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J Stenflo

Publications and source records attributed to J Stenflo.

At least 91 records · Page 5Linked to original sources
PubMed1987

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

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 gamma-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 beta-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 beta-hydroxyasparagine residue formed by hydroxylation of asparagine. The two COOH-terminal EGF-like repeats also contain beta-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).

Amino Acid Sequence↗
PubMed1986

Localization of thrombin cleavage sites in the amino-terminal region of bovine protein S.

Protein S is a vitamin K-dependent plasma protein. It functions as a cofactor to activated protein C in the inactivation of factors Va and VIIIa by limited proteolysis. Protein S is very sensitive to proteolysis by thrombin which reduces its calcium ion binding and leads to a loss of its cofactor activity. We have now determined the sequence of the 100 amino-terminal amino acid residues and localized the thrombin cleavage sites. Protein S contains 11 gamma-carboxyglutamic acid residues in the amino-terminal region (residues 1-36). This part of protein S is highly homologous to the corresponding parts in the other vitamin K-dependent clotting factors, whereas the region between residues 45 and 75 is not at all homologous to the other clotting factors. Thrombin cleaves two peptide bonds in this part of protein S, first at arginine 70 and then at arginine 52. The peptide containing residues 53-70 is released from protein S after thrombin cleavage. The amino-terminal fragment, residues 1-52, is linked to the large carboxyl-terminal fragment by a disulfide bond, which involves cysteine 47. After residue 78, protein S is again homologous to factors IX and X and to proteins C and Z, but not to prothrombin. Position 95 is occupied by a beta-hydroxyaspartic acid residue.

Amino Acid Sequence↗
PubMed1986

Calcium binding of bovine protein S. Effect of thrombin cleavage and removal of the gamma-carboxyglutamic acid-containing region.

Thrombin cleaves protein S at arginine residues 52 and 70 resulting in loss of cofactor activity and reduced Ca2+ ion binding. After thrombin cleavage the NH2-terminal region containing gamma-carboxyglutamic acid (Gla) is linked to the large COOH-terminal fragment by a disulfide bond. Measurements of the rate of disulfide bond reduction by thioredoxin in intact protein S showed that the disulfide bonds are largely inaccessible to thioredoxin in the presence of Ca2+ ions, whereas in the presence of EDTA apparently all of the disulfide bonds are rapidly reduced. Probing the reactivity of the disulfide bonds in thrombin-modified proteins indicated that the thrombin cleavage induces a conformational change in the protein. After thrombin cleavage of protein S, the domain containing gamma-carboxyglutamic acid could be removed by selective reduction with thioredoxin followed by alkylation of the sulfhydryl groups. Ca2+ ion binding was compared in intact protein S, thrombin-modified protein S, and Gla domainless protein S. The intact protein S bound several Ca2+ ions, and the binding was not saturable. Thrombin-modified protein S, whether intact or with the Gla domain removed by selective reduction, bound two to three Ca2+ ions with a KD of 15-20 microM. The Gla domain in thrombin-modified protein S thus does not contribute significantly to the high affinity Ca2+ ion binding. Thrombin cleavage of protein S may be of physiological importance in the regulation of blood coagulation.

1-Carboxyglutamic Acid↗
PubMed1986

Primary structure of bovine vitamin K-dependent protein S.

Protein S is a vitamin K-dependent plasma protein that functions as a cofactor to activated protein C in the inactivation of coagulation factors Va and VIIIa. The nucleotide sequence of a full-length cDNA clone, obtained from a bovine liver library, was determined and the amino acid sequence was deduced. In addition, 95% of the structure was determined by protein sequencing. Protein S consists of 634 amino acids in a single polypeptide chain and has one asparagine-linked carbohydrate side chain. The cDNA sequence showed that the protein has a leader sequence, 41 amino acid residues long. The amino-terminal part of the molecule containing gamma-carboxyglutamic acid is followed by a region, residues 42-75, with two peptide bonds that are very sensitive to cleavage by thrombin. Residues 76-244 have four cysteinerich repeat sequences, each about 40 residues long, that are homologous to the precursor of mouse epidermal growth factor. In contrast to the other vitamin K-dependent plasma proteins, the carboxyl-terminal part of protein S is not homologous to the serine proteases.

Amino Acid Sequence↗
PubMed1986

Isolation and sequence of the cDNA for human protein S, a regulator of blood coagulation.

Protein S is a cofactor of activated protein C; together they function as a regulator of blood coagulation. A human liver cDNA library constructed in bacteriophage lambda gt11 was screened with DNA fragments from a full-length bovine cDNA clone encoding protein S. Several cDNA clones were isolated and sequenced. The combined cDNA sequences encoded the mature protein and 15 residues of the leader sequence when compared to bovine protein S. Human protein S is a single-chain protein consisting of 635 amino acids with 82% homology to bovine protein S. After an NH2-terminal gamma-carboxyglutamic acid-containing region, there is a short region with thrombin-sensitive bond(s), followed by a region with four repeat sequences that are homologous to the precursor of mouse epidermal growth factor. In contrast to the other vitamin K-dependent plasma proteins, the COOH-terminal portion of human protein S does not show any resemblance to serine proteases.

Amino Acid Sequence↗
PubMed1985

Characterization of monoclonal antibodies against human protein C specific for the calcium ion-induced conformation or for the activation peptide region.

Three monoclonal antibodies have been produced that are specific for the activation peptide region in human protein C. These antibodies inhibited the activation of protein C by thrombin and by the thrombin-thrombomodulin complex. A fourth monoclonal antibody specifically recognized the Ca2+-stabilized conformation in protein C. This antibody bound both intact protein C and protein C from which the gamma-carboxyglutamic acid-containing region had been removed by limited proteolysis. These results indicate that this antibody recognizes the conformation in protein C stabilized by Ca2+ bound to the single binding site that is independent of gamma-carboxyglutamic acid.

Animals↗
PubMed1985

Protein C in bovine plasma after warfarin treatment. Purification, partial characterization, and beta-hydroxyaspartic acid content.

Protein C was purified from the plasma of a cow treated with the vitamin K antagonist warfarin. The purified protein appeared not to bind Ca2+ ions in contrast to protein C from an untreated animal. The gamma-carboxyglutamic acid content of the abnormal protein C was reduced to approximately 10% of normal, whereas the beta-hydroxyaspartic acid content was only slightly decreased, suggesting that vitamin K is not involved in the postribosomal hydroxylation of the aspartic acid residue in position 71 of the light chain of protein C. The abnormal and normal proteins were activated at the same rates by thrombin, but normal protein C was more rapidly activated by the thrombin-thrombomodulin complex. Compared to normal protein C, the abnormal one had virtually no anticoagulant activity.

Animals↗
PubMed1985

A radioimmunoassay for protein C.

A simple and dependable radioimmunoassay has been devised to measure protein C in human plasma. From a rabbit antiserum against human protein C, the antibodies recognizing the activation peptide region (residues 1-15) in the heavy chain of protein C were affinity purified for use in the radioimmunoassay. The radiolabelled peptide was used as a tracer. The protein C concentration in healthy subjects was 2.5 +/- 0.9 mg/l (2 SD). In warfarin treated persons the protein C level was reduced to approximately 40% of the normal level. Patients with hereditary protein C deficiency had protein C levels ranging from 0.5 mg/l to 1.6 mg/l.

Amino Acid Sequence↗
PubMed1984

Calcium-binding properties of bovine factor X lacking the gamma-carboxyglutamic acid-containing region.

In bovine protein C normal activation by the thrombin-thrombomodulin complex requires binding of calcium to one high affinity binding site, contained in a protein fragment that lacks the gamma-carboxyglutamic acid (Gla) region (Esmon, N. L., De Bault, L. E., and Esmon, C. T. (1983) J. Biol. Chem. 258, 5548-5553). In this work, the calcium binding to and the conformational change induced by calcium in the corresponding Gla-domainless fragment of bovine factor X, prepared by limited proteolysis by chymotrypsin, were compared with the calcium-binding properties of Gla-domainless protein C. Equilibrium dialysis experiments demonstrated that the proteolytically modified factor X has one high affinity calcium ion-binding site with Kd = 180 microM, a value almost identical to the Kd for the binding of calcium to proteolytically modified protein C. Measurements of the rate of disulfide bond reduction by thioredoxin showed that the disulfide bonds of both factor X and protein C lacking the Gla domains were more rapidly reduced in the absence than in the presence of calcium. Thus, calcium binding induces a conformational change in both proteolytically modified proteins. Calcium binding to Gla-domainless protein C is accompanied by a quenching of the intrinsic tryptophan fluorescence and by changes in the CD spectrum, indicative of perturbation of the environment of aromatic amino acids by the metal ion. However, no such changes were observed with the proteolytically modified factor X. This difference may be due to the fact that one tryptophan residue (in position 84) is present in the light chain of the proteolytically modified protein C but none in the light chain of the modified factor X. The light chain of factor X has beta-hydroxyaspartic acid in position 64 which is homologous to the beta-hydroxyaspartic acid in position 71 in the light chain of protein C. Our results are compatible with the hypothesis that beta-hydroxyaspartic acid is involved in the Ca2+ ion binding.

1-Carboxyglutamic Acid↗
PubMed1984

Beta-hydroxyaspartic acid in vitamin K-dependent plasma proteins from scorbutic and warfarin-treated guinea pigs.

beta- Hydroxyaspartic acid is a rare amino acid, present in all vitamin K-dependent plasma proteins except prothrombin, and is formed by a post-translational hydroxylation of aspartic acid. We have now investigated whether this hydroxylation, like that of proline in collagen, is vitamin C-dependent. The vitamin K-dependent plasma proteins were isolated from normal and scorbutic guinea pig plasma by barium citrate adsorption and the beta- hydroxyaspartic acid content was determined. Compared with normal animals, scorbutic animals showed no significant reduction of beta- hydroxyaspartic acid content. In warfarin-treated animals there was a decreased content of both beta- hydroxyaspartic acid and gamma-carboxyglutamic acid in the barium citrate adsorbed fraction. It was concluded that the post-translational hydroxylation of aspartic acid is unlikely to be vitamin C-dependent.

1-Carboxyglutamic Acid↗
PubMed1984

erythro-beta-Hydroxyaspartic acid in bovine factor IX and factor X.

To localize beta-hydroxyaspartic acid in factor IX and factor X the two proteins were cleaved with cyanogen bromide and trypsin, respectively. Peptides containing beta-hydroxyaspartic acid were isolated and subjected to Edman degradation. The phenylthiohydantoin derivative of beta-hydroxyaspartic acid was identified by HPLC in position 3 in the factor IX fragment and in position 1 in the factor X fragment. This corresponds to position 64 in factor IX and position 63 in the light chain of factor X. The assignments were confirmed by subtractive Edman degradation and with the dansyl method.

Amino Acid Sequence↗
PubMed1983

Beta-hydroxyaspartic acid in vitamin K-dependent proteins.

A method for the quantitation of beta-hydroxyaspartic acid in proteins is described. After hydrolysis in 6 M HCl, the beta-hydroxyaspartic acid released is quantitated on an automatic amino acid analyzer employing a pH 2.0 eluting buffer and postcolumn reaction with o-phthalaldehyde for detection. The sensitivity is about 0.01 nmol. Among vitamin K-dependent proteins, factor IX, factor X, protein C, and protein Z each contain about one residue of beta-hydroxyaspartic acid whereas protein S contains two or three residues. Prothrombin lacks beta-hydroxyaspartic acid as do a number of non-vitamin K-dependent proteins also analyzed.

Amino Acids↗
PubMed1983

Translation and processing of normal (PiMM) and abnormal (PiZZ) human alpha 1-antitrypsin.

Human liver mRNA isolated from subjects phenotyped as homozygous PiMM or PiZZ alpha 1-antitrypsin, was translated in a reticulocyte cell-free system, and alpha 1-antitrypsin identified by immunoprecipitation. In the presence of dog pancreas membranes the translated alpha 1-antitrypsin appeared as a larger product. Treatment with endo-beta-N-glucosaminidase yielded a protein smaller than the reticulocyte translated product, presumably due to removal of the N-terminal signal sequence by membranes and sugar residues by endo-beta-N-glucosaminidase. Quantitation of alpha 1-antitrypsin translated from PiMM and PiZZ livers suggests that both mRNA species were present at the same cellular concentration, and that processing to the core glycosylation stage proceeded at identical rates.

Animals↗
PubMed1983

Inactivation of human coagulation factor V by activated protein C.

Thrombin-activated vitamin K-dependent protein C purified from human plasma has a potent anticoagulant effect on human plasma, whereas its bovine counterpart has a very weak anticoagulant effect on human plasma. This species difference was found to be partly due to a more rapid degradation of human factor Va by human than by bovine activated protein C. In the presence of phospholipid, activated human protein C cleaves several peptide bonds in fragment D (heavy chain of factor Va), whereas in fragment F1F2 (light chain of factor Va) there appears to be only one peptide bond that is slowly cleaved. The degradation of fragment D is accompanied by a parallel loss of factor V activity. With the blood coagulation factor Xa bound to factor Va, fragment D is protected from degradation by activated protein C, and factor Va remains active. Fragment D isolated from factor Va was exposed to activated protein C in the presence of phospholipid and was found not to be degraded. This observation suggests that fragment D of factor V is bound to phospholipid via fragment F1F2.

Amino Acids↗
PubMed1983

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

Previous work has shown that the light chain of protein C, an anticoagulant plasma protein, contains an unusual amino acid [Fernlund, P. & Stenflo, J. (1982) J. Biol. Chem. 257, 12170-12179]. To determine the structure of this amino acid a heptapeptide, CMCys-Ile-X-Gly-Leu-Gly-Gly (residues 69-75 in the light chain), was isolated from enzymatic digests of the light chain. According to automatic Edman sequence analysis, 1H NMR spectroscopy, and mass spectrometry the heptapeptide had beta-hydroxyaspartic acid in its third position, which corresponds to position 71 in the light chain of protein C. Analysis of acid and aminopeptidase M hydrolysates of the heptapeptide showed the beta-hydroxyaspartic acid to be the erythro form. Acid hydrolysis of protein C released approximately equal to 1 mol of beta-hydroxyaspartic acid per mol of protein. The function of this amino acid, which, to the best of our knowledge, has not been found previously in proteins, is unknown.

Amino Acid Sequence↗
PubMed1982

Amino acid sequence of the light chain of bovine protein C.

The amino acid sequence of the light chain of the vitamin K-dependent protein C from bovine plasma is reported together with the supporting data. The sequence was determined by sequenator analysis of intact light chain, which contains 155 amino acid residues, and fragments obtained by chemical and enzymatic degradation of the light chain. All 11 glutamyl residues up to position 44 are carboxylated as gamma-carboxyglutamic acid residues. This part of the molecule proved very homologous to the other vitamin K-dependent plasma proteins. The remaining part (111 residues) is homologous to the corresponding parts in factor IX and factor X but not to prothrombin.

Amino Acid Sequence↗