Site‐directed mutagenesis but not γ‐carboxylation of Glu‐35 in factor VIIa affects the association with tissue factor

Persson, Egon; Nielsen, Lene

doi:10.1016/0014-5793(96)00400-0

Cited by 32 publications

(36 citation statements)

References 19 publications

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“…Gla Content of Recombinant Q100RFVII-Following Edman degradation and direct sequencing, signals were absent in all the residue positions given above up to 29, despite clear and unambiguous responses for other residues as far as position 33. We were also unable to detect a signal at position 35 (the 10th and last Gla position), but the surrounding residue signals were too attenuated to be certain as to the significance of the absence of signal at Glu 35 ; however, it has been stated that a Gla rather than Glu at position 35 does not increase the affinity of FVIIa for TF (15). Thus the level of gamma-carboxylation in Q100RFVII is comparable to that in WTFVII (10).…”

Section: Expression Of Wild-type and Variant Q100rfvii In Mam-mentioning

confidence: 78%

Coagulation Factor VII Gln100 → Arg

Kemball‐Cook¹,

Johnson²,

Takahashi³

et al. 1998

Journal of Biological Chemistry

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We have used recombinant mammalian expression and purification of the factor VII (FVII) variant Gln 100 3 Arg (Q100RFVII) to study FVII deficiency in subjects with this mutation. Q100RFVII was secreted poorly in comparison with wild-type FVII (WTFVII) in a stable mammalian expression system, and purified variant protein was found to have undetectable clotting activity. Following activation by immobilized factor Xa, Q100RFVIIa had amidolytic activity similar to WTFVIIa in the absence of soluble tissue factor (sTF); however, unlike WTFVIIa no typical increase in activity was seen after addition of sTF. In a factor X activation assay using relipidated transmembrane truncated tissue factor (residues 1-243), Q100RFVIIa showed less than 5% of the ability of WTFVIIa to activate factor X.We performed direct binding analysis of WT and Q100RFVII/FVIIa to immobilized sTF using surface plasmon resonance, and severely reduced binding of both non-activated and activated Q100RFVII to sTF was seen, indicating a pronounced defect in tissue factor (TF) interaction with this variant.In the sTF-FVIIa crystal structure the candidate residue Gln 100 is not in contact with TF but is at the epidermal growth factor 2-protease domain interface. We suggest that the mutation results in a global fold change severely reducing tissue factor interaction; mutation of FVII residues not directly involved in the interaction with TF may still result in variant FVII unable to take part in the initiation of coagulation.

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Section: Expression Of Wild-type and Variant Q100rfvii In Mam-mentioning

confidence: 78%

Coagulation Factor VII Gln100 → Arg

Kemball‐Cook¹,

Johnson²,

Takahashi³

et al. 1998

Journal of Biological Chemistry

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“…The wild-type FVII expression plasmid pLN174 [18] was used as template for site-directed mutagenesis using the QuikChange ® II XL Site-Directed Mutagenesis Kit (Stratagene). Plasmids were prepared using a QIAprep spin mini-prep kit (Qiagen), and the mutations were verified by DNA sequencing.…”

Section: Mutagenesis Protein Expression and Purificationmentioning

confidence: 99%

Engineering the substrate and inhibitor specificities of human coagulation Factor VIIa

Larsen

Østergaard

Bjelke

et al. 2007

Biochemical Journal

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The remarkably high specificity of the coagulation proteases towards macromolecular substrates is provided by numerous interactions involving the catalytic groove and remote exosites. For FVIIa [activated FVII (Factor VII)], the principal initiator of coagulation via the extrinsic pathway, several exosites have been identified, whereas only little is known about the specificity dictated by the active-site architecture. In the present study, we have profiled the primary P4-P1 substrate specificity of FVIIa using positional scanning substrate combinatorial libraries and evaluated the role of the selective active site in defining specificity. Being a trypsin-like serine protease, FVIIa had P1 specificity exclusively towards arginine and lysine residues. In the S2 pocket, threonine, leucine, phenylalanine and valine residues were the most preferred amino acids. Both S3 and S4 appeared to be rather promiscuous, however, with some preference for aromatic amino acids at both positions. Interestingly, a significant degree of interdependence between the S3 and S4 was observed and, as a consequence, the optimal substrate for FVIIa could not be derived directly from a subsite-directed specificity screen. To evaluate the role of the active-site residues in defining specificity, a series of mutants of FVIIa were prepared at position 239 (position 99 in chymotrypsin), which is considered to be one of the most important residues for determining P2 specificity of the trypsin family members. This was confirmed for FVIIa by marked changes in primary substrate specificity and decreased rates of antithrombin III inhibition. Interestingly, these changes do not necessarily coincide with an altered ability to activate Factor X, demonstrating that inhibitor and macromolecular substrate selectivity may be engineered separately.

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“…Human recombinant FVIIa was expressed and purified as previously described~Thim et al., 1988;Persson & Nielsen, 1996!. The wild-type FVII expression plasmid, pLN174, contains the FVII cDNA driven by a mouse metallothionein promoter and as a selectable marker dihydrofolate reductase driven by an SV40 promoter.…”

Section: Proteinsmentioning

confidence: 99%

Binding of Zn²⁺ to a Ca²⁺ loop allosterically attenuates the activity of factor VIIa and reduces its affinity for tissue factor

Petersen¹,

Olsen²,

Nielsen³

et al. 2000

Protein Science

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The protease domain of coagulation factor VIIa~FVIIa! is homologous to trypsin with a similar active site architecture. The catalytic function of FVIIa is regulated by allosteric modulations induced by binding of divalent metal ions and the cofactor tissue factor~TF!. To further elucidate the mechanisms behind these transformations, the effects of Zn to FVIIa, which was influenced by the presence of Ca 2ϩ , resulted in decreased amidolytic activity and slightly reduced affinity for TF. After binding to TF, FVIIa was less susceptible to zinc inhibition. Alanine substitutions for either of two histidine residues unique for FVIIa, His216, and His257, produced FVIIa variants with decreased sensitivity to Zn 2ϩ inhibition. A search for putative Zn 2ϩ binding sites in the crystal structure of the FVIIa protease domain was performed by Grid calculations. We identified a pair of Zn 2ϩ binding sites in the Glu210-Glu220 Ca 2ϩ binding loop adjacent to the so-called activation domain canonical to serine proteases. Based on our results, we propose a model that describes the conformational changes underlying the Zn 2ϩ -mediated allosteric down-regulation of FVIIa's activity.

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Site‐directed mutagenesis but not γ‐carboxylation of Glu‐35 in factor VIIa affects the association with tissue factor

Cited by 32 publications

References 19 publications

Coagulation Factor VII Gln100 → Arg

Coagulation Factor VII Gln100 → Arg

Engineering the substrate and inhibitor specificities of human coagulation Factor VIIa

Binding of Zn²⁺ to a Ca²⁺ loop allosterically attenuates the activity of factor VIIa and reduces its affinity for tissue factor

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Site‐directed mutagenesis but not γ‐carboxylation of Glu‐35 in factor VIIa affects the association with tissue factor

Cited by 32 publications

References 19 publications

Coagulation Factor VII Gln100 → Arg

Coagulation Factor VII Gln100 → Arg

Engineering the substrate and inhibitor specificities of human coagulation Factor VIIa

Binding of Zn2+ to a Ca2+ loop allosterically attenuates the activity of factor VIIa and reduces its affinity for tissue factor

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Binding of Zn²⁺ to a Ca²⁺ loop allosterically attenuates the activity of factor VIIa and reduces its affinity for tissue factor