccharide level in the presence of Ca(2+)-ions. To determine binding forces of the carbohydrate-carbohydrate interactions for both synthetic MAF oligosaccharides, atomic force microscopy (AFM) studies were carried out. It turned out that, in the presence of Ca(2+)-ions, the force required to separate the tip and sample coated with a self-assembling monolayer of thiol-spacer-containing beta-D: -GlcpNAc-(1-->3)-alpha-L: -Fucp-(1-->O)(CH(2))(3)S(CH(2))(6)S- was found to be quantized in integer multiples of 30 +/- 6 pN. No binding was observed between the two monolayers in the absence of Ca(2+)-ions. Cd(2+)-ions could partially induce the self-interaction. In contrast, similar AFM experiments with thiol-spacer-containing beta-D: -Galp4,6(R)Pyr-(1-->4)-beta-D: -GlcpNAc-(1-->3)-alpha-L: -Fucp-(1-->O)(CH(2))(3)S(CH(2))(6)S- did not show a binding in the presence of Ca(2+)-ions. Also TEM experiments of gold nanoparticles coated with the pyruvated trisaccharide could not make visible aggregation in the presence of Ca(2+)-ions. It is suggested that the self-interaction between the sulfated disaccharide fragments is stronger than that between the pyruvated trisaccharide.