Aspartic acid 349 in the fourth epidermal growth factor-like structure of human thrombomodulin plays a role in its Ca(2+)-mediated binding to protein C.
The last three consecutive epidermal growth factor (EGF)-like structures of human thrombomodulin constitute the functional domain for protein C-activating cofactor activity and anticoagulant activity. Using site-directed deletion mutagenesis, we found that amino acid Asp349 of TME456, a recombinantly produced protein consisting of EGF-like structures 4, 5, and 6, is essential for retaining full protein C-activating cofactor activity. To investigate the role of Asp349 in the protein C-activating cofactor activity of human thrombomodulin, we have constructed two mutants of TMD123, a recombinantly produced protein consisting of domains D1, D2, and D3 of thrombomodulin, using site-directed point mutagenesis of the thrombomodulin coding sequence. In mutant TMD123A, the Asp349 codon was replaced with an Ala codon and in mutant TMD123E, the Asp349 codon was replaced with a Glu codon. The partially purified mutant proteins were assayed for their protein C-activating cofactor activity at various Ca2+ concentrations. TMD123 and TMD123E protein showed similar high levels of cofactor activity and similar patterns of Ca2+ dependence, while TMD123A had lower cofactor activity and did not show any Ca2+ dependence. We concluded that Asp349 in the fourth EGF-like structure of human thrombomodulin plays a role in its Ca(2+)-mediated binding to protein C.
Zushi
M
M
Bio-Science Laboratory, Asahi Chemical Industry, Japan.
Gomi
K
K
Honda
G
G
Kondo
S
S
Yamamoto
S
S
Hayashi
T
T
Suzuki
K
K
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000235:genetics*; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002384:Catalysis; D002460:Cell Line; D004247:DNA / Q000235:genetics; D004815:Epidermal Growth Factor / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011486:Protein C / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000378:metabolism; D018179:Receptors, Thrombin; D016415:Sequence Alignment; D013917:Thrombin / Q000378:metabolism*
D011486:Protein C; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D001224:Aspartic Acid; D004815:Epidermal Growth Factor; D004247:DNA; D013917:Thrombin; D002118:Calcium