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This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Grinnell, B. W. Articles by Bang, N. U. Search for Related Content PubMed PubMed Citation Articles by Grinnell, B. W. Articles by Bang, N. U. Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Gamma-carboxylated isoforms of recombinant human protein S with different biologic properties BW Grinnell, JD Walls, C Marks, AL Glasebrook, DT Berg, SB Yan and NU Bang Department of Molecular Genetics, Lilly Research Laboratories, Indianapolis, IN 46285. 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. Volume 76, Issue 12, pp. 2546-2554, 12/15/1990 Copyright © 1990 by The American Society of Hematology CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: X. He, L. Shen, B. O. Villoutreix, and B. Dahlback Amino Acid Residues in Thrombin-sensitive Region and First Epidermal Growth Factor Domain of Vitamin K-dependent Protein S Determining Specificity of the Activated Protein C Cofactor Function J. Biol. Chem., October 16, 1998; 273(42): 27449 - 27458. [Abstract] [Full Text] [PDF]

	Copyright © 1990 by American Society of Hematology         Online ISSN: 1528-0020