Carbohydrate-dependent inhibition of protein-carbohydrate interactions

Abstract

Protein-carbohydrate interactions are essential in different physiological and pathologicalcell functions. Cell surface-expressed carbohydrates are important determinants of self andnon-self, as anti-carbohydrate antibodies may mediate rejection following transplantation oforgans between individuals or species. This has had major implications for the attempt tosolve the lack of donor organs by using organs from other species. Further, proteincarbohydrateinteractions mediate the colonization and infection of different hosts bydifferent microbial pathogens and some bacterial toxins. Consequently, the possibility ofinhibiting protein-carbohydrate interactions is of therapeutic interest in a variety ofbiomedical settings. However, protein-carbohydrate interactions are characterized by lowaffinities and although several different strategies have been explored, most attempts toinhibit physiological protein-carbohydrate interactions have had limited success. The reasonfor this is the enhanced affinities of protein-carbohydrate interactions obtained as a result oftheir multivalent binding points, i.e. multiple carbohydrate ligands on one biological entitybinding to multiple receptors on another.Mucins and mucin-type proteins are highly substituted with O-linked glycans. Thisenables a multivalent presentation of their oligosaccharides. Expression of the extracellularpart of the mucin-type protein P-selectin glycoprotein ligand-1 fused to the Fc part of mouseIgG2b (PSGL-1/mIgG2b) in different host cells together with glycosyltransferases known tomediate the biosynthesis of specific carbohydrate epitopes resulted in secretion of PSGL-1/mIgG2b carrying Galalpha1,3Gal, Lewis (Le)b or sialyl(s)-Lex epitopes as determined by massspectrometry and immunostaining (papers I and II). PSGL-1/mIgG2b expressing terminalGalalpha1,3Gal was shown to be an efficient adsorber of xenoreactive Galalpha1,3Gal-bindingantibodies (paper I). In paper III, PSGL-1/mIgG2b was used as a probe to investigate the Oglycanrepertoire in three cell lines commonly used for production of recombinant proteins.The results suggest that, in spite of the human origin of HEK-293 cells, this cell line may notprovide a human-like glycosylation because terminal hex-hex sequences and reactivity withanti-Gal antibodies were detected.Carbohydrate screening of animal milk glycoproteins using Western blotting andimmunostaining showed that complex carbohydrate epitopes such as Lex, sLex, Lea, sLea andLeb were expressed on human, pig and horse milk proteins (paper IV). The presence of theHelicobacter pylori binding carbohydrate epitopes Leb and sLex on pig milk proteins werefurther investigated in paper V. The expression of Leb and sLex were species- and individualspecific,and pig milk expressing these carbohydrate epitopes had an inhibitory effect on H.pylori binding in vitro and in vivo.In conclusion, we have developed a multivalent inhibitor of protein-carbohydrateinteractions. The glycosylation pattern of this mucin-type protein is dependent on the hostcell used for its production, and can be further modified by co-expression of differentglycosyltransferases. The efficient adsorption of xenoreactive antibodies shows the potentialof this mucin-type protein to interfere with physiological multivalent protein-carbohydrateinteractions. The in vivo result of the animal milk study further support the notion thatprotein-bound carbohydrate ligands are good inhibitors of physiological protein-carbohydrateinteractions; an effect most likely due to multivalent presentation of carbohydrate epitopes asdetermined by the protein backbone.