10E4 antigen of Scrapie lesions contains an unusual nonsulfated heparan motif.
The carbohydrate antigen on heparan sulfate recognized by monoclonal antibody 10E4 is uniquely codistributed with the abnormal prion protein, PrP(Sc), even in the earliest detectable brain lesions of scrapie-infected mice. Determining 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. Toward this aim, we have examined preparations of heparan sulfate, with differing 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 fragments examined for 10E4 antigen expression by the neoglycolipid technology. An antigen-positive and two antigen-negative tetrasaccharides were isolated and examined by electrospray mass spectrometry. The antigen-positive tetrasaccharide sequence on heparan sulfate was thus deduced to contain a unique unsulfated motif that includes an N-unsubstituted glucosamine in the sequence, UA-GlcN-UA-GlcNAc. Antibody binding experiments with neoglycolipids prepared from a series of heparin/heparan sulfate disaccharides, and the trisaccharide 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 antigenicity.
Glycosciences Laboratory, Imperial College School of Medicine, Northwick Park Hospital, Watford Road, Harrow, Middlesex HA1 3UJ, United Kingdom.
C
C
Leteux
W
W
Chai
K
K
Nagai
C G
CG
Herbert
A M
AM
Lawson
T
T
Feizi
D000818:Animals; D000911:Antibodies, Monoclonal; D000941:Antigens / Q000737:chemistry / Q000276:immunology; D001666:Binding Sites, Antibody; D002240:Carbohydrate Sequence; D004187:Disaccharides / Q000737:chemistry; D006497:Heparan Sulfate / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry* / Q000276:immunology; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011133:Polysaccharide-Lyases / Q000378:metabolism; D018620:PrPSc Proteins / Q000737:chemistry* / Q000276:immunology*; D011994:Recombinant Proteins / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization
D000911:Antibodies, Monoclonal; D000941:Antigens; D004187:Disaccharides; D006497:Heparan Sulfate; D009844:Oligosaccharides; D010446:Peptide Fragments; D011133:Polysaccharide-Lyases; D018620:PrPSc Proteins; D011994:Recombinant Proteins; C021349:heparitinsulfate lyase