Alanine-scanning mutagenesis of the epidermal growth factor-like domains of human thrombomodulin identifies critical residues for its cofactor activity.
Thrombomodulin (TM) is an endothelial cell surface-bound cofactor in thrombin-dependent formation of activated protein C, a potent anticoagulant. Cofactor activity has been localized to the carboxyl-terminal half of the six epidermal growth factor-like (EGF) domains of TM (TME). To identify residues in TME that are critical for activity, 77 alanine point mutants were made between Cys-333 and Cys-462 by site-directed mutagenesis (all residues except Ala, Cys, Gly, and Pro). Mutants were expressed in Escherichia coli and cofactor activity measured directly in periplasmic extracts obtained by osmotic shock. Critical residues were defined as those which when mutated had less than 25% cofactor activity of a reference TME. Western blots of non-reduced samples confirmed that alanine substitutions did not significantly decrease expression levels or result in the formation of multimers. In EGF4, which is essential for protein C activation by the thrombin-TM complex, critical residues were: Asp-349, Glu-357, Tyr-358, and Phe-376. In EGF5-EGF6, critical residues within a proposed acidic thrombin-binding region were: Glu-408, Tyr-413, Ile-414, Leu-415, Asp-416, Asp-417, Asp-423, Ile-424, Asp-425, and Glu-426. A potential Ca(2+)-binding site, which is comprised of residues Asp-423, Asp-425, Glu-426, Asn-439, Leu-440, and Phe-444, was also identified and overlaps the thrombin-binding region. Asp-461, in the C-loop of EGF6 previously shown to be critical for thrombin binding, was also critical. Asp-398, Asp-400, Asn-402, and Asn-429 in EGF5 were also critical. Thus, rapid alanine-scanning mutagenesis of TME has identified 22 critical residues in the region comprising EGF4-6, which is essential for thrombin binding and protein C activation by the thrombin-TM complex.
Nagashima
M
M
Berlex Biosciences Inc., Richmond, California 94804-0099.
Lundh
E
E
Leonard
J C
JC
Morser
J
J
Parkinson
J F
JF
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011485:Protein Binding; D011486:Protein C / Q000378:metabolism; D017433:Protein Structure, Secondary; D011956:Receptors, Cell Surface / Q000737:chemistry*; D018179:Receptors, Thrombin; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000378:metabolism
D009838:Oligodeoxyribonucleotides; D011486:Protein C; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D013917:Thrombin; D000409:Alanine