Title : Post-translational modifications of human
thrombin-activatable fibrinolysis inhibitor (
TAFI ): evidence for a large shift in the isoelectric point and reduced solubility upon activation
Abstract :
- Thrombin-activable fibrinolysis inhibitor ( TAFI ) is distinct from pancreatic procarboxypeptidase B in several ways
- The enzymatic activity of TAFIa is unstable and decays with a half-life of a few minutes
- During this study, we observed that (i) the isoelectric point (pI) of TAFI shifts dramatically from pH 5 toward pH 8 upon activation and (ii) TAFIa is significantly less soluble than TAFI
- The structural bases for these observations were investigated by characterizing all post-translational modifications, including attached glycans and disulfide connectivity
- The analyses revealed that all five potential N-glycosylation sites were utilized including Asn22, Asn51, Asn63, Asn86 (located in the activation peptide ), and Asn219 (located in the catalytic domain )
- Asn219 was also found in an unglycosylated variant
- Four of the glycans , Asn51, Asn63, Asn86, and Asn219 displayed microheterogeneity, while the glycan attached to Asn22 appeared to be homogeneous
- In addition, bisecting GlcNAc attached to the trimannose core was detected, suggesting an origin other than the liver
- Monosaccharide composition and LC-MS/MS analyses did not produce evidence for O glycosylation
- TAFI contains eight cysteine residues , of which two, Cys69 and Cys383 , are not involved in disulfides and contain free sulfhydryl groups
- The remaining six cystines form disulfides, including Cys156-Cys169, Cys228-Cys252, and Cys243-Cys257
- This pattern is homologous to pancreatic procarboxypeptidase B , and it is therefore unlikely that permutations in the cysteine connectivity are responsible for the enzymatic instability
- LC-MS/MS analyses covering more than 90% of the TAFI amino acid sequence revealed no additional modifications
- When these results are taken together, they suggest that the inherent instability of TAFIa is not caused by post-translational modifications
- However, after activation, TAFIa loses 80% of the attached glycans , generating a large shift in pI and a propensity to precipitate
- These changes are likely to significantly affect the properties of TAFIa as compared to TAFI