PMID: 16445295

 

    Legend: Sugar

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 :
  1. Thrombin-activable fibrinolysis inhibitor ( TAFI ) is distinct from pancreatic procarboxypeptidase B in several ways
  2. The enzymatic activity of TAFIa is unstable and decays with a half-life of a few minutes
  3. 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
  4. The structural bases for these observations were investigated by characterizing all post-translational modifications, including attached glycans and disulfide connectivity
  5. 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 )
  6. Asn219 was also found in an unglycosylated variant
  7. Four of the glycans , Asn51, Asn63, Asn86, and Asn219 displayed microheterogeneity, while the glycan attached to Asn22 appeared to be homogeneous
  8. In addition, bisecting GlcNAc attached to the trimannose core was detected, suggesting an origin other than the liver
  9. Monosaccharide composition and LC-MS/MS analyses did not produce evidence for O glycosylation
  10. TAFI contains eight cysteine residues , of which two, Cys69 and Cys383 , are not involved in disulfides and contain free sulfhydryl groups
  11. The remaining six cystines form disulfides, including Cys156-Cys169, Cys228-Cys252, and Cys243-Cys257
  12. 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
  13. LC-MS/MS analyses covering more than 90% of the TAFI amino acid sequence revealed no additional modifications
  14. When these results are taken together, they suggest that the inherent instability of TAFIa is not caused by post-translational modifications
  15. However, after activation, TAFIa loses 80% of the attached glycans , generating a large shift in pI and a propensity to precipitate
  16. These changes are likely to significantly affect the properties of TAFIa as compared to TAFI