title: Gamma-carboxylated isoforms of recombinant human protein S with different biologic properties.
abstract: Human protein S (HPS), a regulator of hemostasis, is a vitamin K-dependent plasma protein with potential clinical utility. We have obtained high-level expression of the cDNA for HPS in two mammalian cell lines. Both cell lines secreted single chain recombinant HPS (rHPS) in serum-free medium as determined by Western blot analysis. The ability of the rHPS from both cell lines to act as a cofactor for human protein C (HPC) was determined; the rHPS secreted from the human 293 cell line had an activity six times that of the rHPS from the AV12-664 Syrian hamster cell line. Furthermore, the relative specific cofactor activity of rHPS from the 293 cell line was actually 2.5-fold higher than that of single-chain human plasma-derived HPS. Essentially all of the rHPS secreted from the 293 cell line exhibited a calcium-dependent elution profile on anion exchange chromatography, whereas only 25% to 35% of the hamster cell-derived rHPS exhibited this profile. However, the calcium-eluted rHPS from the AV12 cell line had a high specific cofactor activity, equivalent to that of the 293-derived rHPS. A NaCl-elutable rHPS fraction (calcium nondependent) was isolated from the recombinant AV12-664 cell line, further purified, and found to have reduced activity, only 40% that of the calcium-dependent rHPS. The only observable difference in the calcium-dependent and nondependent rHPS molecules was in the content of gamma-carboxyglutamic acid (Gla); the calcium-dependent material contained approximately 10 mol Gla/mol protein whereas the calcium-nondependent material contained only approximately 8 mol Gla/mol of protein. In addition, the calcium-nondependent rHPS had reduced ability to interact with phospholipid vesicles as evidenced by an eightfold increase in the apparent kd. Our data demonstrate the isolation of rHPS with high specific activity, and show that a reduction in as few as two Gla residues dramatically decreases its functional cofactor activity for HPC, due to a reduction in ability to interact with the phospholipid bilayer.
authors: Department of Molecular Genetics, Lilly Research Laboratories, Indianapolis, IN 46285.
authors: B W
authors: BW
authors: Grinnell
authors: J D
authors: JD
authors: Walls
authors: C
authors: C
authors: Marks
authors: A L
authors: AL
authors: Glasebrook
authors: D T
authors: DT
authors: Berg
authors: S B
authors: SB
authors: Yan
authors: N U
authors: NU
authors: Bang
mesh_terms: D015055:1-Carboxyglutamic Acid / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000502:physiology; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D006224:Cricetinae; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006023:Glycoproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007536:Isomerism; D007668:Kidney / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D008647:Mesocricetus; D010743:Phospholipids / Q000032:analysis / Q000378:metabolism; D017293:Protein S; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D006023:Glycoproteins; D010743:Phospholipids; D017293:Protein S; D011994:Recombinant Proteins; D015055:1-Carboxyglutamic Acid; D004247:DNA; D002118:Calcium