title: gamma-Carboxyglutamic acids 36 and 40 do not contribute to human factor IX function.
abstract: The gamma-carboxyglutamic acid (Gla) domains of the vitamin K-dependent blood coagulation proteins contain 10 highly conserved Gla residues within the first 33 residues, but factor IX is unique in possessing 2 additional Gla residues at positions 36 and 40. To determine their importance, factor IX species lacking these Gla residues were isolated from heterologously expressed human factor IX. Using ion-exchange chromatography, peptide mapping, mass spectrometry, and N-terminal sequencing, we have purified and identified two partially carboxylated recombinant factor IX species; factor IX/gamma 40E is uncarboxylated at residue 40 and factor IX/gamma 36,40E is uncarboxylated at both residues 36 and 40. These species were compared with the fully gamma-carboxylated recombinant factor IX, unfractionated recombinant factor IX, and plasma-derived factor IX. As monitored by anti-factor IX:Ca (II)-specific antibodies and by the quenching of intrinsic fluorescence, all these factor IX species underwent the Ca(II)-induced conformational transition required for phospholipid membrane binding and bound equivalently to phospholipid vesicles composed of phosphatidylserine, phosphatidylcholine, and phosphatidylethanolamine. Endothelial cell binding was also similar in all species, with half-maximal inhibition of the binding of 125I-labeled plasma-derived factor IX at concentrations of 2-6 nM. Functionally, factor IX/gamma 36,40E and factor IX/gamma 40E were similar to fully gamma-carboxylated recombinant factor IX and plasma-derived factor IX in their coagulant activity and in their ability to participate in the activation of factor X in the tenase complex both with synthetic phospholipid vesicles and activated platelets. However, Gla 36 and Gla 40 represent part of the epitope targeted by anti-factor IX:Mg(II)-specific antibodies because these antibodies bound factor IX preferentially to factor IX/gamma 36,40E and factor IX/gamma 40E. These results demonstrate that the gamma-carboxylation of glutamic acid residues 36 and 40 in human factor IX is not required for any function of factor IX examined.
authors.affiliation: Division of Hematology-Oncology, New England Medical Center, Boston, Massachusetts 02111, USA.
authors.forename: S
authors.initials: S
authors.lastname: Gillis
authors.forename: B C
authors.initials: BC
authors.lastname: Furie
authors.forename: B
authors.initials: B
authors.lastname: Furie
authors.forename: H
authors.initials: H
authors.lastname: Patel
authors.forename: M C
authors.initials: MC
authors.lastname: Huberty
authors.forename: M
authors.initials: M
authors.lastname: Switzer
authors.forename: W B
authors.initials: WB
authors.lastname: Foster
authors.forename: H A
authors.initials: HA
authors.lastname: Scoble
authors.forename: M D
authors.initials: MD
authors.lastname: Bond
mesh_terms: D015055:1-Carboxyglutamic Acid / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D005164:Factor IX / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D011994:Recombinant Proteins; D015055:1-Carboxyglutamic Acid; D005164:Factor IX