PB0947 - Glu330Gly alters N-linked glycosylation at Asn-329 in Protein C and causes thrombosis

Background: Protein C (PC) deficiency is one of the important risk factors for venous thrombosis. We found a new PC mutation Glu330Gly (E330G) in two unrelated thrombosis patients. E330 is involved in the N-linked glycosylation consensus sequence at Asn329 site.

Aims: We would like to explore how this mutation affects the N329 glycosylation and anticoagulant function of PC.

Methods: We constructed E330G-PC plasmid, expressed the mutant in mammalian cells and purified the recombinant PC with ion affinity and exchange chromatography. SDS-PAGE of PC before and after treatment with PNGase F were used to analyze the glycosylation alteration. The initial rate of PC activation by thrombin was monitored in both absence and presence of soluble thrombomodulin. The anticoagulant properties of APC were analyzed through clotting time (aPTT) assay.

Results: SDS-PAGE showed that the size of alpha-band to E330G-PC was different from that of WT-PC, indicating that E330G mutation altered the N-linked glycosylation of PC. Immunofluorescence assays showed partial retention of E330G-PC in endoplasmic reticulum of cells. Further analysis of the initial rate of PC activation illustrated that E330G-PC was better than WT-PC. But enzyme kinetic analysis indicated that E330G-APC adversely affected the reactivity of the catalytic pocket. Furthermore, E330G-APC showed defective anticoagulant activity in aPTT assay.

Conclusion(s): E330G mutation affected the process of N-linked glycosylation at Asn329, which resulted in the structural abnormality of PC and caused the partial retention of E330G-PC in the endoplasmic reticulum of cells and finally decreased the expression. At the same time, E330G-APC adversely affected the reactivity of the catalytic pocket and anticoagulant activity. So carrying E330G mutation in PROC was the risk factor for venous thrombosis in these two patients.