Vitamin K-Dependent Formation of γ-Carboxyglutamic Acid

Stenflo, Johan; Suttie, J. W.

doi:10.1146/annurev.bi.46.070177.001105

Cited by 363 publications

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“…The vitamin K-dependent ␥-glutamyl carboxylase catalyzes the post-translational conversion of glutamyl residues to ␥-carboxyglutamyl (Gla) 1 residues in precursor proteins that contain the appropriate ␥-carboxylation recognition site within the propeptide of the precursor (1)(2)(3)(4). Among the classes of proteins that contain Gla, the vitamin K-dependent blood coagulation proteins have been most thoroughly studied.…”

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The First γ-Carboxyglutamic Acid-containing Contryphan

Hansson

Eliasson

et al. 2004

Journal of Biological Chemistry

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Contryphans constitute a group of conopeptides that are known to contain an unusual density of post-translational modifications including tryptophan bromination, amidation of the C-terminal residue, leucine, and tryptophan isomerization, and proline hydroxylation. Here we report the identification and characterization of a new member of this family, glacontryphan-M from the venom of Conus marmoreus. This is the first known example of a contryphan peptide carrying glutamyl residues that have been post-translationally carboxylated to ␥-carboxyglutamyl (Gla) residues. The amino acid sequence of glacontryphan-M was determined using automated Edman degradation and electrospray ionization mass spectrometry. The amino acid sequence of the peptide is: Asn-Gla-Ser-Gla-Cys-Pro-D-Trp-His-Pro-Trp-Cys. As with most other contryphans, glacontryphan-M is amidated at the C terminus and maintains the five-residue intercysteine loop. The occurrence of a D-tryptophan residue was confirmed by chemical synthesis and HPLC elution profiles. Using fluorescence spectroscopy we demonstrated that the Gla-containing peptide binds calcium with a K D of 0.63 mM. Cloning of the full-length cDNA encoding glacontryphan-M revealed that the primary translation product carries an N-terminal signal/ propeptide sequence that is homologous to earlier reported contryphan signal/propeptide sequences up to 10 amino acids preceding the toxin region. Electrophysiological experiments, carried out on mouse pancreatic B-cells, showed that glacontryphan-M blocks L-type voltage-gated calcium ion channel activity in a calciumdependent manner. Glacontryphan-M is the first contryphan reported to modulate the activity of L-type calcium ion channels.

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confidence: 99%

The First γ-Carboxyglutamic Acid-containing Contryphan

Hansson

Eliasson

et al. 2004

Journal of Biological Chemistry

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“…The light chain has 11 amino-terminal glutamyl residues that are post-translationally modified in a vitamin K-dependent reaction to form a ␥-carboxyglutamic acid-containing domain or "Gla domain" critical for the binding of calcium ions and phospholipids (14). The Gla domain is followed by two domains homologous to the epidermal growth factor (EGF) precursor, considered important for protein-protein interactions (15).…”

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Role of the Gla and First Epidermal Growth Factor-like Domains of Factor X in the Prothrombinase and Tissue Factor-Factor VIIa Complexes

Thiec

Cherel

Olivier

2003

Journal of Biological Chemistry

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Factor X (FX) has high structure homology with other proteins of blood coagulation such as factor IX (FIX) and factor VII (FVII). These proteins present at their aminoterminal extremity a ␥-carboxyglutamic acid containing domain (Gla domain), followed by two epidermal growth factor-like (EGF1 and EGF2) domains, an activation peptide, and a serine protease domain. After vascular damage, the tissue factor-FVIIa (TF-FVIIa) complex activates both FX and FIX. FXa interacts stoichiometrically with tissue pathway inhibitor (TFPI), regulating TF-FVIIa activity by forming the TF-FVIIa-TFPI-FXa quaternary complex. Conversely, FXa boosts coagulation by its association with its cofactor, factor Va (FVa). To investigate the contribution of the Gla and EGF1 domains of FX in these complexes, FX chimeras were produced in which FIX Gla and EGF1 domains substituted the corresponding domains of FX. The affinity of the two chimeras, FX/FIX(Gla) and FX/FIX(EGF1), for the TF-FVIIa complex was markedly reduced compared with that of wild-type-FX (wt-FX) independently of the presence of phospholipids. Furthermore, the association rate constants of preformed FX/FIX(Gla)-TFPI and FX/FIX(EGF1)-TFPI complexes with TF-FVIIa were, respectively, 10-and 5-fold slower than that of wt-FXa-TFPI complex. Finally, the apparent affinity of FVa was 2-fold higher for the chimeras than for wt-FX in the presence of phospholipids and equal in their absence. These data demonstrate that FX Gla and EGF1 domains contain residues, which interact with TF-FVIIa exosites contributing to the formation of the TF-FVIIa-FX and TF-FVIIa-TFPI-FXa complexes. On the opposite, FXa Gla and EGF1 domains are not directly involved in FVa binding.The blood coagulation cascade consists of a series of enzymatic conversions driven by the formation of complexes between serine proteases and cell membrane-bound cofactors. Human factor X (FX) 1 is one of the serine protease zymogens playing a central role in coagulation processes leading to the formation of a fibrin clot. This is illustrated by the behavior of FX as a substrate or as an enzyme in three essential blood coagulation complexes. First, FX is a natural substrate, as well as factor IX (FIX), of the tissue factor-factor VIIa (TF-FVIIa) complex (1) considered as the initial enzyme complex in the cascade following vascular damage. FX activation by TF-FVIIa results from specific cleavage and release of a 52-residue activation peptide. Activated FX (FXa) can generate a tiny amount of thrombin from prothrombin in an extremely inefficient reaction (2). Tissue factor pathway inhibitor (TFPI) binds to TF-FVIIa-FXa to limit the production of FXa and FIXa by TF-FVIIa (3, 4). Nevertheless, once produced, thrombin and the initially formed FXa activate small quantities of factor V (FV) to FVa and factor VIII (FVIII) to FVIIIa (5-8). The activation of these two cofactors leads to the formation of two other essential procoagulant complexes, both involving FX, at the surface of procoagulant phospholipids in the presence of calcium ions (9),...

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“…1992;Schifldt et al, 1992;Wildgoose et al, 1993). Ca'+ binding to the Gla module is known to be essential for membrane binding (Stenflo and Suttie, 1977) and saturation of the site in the catalytic domain appears to be important for activity and tissue factor binding of factor VIIa , but much is still unknown about the structural and functional effects of Ca2' binding to the different sites. To further investigate the role of Ca" binding to various regions of factor VIIa, we have studied functional and structural properties of factor VIIa and derivatives thereof as a function of the Ca'+ concentration.…”

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confidence: 99%

Structurally and Functionally Distinct Ca²⁺ Binding Sites in the γ‐Carboxyglutamic Acid‐Containing Domain of Factor VIIa

Persson

Petersen

1995

European Journal of Biochemistry

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The structural and functional effects of Ca'+ binding to vitamin-K-dependent coagulation factor VIIa were investigated. Conformational changes with a midpoint around 0.7 mM Ca2+ quenched the intrinsic protein fluorescence of a fragment of factor VIIa comprising only the light chain and this coincided with an increase in factor VIIa amidolytic activity in the absence of tissue factor. Ca'+ binding to sites in factor VIIa and in the fragment with an apparent dissociation constant of 1.3 -1.4 mM induced binding to phospholipids. A similar Caz+ dependency was not observed with factor VIIa lacking the N-terminal 38 or 44 residues of the light chain and the observed effects could thus be attributed to y-carboxyglutamicacid-dependent Ca2+ binding. Mg" appeared to bind to the site(s) of relatively higher affinity since, although it was less efficient than Ca", it stimulated the amidolytic activity and induced quenching of the intrinsic fluorescence. In contrast, Mg'+ did not induce expression of the phospholipid-interactive structure. The binding properties of two monoclonal antibodies that recognized epitopes in the y-carboxyglutamic-acid-rich domain of factor VIIa corroborated the occurrence of two Ca' ' -induced, sequential structural changes and only one of the antibodies recognized the Mg2' -induced structure. Thus Ca2+ binding to the y-carboxyglutamic-acid-containing domain appeared to result in at least two distinct structural transitions with different functional consequences. The two (sets of) sites responsible for the observed effects could be distinguished based upon differences in Ca'+ affinity and metal ion selectivity. The interaction between factor VIIa and tissue factor was monitored by means of a direct binding assay and an amidolytic assay. In both systems, half-maximal Ca'+ enhancement was observed at 0.25 mM. This coincided with a Ca'+-induced conformational change in factor VIIa associated with fluorescence quenching. The same effect on amidolytic activity was observed with the two N-terminally truncated forms of factor VIIa and it is presumably mediated by Ca" binding to a site located in the serine protease part.

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Vitamin K-Dependent Formation of γ-Carboxyglutamic Acid

Cited by 363 publications

References 0 publications

The First γ-Carboxyglutamic Acid-containing Contryphan

The First γ-Carboxyglutamic Acid-containing Contryphan

Role of the Gla and First Epidermal Growth Factor-like Domains of Factor X in the Prothrombinase and Tissue Factor-Factor VIIa Complexes

Structurally and Functionally Distinct Ca²⁺ Binding Sites in the γ‐Carboxyglutamic Acid‐Containing Domain of Factor VIIa

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Vitamin K-Dependent Formation of γ-Carboxyglutamic Acid

Cited by 363 publications

References 0 publications

The First γ-Carboxyglutamic Acid-containing Contryphan

The First γ-Carboxyglutamic Acid-containing Contryphan

Role of the Gla and First Epidermal Growth Factor-like Domains of Factor X in the Prothrombinase and Tissue Factor-Factor VIIa Complexes

Structurally and Functionally Distinct Ca2+ Binding Sites in the γ‐Carboxyglutamic Acid‐Containing Domain of Factor VIIa

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Structurally and Functionally Distinct Ca²⁺ Binding Sites in the γ‐Carboxyglutamic Acid‐Containing Domain of Factor VIIa