Title : Determination of
carbohydrate structures N-linked to soluble
CD154 and characterization of the interactions of
CD40 with
CD154 expressed in Pichia pastoris and Chinese hamster ovary cells
Abstract :
- CD40- CD154 ( CD40 ligand) interactions are essential for the development of protective immunity
- Previous studies have described the CD40 binding site as a shallow groove formed between two monomers of CD154
- However, these studies have not examined the structure or biological function of the carbohydrate on CD154
- Human CD154 contains a single N-linked glycosylation site at asparagine 240
- We have characterized the interactions between CD40 and soluble (s) CD154 in which s CD154 contains different types of carbohydrates
- Detailed carbohydrate analysis revealed high-mannose structures on s CD154 purified from Pichia pastoris, whereas CD154 purified from Chinese hamster ovary E1A contained heterogeneous populations of complex carbohydrates
- s CD154 purified from either system was trimeric, it bound to CD40 with similar affinities of 10-30 nM, and it functionally induced CD69 and CD95 expression on primary B cells
- Together, these results indicate that the presence of varied types of N-linked glycans on asparagine 240 of CD154 does not play a significant role in the CD40- CD154 interactions