BBB seminar: Gary L. Nelsestuen
Enhanced vitamin K-dependent proteins for therapy: Contribution of biophysical concepts to a practical outcome
Gary L. Nelsestuen
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA
Vitamin K is essential for biosynthesis of gamma-carboxyglutamic acid (Gla), a modification of 9 to 13 glutamate residues in the N-terminal region of a larger family of proteins that includes protein C, factor IX and factor VII. Gla is needed for the calcium and membrane binding functions of these proteins and is essential to their action in vivo. These proteins were targeted for special study as they are current pharmaceutical agents used to treat bleeding disorders such as hemophilia, with other likely targets in development (factors IXa and VIIa), or coagulation disorders such as sepsis (activated Protein C). The proteins also show lower affinity for membranes than other proteins of this family, suggesting the potential to enhance their function. The practical challenge for use of these agents is the cost of producing proteins in special animal cell lines that results in drug costs of tens of thousands of dollars per treatment. This can run to hundreds and, in a few cases, a million dollars per year to provide hemostasis for some individuals. High cost also restricts the use of these reagents to life threatening situations. A very long-standing academic challenge has been to understand how the Gla-containing domain interacts with the membrane.
This seminar will show how the practical and academic challenges converged to generate site-directed mutants with enhanced membrane affinity. The mutations occur at positions that were not considered by popular opinion to be involved in membrane association. Free energy arguments can be used to suggest a direct role for the modified residues in membrane contact, but proof remains elusive. Most important has been that the proteins with high membrane affinity show higher functional activity. The increase can be up to 200-fold for some modifications of factor VIIa. Detection of enhanced function faced another theoretical challenge, however, as the reactions proceeded at the rate of particle collision in solution, resulting in the inability to detect enhanced function with 'off-the-shelf' coagulation reagents. Insight into collision-limited reactions provided assays that detected enhanced function. Chemical derivatives of active site-blocked factor VIIa, another inhibitor of coagulation, have also been produced with up to 2000-fold enhanced function. Overall, this rather extended study has ultimately resulted in a juncture of what may seem highly academic matters and practical outcomes to produce enhanced proteins for possible drug development.
Host: Eirik Søfteland, Department of Biomedicine
Professor Gary Nelsestuen, first holder of the Samuel Kirkwood Chair of Biochemistry and Director at The Center for Mass Spectrometry and Proteomics at the University of Minnesota, Minneapolis, USA, is a leading scientist in membrane-protein interactions and proteomics.
His proteomics work, in collaboration with several research groups in the USA and abroad, has recently focused on plasma profiling of individuals, identification of human neutrophil alpha-defensins in chronic lung allograft rejection and early plasma protein changes in sepsis.
For many years Gary Nelsestuen has also researched into vitamin K-dependent coagulation proteins to elucidate their membrane-binding biochemical properties. This has led to the generation of mutants with enhanced membrane affinity and potential for drug development.