Membrane binding properties of the factor IX gamma-carboxyglutamic acid-rich domain prepared by chemical synthesis.
The fully gamma-carboxylated peptides based upon the complete and truncated Gla/aromatic amino acid stack domains of human Factor IX were prepared by solid phase peptide synthesis using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry. A 47-residue peptide Factor IX-(1-47) and a 42-residue peptide Factor IX-(1-42), both containing 12 residues of L-gamma-carboxyglutamic acid, were purified by high performance liquid chromatography and oxidized to form the disulfide bond. Quantitative gamma-carboxyglutamic acid analysis of Factor IX-(1-47) and Factor IX-(1-42) indicated the presence of 12.1 and 11.2 gamma-carboxyglutamic acid residues/mol of peptide, respectively; no glutamic acid was detected. As monitored by fluorescence quenching, calcium ions induced the prototypical conformational transition in Factor IX-(1-47), but not in Factor IX-(1-42), that is observed with Factor IX. Half-maximal quenching of the intrinsic fluorescence of Factor IX-(1-47) was observed at Ca(II) concentrations of about 50 microM. Factor IX-(1-47) bound to the conformation-specific antibodies, anti-Factor IX:Mg(II) and anti-Factor IX:Ca(II)-specific in the presence of metal ions. Factor IX-(1-47) bound to phospholipid membranes, as monitored by energy transfer from intrinsic fluorophores to dansyl (5-dimethylaminonaphthalene-1-sulfonyl)-phosphatidylethanolamine incorporated into a lipid bilayer composed of phosphatidylserine:phosphatidylcholine. In contrast, Factor IX-(1-42) bound poorly to these same membranes. Factor IX-(1-47) did not inhibit Factor XIa activation of Factor IX but did inhibit the activation of Factor X by Factor IXa bound to Factor VIII in the presence of calcium ions and phospholipid. These results show that phospholipid membrane binding is a property of the Gla/aromatic amino acid stack domain and that the Factor IX-(1-47) peptide, prepared by chemical synthesis, preserves the membrane binding properties and the metal-induced conformational transitions observed in native Factor IX. These results indicate that Factor IX-(1-47) but not Factor IX-(1-42) is a suitable model for structural studies of Factor IX-membrane interaction.
Jacobs
M
M
Center for Hemostasis and Thrombosis Research, New England Medical Center, Boston, Massachusetts.
Freedman
S J
SJ
Furie
B C
BC
Furie
B
B
D015055:1-Carboxyglutamic Acid / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002118:Calcium / Q000378:metabolism; D005164:Factor IX / Q000138:chemical synthesis / Q000276:immunology / Q000378:metabolism*; D005169:Factor VIII / Q000378:metabolism; D005170:Factor X / Q000378:metabolism; D015945:Factor XIa / Q000378:metabolism; D005470:Fluorometry; D006801:Humans; D008051:Lipid Bilayers / Q000378:metabolism*; D015335:Molecular Probes; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship
D000596:Amino Acids; D008051:Lipid Bilayers; D015335:Molecular Probes; D010446:Peptide Fragments; C089943:factor IX (1-47); D015055:1-Carboxyglutamic Acid; D005169:Factor VIII; D005164:Factor IX; D005170:Factor X; D015945:Factor XIa; D002118:Calcium