Proteolytic formation and properties of a fragment of protein C containing the gamma-carboxyglutamic acid rich domain and the EGF-like region.
The function of the epidermal growth factor (EGF) like domains in the vitamin K dependent plasma proteins is largely unknown. In order to elucidate the function of these domains in protein C, we have devised a method to isolate the EGF-like region from the light chain connected to the NH2-terminal region, containing the gamma-carboxyglutamic acid (Gla) residues. This was accomplished by tryptic cleavage of protein C that had been reversibly modified with citraconic anhydride to prevent cleavage at the lysine residue (in position 43) that is located between the two regions. The isolated fragment consists of residues 1-143 from the light chain of protein C connected by a disulfide bond to residues 108-131 from the heavy chain. Upon Ca2+ binding to the isolated Gla-EGF fragment from bovine protein C, the tryptophan fluorescence emission was quenched in a manner indicating binding to at least two classes of binding sites. These were presumably the Gla-independent Ca2(+)-binding site located in the EGF-like region and the lower affinity sites in the Gla region. A comparison with the tryptophan fluorescence quenching that occurred upon Ca2+ binding to the separately isolated EGF-like and Gla regions suggested that the EGF-like region influenced the structure and Ca2+ binding of the Gla region. The isolated Gla-EGF fragment functioned as an inhibitor of the anticoagulant effect of activated protein C in a clotting assay, whereas no inhibition was observed with either the Gla region or the EGF-like region.
Ohlin
A K
AK
Department of Clinical Chemistry, University of Lund, Malmö General Hospital, Sweden.
Björk
I
I
Stenflo
J
J
D015055:1-Carboxyglutamic Acid; D000818:Animals; D001777:Blood Coagulation / Q000502:physiology; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D004815:Epidermal Growth Factor; D015394:Molecular Structure; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000302:isolation & purification / Q000502:physiology; D011485:Protein Binding; D011486:Protein C / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000502:physiology; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
D010446:Peptide Fragments; D011486:Protein C; D015055:1-Carboxyglutamic Acid; D004815:Epidermal Growth Factor; D002118:Calcium