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Related Experiment Videos

gamma-Carboxyglutamic acids 36 and 40 do not contribute to human factor IX function

S Gillis1, B C Furie, B Furie

  • 1Division of Hematology-Oncology, New England Medical Center, Boston, Massachusetts 02111, USA.

Protein Science : a Publication of the Protein Society
|January 1, 1997
PubMed
Summary

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The gamma-carboxylation of glutamic acid residues 36 and 40 in human factor IX is not essential for its blood coagulation function or phospholipid binding. These specific residues are not required for factor IX

Area of Science:

  • Biochemistry
  • Hematology
  • Molecular Biology

Background:

  • Vitamin K-dependent proteins, including coagulation factor IX, undergo gamma-carboxylation of glutamic acid residues.
  • Factor IX possesses unique gamma-carboxyglutamic acid (Gla) residues at positions 36 and 40, in addition to the conserved Gla residues in its Gla domain.
  • The functional significance of these unique Gla residues in factor IX remains to be fully elucidated.

Purpose of the Study:

  • To investigate the functional importance of gamma-carboxyglutamic acid (Gla) residues at positions 36 and 40 in human factor IX.
  • To determine if these unique Gla residues are necessary for factor IX's calcium-dependent conformational changes, phospholipid binding, endothelial cell binding, and coagulant activity.
  • To assess the role of Gla 36 and Gla 40 in the epitope recognized by specific anti-factor IX antibodies.

Related Experiment Videos

Main Methods:

  • Generation and purification of recombinant human factor IX species with specific uncarboxylated residues (factor IX/gamma 40E and factor IX/gamma 36,40E).
  • Biochemical characterization using ion-exchange chromatography, peptide mapping, mass spectrometry, and N-terminal sequencing.
  • Assessment of calcium-dependent conformational changes, phospholipid binding, endothelial cell binding, and coagulant activity using various assays and anti-factor IX antibodies.

Main Results:

  • Partially carboxylated recombinant factor IX species (factor IX/gamma 40E and factor IX/gamma 36,40E) were successfully purified and identified.
  • All tested factor IX species, including those lacking Gla 36 and 40, exhibited normal calcium-induced conformational transitions and equivalent binding to phospholipid vesicles.
  • Coagulant activity and participation in factor X activation within the tenase complex were similar across all factor IX variants.
  • Antibody binding studies revealed that Gla 36 and Gla 40 are part of the epitope for anti-factor IX:Mg(II)-specific antibodies.

Conclusions:

  • The gamma-carboxylation of glutamic acid residues 36 and 40 in human factor IX is not required for its essential functions, including phospholipid binding and coagulant activity.
  • These unique Gla residues do not influence the calcium-dependent conformational changes necessary for factor IX's procoagulant functions.
  • Gla 36 and Gla 40 are important for antibody recognition, specifically for antibodies targeting the Mg(II)-dependent epitope of factor IX.