Biochemical importance of glycosylation in thrombin activatable fibrinolysis inhibitor.
Activated Thrombin Activatable Fibrinolysis Inhibitor (TAFIa) exerts an antifibrinolytic effect by removing C-terminal lysines from partially degraded fibrin. These lysines are essential for a rapid conversion of plasminogen to plasmin by tissue type plasminogen activator. TAFI is heavily glycosylated at Asn22, Asn51, Asn63, and Asn86. Although the glycans occurring at the glycosylation sites have previously been identified, the biochemical role of these glycans is not known yet. Therefore, we have determined the biochemical importance of the glycosylation in TAFI. Four single, 6 double, 4 triple, and 1 quadruple mutant, in which asparagine was replaced by glutamine, were constructed and transfected into HEK293T cells. Based on the determination of antigen and activity levels on conditioned medium, 4 single and 1 triple mutant were purified and their biochemical properties were determined. The glycosylation knockout mutants did neither reveal an altered fragmentation pattern nor differences in TAFIa stability, but TAFI-N51Q, TAFI-N63Q, and TAFI-N22Q-N51Q-N63Q revealed a decreased TAFIa activity, an increased intrinsic catalytic activity of the zymogen, and a decreased antifibrinolytic potential compared with TAFI-wild-type, whereas TAFI-N22Q and TAFI-N86Q revealed an increased antifibrinolytic potential probably because of an increased catalytic efficiency toward the physiological substrate. From these data it can be concluded that mainly the glycosylation at Asn86 contributes to the biochemical characteristics of TAFI. Furthermore we provide evidence that the activation peptide stays in close proximity to the TAFIa moiety after activation.
Buelens
Karlien
K
Laboratory for Pharmaceutical Biology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Campus Gasthuisberg, O&N2, PB 824, Herestraat 49, B-3000 Leuven, Belgium.
Hillmayer
Kerstin
K
Compernolle
Griet
G
Declerck
Paul J
PJ
Gils
Ann
A
D019943:Amino Acid Substitution; D000818:Animals; D000933:Antifibrinolytic Agents / Q000378:metabolism*; D025901:Carboxypeptidase B2 / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004789:Enzyme Activation / Q000235:genetics; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism*; D005337:Fibrin / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D020125:Mutation, Missense; D011499:Protein Processing, Post-Translational / Q000235:genetics; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D000933:Antifibrinolytic Agents; D004792:Enzyme Precursors; D011994:Recombinant Proteins; D005337:Fibrin; D025901:Carboxypeptidase B2