A method for specific chemical modification of γ-carboxyglutamic acid residues in proteins

Wright, Steven F.; Bourne, Carolyn; Hoke, Randal A.; Koehler, Karl A.; Hiskey, Richard G.

doi:10.1016/0003-2697(84)90392-0

Cited by 19 publications

(8 citation statements)

References 15 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Following electrophoresis, the proteins were visualized by staining with Coomassie Brilliant Blue. The y-carboxyglutamic acid (Gla) content of factor VII/ VII a and factor VII gla-peptide preparations were determined according to Wright et al (7). NH2-terminal sequence analyses were carried out by automated Edman degradation using an Applied Biosystems 470A gasphase sequencer.…”

Section: General Methodsmentioning

confidence: 99%

The Role of Phospholipids and the Factor VII Gla-Domain in the Interaction of Factor VII with Tissue Factor

et al. 1992

View full text Add to dashboard Cite

SummaryWhether or not the factor VII Gla-domain is involved in the high-affinity interaction of factor VII and tissue factor via calcium-dependent interactions with surrounding phospholipids is unknown. To investigate this, we have purified the factor VII Gla-peptide (FVII-GP) from digested recombinant human factor VII a and assessed its effect on factor VII: tissue factor interactions. FVII-GP inhibited the activation of factor X by factor Vila in the presence of either soluble or cell surface tissue factor halfmaximally at 0.5 μM and 2.7 μM, respectively. However, FVII-GP failed to inhibit the specific binding of factor Vila to cell-surface tissue factor, and did not inhibit the ability of tissue factor to stimulate the amidolytic activity of factor Vila. Unrelipidated tissue factor apoprotein stimulated the amidolytic activity of factor Vila to the same extent as relipidated tissue factor apoprotein. These findings suggest that the factor VII Gla-domain does not directly interact with tissue factor, but rather is important for calcium binding and concomitant expression of other factor VII epitopes necessary for tissue factor recognition and binding. To test this hypothesis, we have prepared a monoclonal antibody against a putative factor VII epitope that participates in the interaction of factor VII with cell-surface tissue factor (peptide 195-206) and assessed its ability to bind to factor VII in the presence and absence of calcium. Binding of this monoclonal antibody (PW-4) to intact factor VII a was calcium-dependent and could be inhibited in a dose-dependent manner by peptide 195-206. The antibody reacted with Gla-domainless factor Vila, but only 37% as compared to intact factor Vila. In addition, PW4 as well as its Fab’ fragment, inhibited factor Vila binding to cell-surface tissue factor. These studies indicate that the factor VII Gla-domain does not provide structural elements that contribute to the formation of a stable factor VII/VII a-tissue factor binary complex. The factor VII Gla-domain appears to be necessary, however, in binding calcium ions and inducing a calcium-dependent conformational change in factor VII/VII a that expresses one or more neoepitopes that participates in the interaction of factor VII/VII a with the extracellular domain of tissue factor apoprotein.

show abstract

Section: General Methodsmentioning

confidence: 99%

The Role of Phospholipids and the Factor VII Gla-Domain in the Interaction of Factor VII with Tissue Factor

et al. 1992

View full text Add to dashboard Cite

show abstract

“…Hydrolyses of 4-vinylpyridine-treated samples were performed by 4 M methanesulfonic acid or 3 M mercaptoethanesulfonic acid at 110 °C for 24 h as described above. Gla residues were converted to 7-methyleneglutamic acid (Wright et al, 1984), and such samples were hydrolyzed in 3 M mercaptoethanesulfonic acid at 110 °C for 24 h.…”

Section: Methodsmentioning

confidence: 99%

Amino acid sequence and posttranslational modifications of human factor VIIa from plasma and transfected baby hamster kidney cells

Thim

Bjoern²,

Christensen³

et al. 1988

Biochemistry

290

186

View full text Add to dashboard Cite

Blood coagulation factor VII is a vitamin K dependent glycoprotein which in its activated form, factor VIIa, participates in the coagulation process by activating factor X and/or factor IX in the presence of Ca2+ and tissue factor. Three types of potential posttranslational modifications exist in the human factor VIIa molecule, namely, 10 gamma-carboxylated, N-terminally located glutamic acid residues, 1 beta-hydroxylated aspartic acid residue, and 2 N-glycosylated asparagine residues. In the present study, the amino acid sequence and posttranslational modifications of recombinant factor VIIa as purified from the culture medium of a transfected baby hamster kidney cell line have been compared to human plasma factor VIIa. By use of HPLC, amino acid analysis, peptide mapping, and automated Edman degradations, the protein backbone of recombinant factor VIIa was found to be identical with human factor VIIa. Neither recombinant factor VIIa nor human plasma factor VIIa was found to contain beta-hydroxyaspartic acid. In human plasma factor VIIa, the 10 N-terminally located glutamic acid residues were found to be fully gamma-carboxylated whereas 9 full and 1 partial gamma-carboxylated residues were found in the corresponding positions of the recombinant factor VIIa molecule. Asparagine residues 145 and 322 were found to be fully N-glycosylated in human plasma factor VIIa. In the recombinant factor VIIa, asparagine residue 322 was fully glycosylated whereas asparagine residue 145 was only partially (approximately 66%) glycosylated. Besides minor differences in the sialic acid and fucose contents, the overall carbohydrate compositions were nearly identical in recombinant factor VIIa and human plasma factor VIIa.(ABSTRACT TRUNCATED AT 250 WORDS)

show abstract

“…Several groups have used a modifying reaction that converts 7-carboxyglutamic acid (Gla) residues to 7-methyleneglutamic acid to study the role of the Gla amino acids. The modification of the Gla residues in prothrombin fragment 1 resulted in changes in its interaction with calcium (Wright et al, 1984). Modification of the Gla residues in factor X resulted in the inhibition of its activation by a snake venom activator (Sherrill et al, 1984), and conversion of the Gla residues in factor IX resulted in the loss of activity (Straight et al, 1984).…”

mentioning

confidence: 99%

Properties of chemically modified protein S: the effect of the conversion of .gamma.-carboxyglutamic acid to .gamma.-methyleneglutamic acid on functional properties

Walker¹

1986

Biochemistry

View full text Add to dashboard Cite

Protein S, the protein cofactor for activated protein C in the proteolytic inactivation of factor Va, was chemically modified with a mixture of morpholine and formaldehyde. This treatment resulted in the conversion of the gamma-carboxyglutamic acid (Gla) residues of this vitamin K dependent protein to gamma-methyleneglutamic acid. With a 10,000-fold molar excess of morpholine and formaldehyde over protein S it was found that between 10 and 11 Gla residues could be modified. The degree of modification was proportional to the concentration of the modifying reagents used. The modification of as few as two residues resulted in the 70% loss of activity. Calcium inhibited the modification of several residues. In the presence of 3.2 mM calcium ion, a derivative with 2.5 residues modified was prepared that appeared to have full activity. Modification of protein S resulted in the alteration of a number of its properties. The quenching of intrinsic fluorescence by calcium decreased. The quenching effect of terbium ions was also decreased. However, the modified protein and the native protein were equivalent when protein-dependent terbium fluorescence was measured. When modified, protein S would no longer bind to phospholipid vesicles. Finally, the ability of protein S to self-associate was decreased by modification. These findings suggest that the gamma-carboxyglutamic acid residues of protein S may play several roles in the maintenance of structure.

show abstract

A method for specific chemical modification of γ-carboxyglutamic acid residues in proteins

Cited by 19 publications

References 15 publications

The Role of Phospholipids and the Factor VII Gla-Domain in the Interaction of Factor VII with Tissue Factor

The Role of Phospholipids and the Factor VII Gla-Domain in the Interaction of Factor VII with Tissue Factor

Amino acid sequence and posttranslational modifications of human factor VIIa from plasma and transfected baby hamster kidney cells

Properties of chemically modified protein S: the effect of the conversion of .gamma.-carboxyglutamic acid to .gamma.-methyleneglutamic acid on functional properties

Contact Info

Product

Resources

About