scholarly journals Amino Acid Residues in Thrombin-sensitive Region and First Epidermal Growth Factor Domain of Vitamin K-dependent Protein S Determining Specificity of the Activated Protein C Cofactor Function

1998 ◽  
Vol 273 (42) ◽  
pp. 27449-27458 ◽  
Author(s):  
Xuhua He ◽  
Lei Shen ◽  
Bruno O. Villoutreix ◽  
Björn Dahlbäck
Keyword(s):  
Amino Acid ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Protein C ◽  
Activated Protein C ◽  
Protein S ◽  
Amino Acid Residues ◽  
Epidermal Growth Factor Domain ◽  
Dependent Protein ◽  
Epidermal Growth

Functional Consequences of Mutations in Amino Acid Residues that Stabilize Calcium Binding to the First Epidermal Growth Factor Homology Domain of Human Protein C

1996 ◽  
Vol 76 (05) ◽  
pp. 720-728 ◽  
Author(s):  
Jie-Ping Geng ◽  
Chong-Hui Cheng ◽  
Francis J Castellino
Keyword(s):  
Amino Acid ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Binding Site ◽  
Calcium Binding ◽  
Protein C ◽  
Human Protein ◽  
Amino Acid Residues ◽  
Factor Va ◽  
Epidermal Growth

SummaryCharge-to-alanine mutations of three amino acid residues, viz., D46, D48, and D/Hya71, which are known to be important in stabilizing Ca2+ binding to epidermal growth factor (EGF) domains of vitamin K-de-pendent blood coagulation proteins, have been engineered into recombinant human protein C (r-PC), The resulting variants were then employed to assess the importance of this Ca2+ binding site in the activation properties of r-PC and in the activity of activated protein C (APC). Another mutation, of D48 to E, was constructed in order that a more conservative mutation at the Ca2+ binding site could be similarly examined.The mutant proteins were fully processed with regard to proper signal peptide cleavage, γ-carboxylation, and β-hydroxylation, except, of course, for the D71A mutant in this latter case. The D48E variant possessed an additional residue of γ-carboxyglutamic acid (Gla), showing that E48 was γ-carboxylated. All of the mutants were reactive against a monoclonal antibody (MAb) specific for a Ca2+-dependent epitope within the amino-terminus of the Gla domain of r-PC, demonstrating that a proper Ca2+-dependent conformation was adopted in this region of the protein. None of the mutants, except for [D48γ]r-PC, were reactive against another Ca2+-dependent MAb which possessed specificity for Ca2+ binding to the EGF1 region of PC-this being the area of the protein that contained the mutated residues. These data strongly suggest that the alanine mutations present at D46, D48, and D71 diminished Ca2+ binding to the EGF1 domain of r-PC.Steady state kinetic analysis demonstrated that determinants for the Ca2+-dependent inhibition of the thrombin (flla)-catalyzed activation of r-PC, and for the kinetic recognition of the flla/thrombomodulin complex, were not dependent on the integrity of the Ca2+ sites present in EGF1. The lone exception was [D48γ]r-PC, which did not undergo inhibition by Ca2+, an effect likely due to the potential for altered coordination of Ca2+ due to the Gla insertion, rather than to a dependency on D48. Plasma-based anticoagulant assays, as well as individual factor Va and factor Villa inactivation assays, showed that only [D71A]r-APC possessed a significantly reduced activity compared to wild-type r-APC. These observations suggest that D/Hya71 is likely an important determinant for activity of APC toward its physiological substrates, factor Va and factor Villa.

scholarly journals Conformational changes in activated protein C caused by binding of the first epidermal growth factor-like module of protein S

2000 ◽  
Vol 349 (3) ◽  
pp. 757-764 ◽  
Author(s):  
Tilman M. HACKENG ◽  
Subramanian YEGNESWARAN ◽  
Arthur E. JOHNSON ◽  
John H. GRIFFIN
Keyword(s):  
Energy Transfer ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Active Site ◽  
Protein C ◽  
Activated Protein C ◽  
Direct Interaction ◽  
Protein S ◽  
N Terminus ◽  
Epidermal Growth

The first epidermal growth factor-like module of human plasma protein S (EGF1, residues 76–116) was chemically synthesized and tested for its ability to inhibit the anticoagulant cofactor activity of protein S for the anticoagulant protease, activated protein C (APC). EGF1 completely inhibited the stimulation of APC activity by protein S in plasma coagulation assays, with 50% inhibition at approx. 1µM EGF1, suggesting direct binding of EGF1 to APC. To investigate a direct interaction between EGF1 and APC, fluorescence resonance energy transfer (FRET) experiments were employed. APC labelled in the active site with fluorescein as the donor, and phospholipid vesicles containing octadecylrhodamine as the acceptor, showed that EGF1 association with APC caused an increase in energy transfer consistent with a relocation of the active site of APC from 94Å (9.4nm) to 85Å above the phospholipid surface (assuming κ2 = 2/3). An identical increase in energy transfer between the APC active site-bound fluorescein and phospholipid-bound rhodamine was obtained upon association of protein S or protein S–C4b-binding protein complex with APC. The latter suggests the presence of a ternary complex of protein S–C4b-binding protein with APC on the phospholipid surface. To confirm a direct interaction of EGF1 with APC, rhodamine was covalently attached to the α-N-terminus of EGF1, and binding of the labelled EGF1 to APC was directly demonstrated using FRET. The data suggested a separation between the active site of APC and the N-terminus of EGF1 of 76Å (κ2 = 2/3), placing the APC-bound protein S-EGF1 close to, but above, the phospholipid surface and near the two EGF domains of APC. Thus we provide direct evidence for binding of protein S-EGF1 to APC and show that it induces a conformational change in APC.

Protein S Tokushima: abnormal molecule with a substitution of Glu for Lys-155 in the second epidermal growth factor-like domain of protein S

Blood ◽  
1994 ◽  
Vol 83 (3) ◽  
pp. 683-690 ◽  
Author(s):  
T Hayashi ◽  
J Nishioka ◽  
T Shigekiyo ◽  
S Saito ◽  
K Suzuki
Keyword(s):  
Molecular Weight ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Activated Protein C ◽  
Protein S ◽  
Gene Encoding ◽  
Calcium Dependent ◽  
Epidermal Growth ◽  
Cofactor Activity ◽  
Variant Protein

A 29-year-old female patient with heterozygous congenital protein S deficiency suffering from thrombotic disease had normal levels of both total and free protein S antigen (70% and 65%, respectively), but low cofactor activity (31%) for activated protein C, indicating that she had a variant of protein S, protein S Tokushima. Western blotting using the polyclonal anti-protein S antibody showed that approximately half of the patient's protein S appeared to be the variant with a higher molecular weight than normal protein S. The partially purified variant protein S bound neither to the monoclonal antibody recognizing calcium- dependent conformation of protein S nor to the antibody recognizing the thrombin-sensitive domain of protein S. Among the exons from II to XV of the patient's protein S gene encoding from the NH2-terminal end to the COOH-terminal end of protein S, only one missense mutation (A to G) was found in exon VI of the protein S alpha-gene, which results in amino acid substitution of Glu(GAG) for Lys-155(AAG) in the second epidermal growth factor-like domain of protein S. The recombinant protein S Tokushima expressed in BHK cells had a slightly higher molecular weight than the recombinant normal one, did not bind to the antibody specific for the thrombin-sensitive domain, and did not show the cofactor activity. These findings suggest that the protein S Tokushima molecule is structurally and functionally a variant of protein S, and that this variant protein S is the cause of severe thrombosis in this patient.

scholarly journals Calcium-Binding Properties of the Third and Fourth Epidermal-Growth-Factor-Like Modules in Vitamin-K-Dependent Protein S

1997 ◽  
Vol 248 (1) ◽  
pp. 163-170 ◽  
Author(s):  
Yvonne Stenberg ◽  
Karin Julenius ◽  
Ingrid Dahlqvist ◽  
Torbjorn Drakenberg ◽  
Johan Stenflo
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Vitamin K ◽  
Calcium Binding ◽  
Protein S ◽  
Binding Properties ◽  
The Third ◽  
Dependent Protein ◽  
Epidermal Growth

Biosynthesis of epidermal growth factor having nonprotein amino acid residues by Escherichia coli

Amino Acids ◽  
1990 ◽  
pp. 193-200
Author(s):  
Hiroshi Koide ◽  
Mitsuko Oishi ◽  
Takanori Oka ◽  
Tetsuo Miyake ◽  
Toru Fuwa ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Amino Acid Residues ◽  
Nonprotein Amino Acid ◽  
Epidermal Growth
Sign in / Sign up

Export Citation Format

Share Document