Page 190 - 2020_08-Haematologica-web

Z. Hao et al.
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Figure 1. Effect of propeptides on coagulation factor carboxylation. (A) Sequence alignment of propeptides of vitamin K-dependent (VKD) coagulation factors and bone Gla protein (BGP), and their relative Kd values for gamma-glutamyl carboxylase (GGCX). Highly conserved residues are indicated by bold letters. The Kd values of different propeptides (adapted from Higgins-Gruber et al.23) are a relative measure of propeptides’ affinity for GGCX. The underlined Kd values correspond to the propeptides used in this study. (B) Carboxylation efficiency of the reporter-protein in HEK293 cells as directed by different propeptides. Reporter-proteins with differ- ent propeptides were transiently expressed in HEK293 cells, and the transfected cells were cultured in complete medium containing 11 mM vitamin K. The carboxy- lated reporter-protein in the cell culture medium was determined by ELISA.(C) Domain structure of the reporter-protein, factor IX gla-protein C (FIXgla-PC), with dif- ferent propeptides. The propeptide is proteolytically removed after carboxylation to form the mature protein. (D) Expression of uncarboxylated reporter-proteins with different propeptides as in (B). Reporter-proteins were transiently expressed in HEK293 cells and the transfected cells were incubated in complete medium contain- ing 5 mM warfarin. The total amount of uncarboxylated reporter-protein in the cell culture medium was determined by ELISA.
tein fusions were transiently expressed in COS-7 cells on cover- slips. For the localization of carboxylated reporter-proteins at dif- ferent cell organelles, fluorescent protein-tagged cell organelle marker protein fusions, and the corresponding marker protein fused with FIXgla, were transiently co-expressed in COS-7 cells. Transfected cells were cultured with 11 mM vitamin K for 48 h, fixed with 4% paraformaldehyde, permeabilized with 0.20% Triton X-100, and immuno-stained with corresponding antibod- ies. The cell nuclei were stained with 2 mM Hoechst 33342.
Results
Contribution of the propeptide to coagulation factor carboxylation
To explore the role of the propeptide on coagulation fac- tor carboxylation in a cellular milieu, we used our recently established mammalian cell-based assay.20 Propeptides with different affinities for GGCX (Figure 1A) were fused to the N-terminus of the chimeric reporter-protein FIXgla- PC (Figure 1C). These chimeric fusion proteins were tran- siently expressed in HEK293 cells, and the efficiency of their carboxylation was determined by ELISA.20 We select- ed propeptides of FX, FIX, PC, and BGP for this study. Results from our cell-based study show that BGP propep- tide cannot direct reporter-protein carboxylation (Figure 1B), which agrees with results from in vitro studies.14-16 In
addition, FIX propeptide is the most efficient propeptide for reporter-protein carboxylation, which is approximate- ly 10-fold higher than that of FX propeptide and 2.5-fold higher than PC propeptide. However, this cell-based result of carboxylation of propeptide attached protein substrate is different from the previous in vitro study showing that all of the propeptides stimulated carboxylation of the non- covalently linked substrate to a similar extent.23
To confirm that the significantly decreased production of carboxylated reporter-proteins, directed by the propep- tides of FX and BGP, was not due to the effect of the propeptide on reporter-protein expression, we determined the expression levels of the reporter-proteins in cell culture medium without carboxylation by feeding the cells with warfarin. The reporter-protein has similar expression lev- els when fused to different coagulation factor propeptides (FX, FIX, and PC) (Figure 1D). However, a significant amount of uncarboxylated reporter-protein was produced when BGP propeptide was used. Nevertheless, these results suggest that the significant difference in reporter- protein carboxylation, when fused to different propep- tides (Figure 1B), is not due to the effect of the propeptide on reporter-protein expression, but rather to its carboxyla- tion.
The unaffected expression and non-carboxylation char- acteristics of the reporter-protein, when fused to BGP propeptide, were confirmed further by immunofluores-
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haematologica | 2020; 105(8)