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The normal PrP(C) (cellular prion protein) contains sLe(X) [sialyl-Le(X) (Lewis X)] and Le(X). sLe(X) is a ligand of selectins. To examine whether PrP(C) is a ligand of selectins, we generated three human PrP(C)-Ig fusion proteins: one with Le(X), one with sLe(X), and the other with neither Le(X) nor sLe(X). Only Le(X)-PrP(C)-Ig binds E-, L- and P-selectins. Binding is Ca(2+)-dependent and occurs with nanomolar affinity. Removal of sialic acid on sLe(X)-PrP(C)-Ig enables the fusion protein to bind all selectins. These findings were confirmed with brain-derived PrP(C). The selectins precipitated PrP(C) in human brain in a Ca(2+)-dependent manner. Treatment of brain homogenates with neuraminidase increased the amounts of PrP(C) precipitated. Therefore the presence of sialic acid prevents the binding of PrP(C) in human brain to selectins. Hence, human brain PrP(C) interacts with selectins in a manner that is distinct from interactions in peripheral tissues. Alternations in these interactions may have pathological consequences.

Mutation in the prion gene, PRNP, accounts for approx. 10-15% of human prion diseases. However, little is known about the mechanisms by which a mutant prion protein (PrP) causes disease. We compared the biochemical properties of a wild-type human prion protein, rPrP(C) (recombinant wild-type PrP), which has five octapeptide-repeats, with two recombinant human prion proteins with insertion mutations, one with three more octapeptide repeats, rPrP(8OR), and the other with five more octapeptide repeats, rPrP(10OR). We found that the insertion mutant proteins are more prone to aggregate, and the degree and kinetics of aggregation are proportional to the number of inserts. The octapeptide-repeat and alpha-helix 1 regions are important in aggregate formation, because aggregation is inhibited with monoclonal antibodies that are specific for epitopes in these regions. We also showed that a small amount of mutant protein could enhance the formation of mixed aggregates that are composed of mutant protein and wild-type rPrP(C). Accordingly, rPrP(10OR) is also more efficient in promoting the aggregation of rPrP(C) than rPrP(8OR). These findings provide a biochemical explanation for the clinical observations that the severity of the disease in patients with insertion mutations is proportional to the number of inserts, and thus have implications for the pathogenesis of inherited human prion disease.