Functional role of O-linked and N-linked glycosylation sites present on the activation peptide of factor X.

Abstract

There are two O-linked and two N-linked glycosylation sites on the activation peptide of factor X (FX) involving residues Thr-17, Thr-29, Asn-39 and Asn-49. The purpose of this study was to explore the contribution of carbohydrates of the FX activation peptide to zymogen recognition by physiological activators. The putative glycosylation sites were substituted individually or in combinations with Ala and mutants were expressed in mammalian cells. The entire activation peptide up to the P3 residue was deleted in another construct. It was discovered that activation of zymogen mutants by both FVIIa and FIXa on negatively charged phospholipid vesicles has been improved 2-40-fold independent of a cofactor. These mutants were activated with slightly lower catalytic efficiency (k(cat)/K(m)) by FVIIa in the extrinsic Xase complex, though both K(m) and k(cat) constants for mutants were elevated. With the exception of approximately 3-fold improvement in the activation of N49A, the catalytic efficiency of FIXa toward mutants was decreased 2-5-fold in the intrinsic Xase complex. The carbohydrate chains of the FX activation peptide play an important role in restricting the specificity of zymogen recognition by both FVIIa and FIXa, thereby preventing the cofactor-independent activation of FX by these proteases. On the other hand, the carbohydrates contribute to the cofactor-dependent recognition of the zymogen by both extrinsic and intrinsic Xase complexes.