Characterization of γ-Carboxyglutamic Acid Residue 21 of Human Factor IX†

Biochemistry ◽  
1996 ◽  
Vol 35 (32) ◽  
pp. 10321-10327 ◽  
Author(s):  
Alisa S. Wolberg ◽  
Leping Li ◽  
Wing-Fai Cheung ◽  
Nobuko Hamaguchi ◽  
Lee G. Pedersen ◽  
...  
Keyword(s):  
Human Factor ◽  
Factor Ix ◽  
Human Factor Ix ◽  
Carboxyglutamic Acid

scholarly journals Expression and characterization of human factor IX. Factor IXthr-397 and factor IXval-397

1991 ◽  
Vol 266 (23) ◽  
pp. 15213-15220
Author(s):  
N. Hamaguchi ◽  
P.S. Charifson ◽  
L.G. Pedersen ◽  
G.D. Brayer ◽  
K.J. Smith ◽  
...  
Keyword(s):  
Human Factor ◽  
Factor Ix ◽  
Human Factor Ix

scholarly journals Construction, Rescue, and Characterization of Vectors Derived from Ovine Atadenovirus

2003 ◽  
Vol 77 (22) ◽  
pp. 11941-11951 ◽  
Author(s):  
Peter Löser ◽  
Christian Hofmann ◽  
Gerald W. Both ◽  
Wolfgang Uckert ◽  
Moritz Hillgenberg
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Human Factor ◽  
Insertion Site ◽  
Factor Ix ◽  
Vector System ◽  
Virus Rescue ◽  
Human Factor Ix

ABSTRACT Gene transfer vectors derived from ovine atadenovirus type 7 (OAdV) can efficiently infect a variety of mammalian cells in vitro and in vivo to deliver and express transgenes. However, early OAdV vectors were designed on human mastadenovirus principles prior to the complete characterization of OAdV genes and transcripts. The distinctive arrangement of the OAdV genome has suggested ways to improve OAdV vector design and utility. We therefore developed a cosmid-based approach that allows efficient construction of recombinant ovine atadenovirus genomes in which the transgene is inserted at one of three sites. Viruses were rescued by transfection of viral DNA into a new ovine fetal skin fibroblast producer cell line, HVO156. The suitability of the three insertion sites was compared with respect to virus rescue efficiency, gene expression levels, and genetic stability of the vectors. We found that one vector with a transgene inserted at site 1, between the pVIII and fiber genes, was unstable. Only one vector that carried a transgene at site 2, near the right end of the genome, together with a nearby deletion was rescued. In contrast, several vectors with different transgenes inserted in site 3, between the E4 and RH transcription units, were repeatedly rescued, and these vectors were stable over at least four passages. Transgene orientation in site 3 had only little effect on expression. Finally, a vector carrying a human factor IX cDNA at site 3, when administered intravenously, produced nearly physiological levels of human factor IX in mice. The availability of an efficient method for vector construction and the identification of a new insertion site for virus rescue and gene expression substantially enhance the utility of the OAdV vector system.

The endothelial cell binding determinant of human factor IX resides in the .gamma.-carboxyglutamic acid domain

Biochemistry ◽  
1992 ◽  
Vol 31 (6) ◽  
pp. 1806-1808 ◽  
Author(s):  
John R. Toomey ◽  
Kenneth J. Smith ◽  
Harold R. Roberts ◽  
Darrel W. Stafford
Keyword(s):  
Endothelial Cell ◽  
Human Factor ◽  
Factor Ix ◽  
Cell Binding ◽  
Human Factor Ix ◽  
Carboxyglutamic Acid ◽  
Acid Domain

Purification and characterization of human factor IX

1975 ◽  
Vol 7 (3) ◽  
pp. 451-459 ◽  
Author(s):  
L-O. Andersson ◽  
H. Borg ◽  
M. Miller-Andersson
Keyword(s):  
Human Factor ◽  
Factor Ix ◽  
Purification And Characterization ◽  
Human Factor Ix

scholarly journals Expression and characterization of human factor IX and factor IX-factor X chimeras in mouse C127 cells.

1990 ◽  
Vol 265 (1) ◽  
pp. 144-150 ◽  
Author(s):  
S W Lin ◽  
K J Smith ◽  
D Welsch ◽  
D W Stafford
Keyword(s):  
Human Factor ◽  
Factor Ix ◽  
Factor X ◽  
Human Factor Ix

RECOGNITION SITE DIRECTING GAMMA-CARBOXYLATION RESIDES ON THE PROPEPTIDES OF FACTOR IX AND PROTRROMBIN

1987 ◽  
Author(s):  
B C Furis ◽  
M J Jorgensem ◽  
M J Rabiet ◽  
A B Contor ◽  
C L Brown ◽  
...  
Keyword(s):  
Vitamin K ◽  
Human Factor ◽  
Chinese Hamster ◽  
Factor Ix ◽  
Absolute Amount ◽  
Native Structure ◽  
Mutant Proteins ◽  
Specific Antibodies ◽  
Human Factor Ix ◽  
Carboxyglutamic Acid

Factor IX and prothrombin vitamin K-dependent proteins that participate in blood coagulation undergo post-translationalmodification in which glutamic acid residues in the amino terminus of the protein are converted to gamma-carboxyglutamic acid residues. This modification confers divalent metal ion binding ability upon the proteins.As a consequence of binding divalent metal ions these proteins undergoconformational changes necessary for biological function.The vitamin K-dependent proteins are synthesized with an NH2-terminal extension. The region distal to the NH2-terminus of the mature protein is a prototypic signal sequence while the proximal region is a propeptide with homology among the vitamin K-dependent proteins. The boundary between the pre and pro sequences has been established for factor IX by analysis of three naturally occurring factor IX mutants factor IX Cambridge factor IX Oxford-3 and factor IX San Dimas, in which processing is incomplete.For human factor IX the propeptide extends from residue -18 to -1. The homology among the propeptides of vitamin K-dependent proteins suggests that the propeptide may designate adjacent gamma-carboxyglutamic acids for carboxylation. To test this hypothesis alterations in sequence were introduced into the propeptide region of human factor IX cDNA by oligonucleotide directed site specific mutagenesis.Mutated genes were expressed in Chinese hamster ovary cells. Rapid and efficient isolationof the mutant proteins by immunoaffinity chromatography permitted detailed analysis of the mutants on quantities of protein easily obtainable at low expression levels. The extent of gamma-carboxylation was assessed by the ability of the mutant proteins to interact with conformation specific antibodies directed against the gamma-carboxyglutamic acid-dependent metal stabilized native structure of factor IX as well as by direct amino acid analysis. Unmodified recombinant factor IX contained, on average, 9 gamma-carboxyglutamic acid residues, as compared to 12 for plasma factor IX. About 70% of the recombinant wild type factor IX bound to the conformation specific antibodies. Deletion of the propiece or point mutations at residues -10 or -16 led to secretion of uncarboxylated factor IX unreaotive with antibodies specific for the native structure but with the NH2-terminus of mature factor IX. In order to assess the universality of these observations we have recently cloned human prothrombin cDNA and expressed the gene in the same Chinese hamster ovary cell system used for factor IX. In contrast to factor IX, at low levels ofexpressionof the prothrombin gene, the prothrombin is fully carboxylated relative to a plasma prothrombin standard.The recombinant prothrombin exhibits the same specific clotting activity as plasma derivedprothrombin and is fully native as evaluated by conformation specific antibodies. At high levels of expression the capacityof the cells to carboxylate prothrombin can be exceeded leading to secretion of under carboxylated prothrombin. However, the absolute amount of fully carboxylated prothrombin that can be produced in this system appears to be a least fivefold greater that the absolute amount of highly carboxylated factor IX that can be synthesized.The elimination of carboxylation observed upon mutation of the propiece of factor IX suggest that the propiece contains a recognition element required for carboxylation of the protein. Assignment of a functional role to the propiece of factor IX represents the first determination of function for any pro sequence. It is anticipated that extension of these studies to prothrombin will demonstrate that this recognition signal is used by all the members of this class of proteins. In order to determine if the propiece is sufficient to designate a protein for gamma-carboxylation we are currently constructing chimeric proteins incorporating the propieceof prothrombin into the cDNA of normally uncarboxylated proteins.

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