Figure 7. Selectin–Ig proteins do not react with brain-derived PrP^C lacking N-linked glycans.

(A) Brain homogenate was treated with PBS or with PNGase F and then subjected to precipitation with either selectin–Ig or NCAM–Ig fusion proteins. The precipitated proteins were then separated by SDS/PAGE and immunoblotted with anti-PrP^C mAb, 8H4. Treatment with PNGase F greatly reduced the amounts of PrP^C precipitated by the three selectin–Ig fusion proteins. Therefore N-linked glycans on PrP^C are critical for binding of selectin–Ig to PrP^C. (B) Human brain homogenates were treated with PBS or neuraminidase. After treatment, proteins were separated by SDS/PAGE and then immunoblotted with anti-PrP^C mAb, 8H4. Neuraminidase treatment reduced the apparent molecular mass of PrP^C as compared with the non-treated PrP^C proteins. (C) ELISA plates were precoated with anti-PrP^C mAb, 8H4. Equal amounts of PBS-treated control homogenates or neuraminidase-treated brain homogenates were added onto the ELISA plate. An anti-CD15 mAb or anti-CD15s mAb were then added to react with the bound PrP^C species. The bound anti-CD15 or anti-CD15s antibodies were detected with a goat anti-mouse IgM–HRP. Anti-CD15 mAb reacted more strongly with neuraminidase-treated brain homogenate. On the other hand, binding of anti-CD15s antibody was reduced in neuraminidase-treated brain homogenates. Results are the means±S.D. for two experiments (n=12 for each treatment). P-values represent comparisons between samples treated with or without neuraminidase. (D) PBS-treated or neuraminidase-treated brain homogenates were precipitated with E-selectin–Ig or NCAM–Ig. Precipitated proteins were then separated by SDS/PAGE and immunoblotted with anti-PrP^C mAb, 8H4. E-selectin–Ig precipitated more PrP^C protein from neuraminidase-treated brain homogenate than PBS-treated brain homogenate. The neuraminidase-treated, E-selectin–Ig-precipitated PrP^C proteins also migrated slightly faster than the PBS-treated controls. I. P., immunoprecipitated.