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Author et al.
still attached to the carboxylated protein (Figure 5). These mutant proteins cannot be recognized by a calcium- dependent conformational specific antibody, suggesting a loss of function.41 However, contradictory results exist on whether these mutations affect FIX carboxylation. It has been shown that proFIX purified from hemophilia patients, carrying mutations at -4, is properly carboxylat- ed,30,34 others have shown that proFIX with a mutation at -4 in a hemophilia patient is only partially carboxylated.35 This discrepancy may result from the dif- ferent approaches used in determining the extent of car- boxylation. Nevertheless, it has been confirmed that these mutations interfere with propeptide cleavage, and there- fore affect the function of FIX (mainly by destabilizing the calcium-binding conformation),34 impair FIX binding to the lipid membrane, and affect FIX activity for coagula- tion.29,41
In contrast to numerous studies which indicate that the propeptide of BGP is not required for GGCX binding and its carboxylation,14-16 results from this study show that the removal of BGP propeptide dramatically decreased BGP carboxylation (Figure 6D). This result is consistent with previous studies showing that a non-covalently attached propeptide stimulates BGP carboxylation and a covalently
attached propeptide directs complete carboxylation of BGP.42,43 In addition, consistent with previous studies, our results show that BGP propeptide cannot direct coagula- tion factor carboxylation (Figures 1B and 2). Together, these results suggest that BGP may have a different mech- anism for carboxylation than coagulation factors. This supports observations that vitamin K intake affects the carboxylation of coagulation factors and of BGP in differ- ent ways.44-47 Further studies are ongoing to clarify the mechanistic differences in the carboxylation of a variety of VKD proteins. Results from these studies will continue to provide insights into efficiently controlling one physiolog- ical process without affecting the other.
Acknowledgments
The authors would like to thank Dr. Paul Bajaj from the University of California, Los Angeles for helpful discussions and for providing the Ca2+-dependent monoclonal antibody against carboxylated Gla domain of protein C.
Funding
This work is supported by grant HL131690 from the National Institutes of Health (to JKT and DWS).
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