Effect of citrullination on the function and conformation of antithrombin.
Antithrombin is an anticoagulant serpin with conformational sensitivity. Mutations and environmental factors may induce its polymerization by a mechanism involving domain swapping, which still requires verification. We have evaluated the functional and conformational effects on antithrombin of citrullination, a post-translational modification catalyzed by peptidylarginine deiminase (PAD), which changes arginine to citrulline. Purified antithrombin (native and latent forms) and plasma were incubated with PAD in the presence and absence of heparin. Citrullines were identified by proteomic analysis. Anti-activated factor X activity determination, IEF, SDS/PAGE and native PAGE were performed. The cleavage pattern with the metalloprotease AspN was studied, and its target residues were identified by Edman sequencing. We confirmed that citrullination of antithrombin abolished its activity; this abolition of activity was accelerated by heparin, which facilitated the early citrullination of Arg393 (P1 residue). Proteomic analyses revealed nine additional citrullines that caused a significant decrease in its electrostatic potential (from 5.95 to 5.06). It was demonstrated that citrullination of antithrombin caused its polymerization. The observation that these polymers, like heat-generated polymers, are cleaved by AspN in helix I is compatible with their linkage by domain swapping from strand 5 to strand 4 of beta-sheet A.
Centro Regional de Hemodonación, University of Murcia, Murcia, Spain.
A
A
Ordóñez
I
I
Martínez-Martínez
F J
FJ
Corrales
C
C
Miqueo
A
A
Miñano
V
V
Vicente
J
J
Corral
D000991:Antithrombins / Q000737:chemistry* / Q000378:metabolism*; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D002956:Citrulline / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006867:Hydrolases / Q000378:metabolism; D008958:Models, Molecular; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D000076342:Protein-Arginine Deiminases; D013329:Structure-Activity Relationship
D000991:Antithrombins; D002956:Citrulline; D001120:Arginine; D006867:Hydrolases; D000076342:Protein-Arginine Deiminases