The vitamin K-dependent blood coagulation proteins undergo co- or post-translational processing that includes gamma carboxylation. This protein modification is required for calcium-dependent membrane binding. The propeptide of the molecule contains the gamma carboxylation recognition site which directs gamma carboxylation, and a propeptide cleavage consensus sequence. The current proposal aims to define the consensus sequence for the gamma carboxylation recognition site using combinatorial chemical peptide synthesis and combinatorial phage display. The three dimensional structure of a synthetic fully carboxylated profactor IX analog will be solved to determine the structure of the propeptide and its relationship to the Gla domain. To prove that the propeptide is sufficient to direct gamma carboxylation, in vivo carboxylation of chimeras of prothrombin propeptide joined to truncated P-selectin and PSGL-1 will be studied to assess carboxylation of glutamic acids in proteins that normally do not undergo gamma carboxylation. The cDNA encoding a protein required for gamma carboxylation in a CHO cell line characterized by defective carboxylation but with normal carboxylase activity will be identified by expression cloning. The physiological roles of furin and proprotein convertase 7 (PC7) in propeptide cleavage of the vitamin K-dependent proteins will be determined using furin deficient CHO cells, and experiments will be performed to identify the consensus sequence of PC7-mediated peptide bond cleavage. Experiments in this proposal will extend understanding of the role of the propeptide in vitamin K-dependent carboxylation and the identification of the enzymes that cleave the propeptide during the biosynthesis of these gamma carboxyglutamic acid-containing proteins. Gamma carboxylation and propeptide cleavage are the posttranslational events that limit the expression of the biologically active recombinant proteins and define protein expression levels for gene therapy of hemophilia B. Detailed knowledge of these processes will improve our understanding of the biology of these proteins and has potential for improvements in hemophilia therapy.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL038216-14
Application #
6056194
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1987-04-01
Project End
2002-08-31
Budget Start
1999-09-01
Budget End
2000-08-31
Support Year
14
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Beth Israel Deaconess Medical Center
Department
Type
DUNS #
076593722
City
Boston
State
MA
Country
United States
Zip Code
02215
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Rigby, A C; Baleja, J D; Furie, B C et al. (1997) Three-dimensional structure of a gamma-carboxyglutamic acid-containing conotoxin, conantokin G, from the marine snail Conus geographus: the metal-free conformer. Biochemistry 36:6906-14
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