beta-Hydroxyaspartic acid and beta-hydroxyasparagine residues in recombinant human protein S are not required for anticoagulant cofactor activity or for binding to C4b-binding protein.
Among the vitamin K-dependent plasma proteins, only protein S contains the post-translationally modified amino acid erythro-beta-hydroxyasparagine (Hyn). Protein S also contains erythro-beta-hydroxyaspartic acid (Hya). The function of these unusual amino acids, located in the epidermal growth factor-like domains, is unknown. To determine if these post-translational modifications contribute to the functional integrity of human protein S (HPS), recombinant human protein S lacking Hya and Hyn (rHPSdesHya/Hyn) was purified from the medium of human kidney 293 cells that were transfected with HPS cDNA and grown in the presence of the hydroxylase inhibitor 2,2'-dipyridyl. Solution-phase equilibrium binding studies revealed that rHPSdesHya/Hyn binds C4b-binding protein (C4BP) in a manner indistinguishable from recombinant HPS and plasma-derived HPS, exhibiting a Kd in the presence of 2 mM CaCl2 of approximately 0.7 nM and a Kd in the presence of 4 mM EDTA approximately 10-fold higher. In a purified component system, rHPSdesHya/Hyn displayed normal anticoagulant cofactor activity in the activated protein C-catalyzed inactivation of coagulation factor Va bound in the prothrombinase complex. In addition, digestion of rHPSdesHya/Hyn with thrombin in the presence of EDTA appeared normal, and 2 mM CaCl2 prevented the cleavage. Together these results suggest that the post-translational modifications of Asn and Asp residues are not necessary for the macromolecular or Ca2+ interactions associated with the anticoagulant and C4BP binding characteristics of HPS.
Nelson
R M
RM
Department of Biochemistry, University of Vermont College of Medicine, Burlington 05405-0068.
VanDusen
W J
WJ
Friedman
P A
PA
Long
G L
GL
D000925:Anticoagulants; D001216:Asparagine / Q000031:analogs & derivatives* / Q000737:chemistry; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D003169:Complement Inactivator Proteins; D004252:DNA Mutational Analysis; D015943:Factor Va / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D011485:Protein Binding; D017293:Protein S; D011994:Recombinant Proteins / Q000737:chemistry; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000378:metabolism
D000925:Anticoagulants; D002352:Carrier Proteins; D003169:Complement Inactivator Proteins; D006023:Glycoproteins; D017293:Protein S; D011994:Recombinant Proteins; C050931:3-hydroxyasparagine; C011274:3-hydroxyaspartic acid; D001224:Aspartic Acid; D015943:Factor Va; D001216:Asparagine; D013917:Thrombin; D002118:Calcium