beta-Hydroxyaspartic acid in vitamin K-dependent protein C.

Drakenberg, Torbjörn; Fernlund, Per; Roepstorff, Peter; Stenflo, Johan

doi:10.1073/pnas.80.7.1802

Cited by 137 publications

(72 citation statements)

References 31 publications

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“…3-Hydroxyaspartate (or the related (3-hydroxyasparagine) has been characterized as a post-translationally modified residue in a number of plasma proteins, including factor VII, factor IX, factor X, protein C, protein S and protein Z, and complement Clr (Drakenberg et al, 1983;McMullen et al, 1983a,b;Fernlund and Stenflo, 1983;Sugo et al, 1984a;H0jrup et al, 1985;Hagen et al, 1986;Stenflo et al, 1987;Arlaud et al, 1987). In each case the (-hydroxyaspartate is found in a domain homologous to epidermal growth factor (EGF).…”

Section: Introductionmentioning

confidence: 99%

The role of beta-hydroxyaspartate and adjacent carboxylate residues in the first EGF domain of human factor IX.

et al. 1988

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We describe a system for the expression of human coagulation factor IX in dog kidney cells in tissue culture, in which the post-translational modifications and the biochemical activity are indistinguishable from factor IX synthesized in vivo. This system has been used to express eight different point mutations of human factor IX in the first epidermal growth factor domain in order to study the role of F3-hydroxyaspartate at residue 64, and the adjacent carboxylate residues at positions 47, 49 and 78. We conclude that this domain is essential for factor IX function and suggest that Ca2+ binds to carboxylate ions in this domain and stabilizes a conformation necessary for the interaction of factor IXa with factor X, factor VIII and phospholipid in the next step of the clotting cascade.

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Section: Introductionmentioning

confidence: 99%

The role of beta-hydroxyaspartate and adjacent carboxylate residues in the first EGF domain of human factor IX.

et al. 1988

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“…An unusual amino acid, erythro-3-hydroxyaspartic acid, was recently found in the NH2-terminal EGF homologous domain in protein C (27) and in factors VII, IX, and X (18,19,(27)(28)(29). It is formed by postribosomal hydroxylation of aspartic acid.…”

mentioning

confidence: 99%

beta-Hydroxyasparagine in domains homologous to the epidermal growth factor precursor in vitamin K-dependent protein S.

Stenflo

Lundwall

Dahlbäck

1987

Proc. Natl. Acad. Sci. U.S.A.

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158

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Vitamin K-dependent protein S is involved in the regulation of blood coagulation. It is a 75-kDa single chain protein with an NH2-terminal y-carboxyglutamic acid-containing domain followed by a thrombin-sensitive region and four domains arranged in tandem, each of which is homologous to the epidermal growth factor (EGF) precursor. The NH2-terminal EGF-like domain contains (3-hydroxyaspartic acid, which has been identified in vitamin K-dependent proteins. The following EGF-like repeat has a very pronounced sequence homology (10 consecutive residues identical) to one of the EGF-like units in the EGF precursor. We now show that, in protein S, this EGF-like repeat has one P-hydroxyasparagine residue formed by hydroxylation of asparagine. The two COOH-terminal EGF-like repeats also contain P-hydroxyasparagine, an amino acid not previously found in proteins. Sequence comparisons have enabled us to identify a consensus sequence that seems to be required by the hydroxylase(s).Protein S is an anticoagulant vitamin K-dependent plasma protein that functions as a cofactor to activated protein C in the degradation of coagulation factors . Unlike the other vitamin K-dependent plasma proteins, it is not homologous to the seine proteases (8,9). The NH2-terminal vitamin K-dependent region in protein S is followed by a small region with two thrombin-sensitive bonds and four consecutive regions that are homologous to the epidermal growth factor (EGF) (8,9). EGF is a 53-amino acid peptide with three intramolecular disulfide bonds (10, 11). It is formed by proteolytic cleavage of the COOH-terminal part of a large precursor that in its NH2-terminal part contains nine EGF-like units (12, 13). Such units have also been found in other proteins, notably all ofthe vitamin K-dependent plasma proteins except prothrombin (8,9,(14)(15)(16)(17)(18)(19) EXPERIMENTAL PROCEDURES Peptide Isolation. Bovine protein S was purified (3), completely reduced and carboxymethylated with iodo[2-'4C]acetic acid, and then citraconylated in 6 M guanidine hydrochloride (15). After digestion with trypsin (1%, wt/wt) for 1 hr at 37°C and inactivation of the enzyme with diisopropylphosphofluoridate, the material was chromatographed on a column (1.6 x 89 cm) packed with Sephacryl S-200 and equilibrated with 50 mM Tris HCl/0.1 M NaCl, pH 7.5, containing 6 M guanidine hydrochloride. The first major peak from the column (Kav = 0.43) contained a fragment previously characterized (residues 71-275; ref. 8), which was homogeneous on NaDodSO4/polyacrylamide gel electrophoresis. The fragment (4 mg) was treated with 10% formic acid to remove the lysine blocking groups, lyophilized, and then digested with the lysine-specific endopeptidase (Wako Chemicals, Neuss, F.R.G.; 3% enzyme, wt/wt) in 0.1 M NH4HCO3 for 3 hr at 37°C. The dried material was dissolved in 0.1% trifluoroacetic acid and chromatographed on an Aquapore RP 300 column (Brownlee Lab, Santa Clara, CA) equilibrated with 0.1% trifluoroacetic acid. A gradient, 0-60% (vol/vol) acetonitrile in 0.1% trifluoroacet...

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“…This sequence is nearly identical to that in bovine factor VII in which the aspartic acid is present as ,&hydroxyaspartic acid (3). Furthermore, P-hydroxyaspartic acid is present in essentially the same position in factor IX, factor X, and protein C (3,42). Thus, it is extremely likely that human factor VII also contains /3-hydroxyaspartic acid at residue 63 in the first potential growth factor domain.…”

Section: Results and Discussio]mentioning

confidence: 92%

Characterization of a cDNA coding for human factor VII.

Hagen¹,

Gray²,

O׳Hara³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

404

211

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Factor VII is a precursor to a serine protease that is present in mammalian plasma. In its activated form, it participates in blood coagulation by activating factor X and/or factor IX in the presence of tissue factor and calcium. Clones coding for factor VII were obtained from two cDNA libraries prepared from poly(A) RNA from human liver and Hep G2 cells. The amino acid sequence deduced from the cDNAs indicates that factor VII is synthesized with a prepro-leader sequence of 60 or 38 amino acids. The mature protein that circulates in plasma is a single-chain polypeptide composed of 406 amino acids. The amino acid sequence analysis of the protein and the amino acid sequence deduced from the cDNAs indicate that factor VII is converted to factor VII8 by the cleavage of a single internal bond between arginine and isoleucine. This results in the formation of a light chain (152 amino acids) and a heavy chain (254 amino acids) that are held together by a disulfide bond. The light chain contains a -carboxyglutamic acid (Gla) domain and two potential epidermal growth factor domains, while the heavy chain contains the serine protease portion of the molecule. Factor VII shows a high degree of amino acid sequence homology with the other vitamin K-dependent plasma proteins.

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beta-Hydroxyaspartic acid in vitamin K-dependent protein C.

Cited by 137 publications

References 31 publications

The role of beta-hydroxyaspartate and adjacent carboxylate residues in the first EGF domain of human factor IX.

The role of beta-hydroxyaspartate and adjacent carboxylate residues in the first EGF domain of human factor IX.

beta-Hydroxyasparagine in domains homologous to the epidermal growth factor precursor in vitamin K-dependent protein S.

Characterization of a cDNA coding for human factor VII.

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