10E4 antigen of scrapie lesions contains an unusual nonsulfated heparan motif

The carbohydrate antigen on heparan sulfate recognized by monoclonal antibo dy 10E4 is uniquely codistributed with the abnormal prion protein, PrPSc, e ven in the earliest detectable brain lesions of scrapie-infected mice. Dete rmining the chemical structure of 10E4 antigen is, therefore, an important aspect of structure elucidation of scrapie lesions, and a prerequisite for designing experiments to understand its role in scrapie pathogenesis, Towar d this aim, we have examined preparations of heparan sulfate, with differin g sulfate contents, for binding by 10E4 antibody. The highest antigenicity was observed in a preparation (HS-1) with the lowest sulfate content. HS-1 was partially depolymerized with heparin lyase III, and oligosaccharide fra gments examined for 10E4 antigen expression by the neoglycolipid technology . An antigen-positive and two antigen-negative tetrasaccharides were isolat ed and examined by electrospray mass spectrometry, The antigen-positive tet rasaccharide sequence on heparan sulfate was thus deduced to contain a uniq ue unsulfated motif that includes an N-unsubstituted glucosamine in the seq uence, UA-GlcN-UA-GlcNAc. Antibody binding experiments with neoglycolipids prepared from a series of heparin/heparan sulfate disaccharides, and the tr isaccharide derived from the antigen-positive tetrasaccharide after removal of the terminal hexuronic acid, show that both the penultimate glucosamine and the outer nonsulfated hexuronic acid are important for 10E4 antigenici ty.