Structure-function studies of plasminogen activator inhibitor-1 (PAI-1) have previously been performed mostly with non-glycosylated material expressed in E. coli. We have now studied the importance of PAI-1 glycosylation for its functional properties. PAI-1 has 3 potential sites for N-linked glycosylation. Biochemical analysis of PAI-1 variants with substitutions of the Asn residues in each of these sites and expression in human embryonic kidney 293 (HEK293) cells showed that only Asn211 and Asn 267, but not Asn331 are glycosylated, and revealed a differential composition of the carbohydrate attached at the 2 sites. Analysing the susceptibility of glycosylated and non-glycosylated PAI-1 to activity neutralisation by monoclonal antibodies, we found that the IC50-values for neutralisation by some monoclonal antibodies differed strongly between glycosylated and non-glycosylated PAI-1. The most susceptible PAI-1 variant was not necessarily the one used when raising the antibody. This and other observations indicated that the carbohydrate moieties or the glycosylation sites are unlikely to be part of the epitopes for these antibodies. The antibody susceptibility characteristic for non-glycosylated PAI-1 could be conferred upon PAI-1 expressed in HEK293 cells by mutational inactivation of one or the other glycosylation site. These findings reveal a novel functional role for glycosylation of a serpin. The glycosylation sites are localised between a-helix H and b-strand 2C and b-strand 3C and a-helix F, respectively. We hypothesise that the carbohydrate moieties affect antibody neutralisation by affecting the movements of b-sheet C and thereby those of the reactive centre loop. Our results indicate that non-glycosylated PAI-1 may not be suited for studying the mechanism of action of PAI-1 neutralising compounds aimed for use with mammalian cells.
|Publication status||Published - 2001|
|Event||8th International Workshop on Molecular and Cellular Biology of Plasminogen Activation - Wyoming, United States|
Duration: 9 Jul 2001 → 13 Jul 2001
Conference number: 8
|Workshop||8th International Workshop on Molecular and Cellular Biology of Plasminogen Activation|
|Period||09/07/2001 → 13/07/2001|