Scientific Report 2003-2004 - Cleveland Clinic Lerner Research ...

THE BERKNERLABORATORYPOSTDOCTORAL FELLOWSWen Qian, Ph.D.Mark Rishavy, Ph.D.TECHNICAL ASSOCIATEKevin HallgrenKathleen L. Berkner, Ph.D.The Department of Molecular CardiologyInsight into Carboxylation of Vitamin K-DependentProteins Offers Hope for DesigningCoagulation Therapies, Hemophiliac HelpWe are interested in how vitamin K-dependent (VKD) proteins are carboxylated to render them active and in therole vitamin K plays in different biologicalsystems. Carboxylation involves the conversionof clusters of Glu residues to gamma-carboxylatedGlu residues in VKD proteins, using vitaminK as a cofactor. This modification generatescalcium-binding modules in VKDproteins, allowing them to exert theireffects in hemostasis, calcium homeostasis,bone development, cellular proliferation,vascular remodeling and signaltransduction. The enzyme that modifiesthe VKD proteins, gamma-carboxylase,resides in the endoplasmic reticulum,and carboxylation occurs during thesecretion of VKD proteins by amechanism that is poorly understood.Our recent work has revealed insightsinto the molecular mechanism forconversion of Glu residues to gammacarboxylatedGlu residues. In studiesthat represent a breakthrough formapping functional residues of thecarboxylase, we identified the active siteand implicated a mechanism ofsubstrate-regulated catalysis. Thesestudies are a critical first step towarddesigning superior anticoagulants thatdirectly target the carboxylase, ratherthan the indirect and nonspecific onescurrently in use. VKD proteins requiremultiple Glu to gamma-carboxylatedGlu conversions for their activity; forexample, 12 such conversions occur in thehemostatic VKD protein factor IX. We recentlyshowed that the carboxylase uses a processivemechanism to effect the multiple carboxylations,Pudota, B.N., Miyagi, M., Hallgren, K.W., West, K.A., Crabb, J.W., Misono, K.S., andK.L. Berkner (2000) Identification of the vitamin K-dependent carboxylase active site:Cys-99 and Cys-450 are required for both epoxidation and carboxylation. Proc. Natl.Acad. Sci. USA 97:13033-13038.Berkner, K.L. (2000) The vitamin K-dependent carboxylase. J. Nutr. 130:1877-1880.Stenina, O., Pudota, B.N., McNally, B.A., Hommema, E.L., and K.L. Berkner (2001)Tethered processivity of the vitamin K-dependent carboxylase: factor IX is efficientlymodified in a mechanism which distinguishes Gla’s from Glu’s and which accounts forcomprehensive carboxylation in vivo. Biochemistry 40:10301-10309.Pudota, B.N., Hommema, E.L., Hallgren, K.W., McNally, B.A., Lee, S., and K.L. Berkner(2001) Identification of sequences within the gamma-carboxylase that represent a novelcontact site with vitamin K-dependent proteins and that are required for activity. J. Biol.Chem. 276:46878-46886.Hallgren, K.W., Hommema, E.L., McNally, B.A., and K.L. Berkner (2002) Carboxylaseoverexpression effects full carboxylation but poor release and secretion of factor IX:implications for the release of vitamin K-dependent proteins. Biochemistry 41:15045-15055.i.e., all modifications occur as a consequence of asingle binding event. We are now determininghow processive carboxylation is accomplished,including whether a novel, second site of contactbetween VKD proteins and the carboxylase wediscovered is important for processivity.Our studies showing efficient processivityin vitro are of interest with regard to the poorefficiency of carboxylation that is sometimesobserved in vivo. Although normal physiologicalconditions result in full VKD protein carboxylation,there are instances when carboxylation issaturated, for example, in the expression ofrecombinant VKD proteins in mammalian cells.At low production levels, fully carboxylatedproteins are secreted, but at increased levels,undercarboxylated proteins are observed. Thislimitation has confounded the production ofVKD proteins, many of which have therapeuticpotential. The reason for such saturation is notunderstood, in part because the events involvedin VKD protein carboxylation and processing bythe secretory machinery are poorly defined. VKDprotein carboxylation places exceptional demandson the quality control mechanisms that facilitatesecretion: protein assembly is unusual because anenzyme-substrate complex is formed, and thecomplex must achieve full VKD proteincarboxylation. In addition, multiple VKDproteins (e.g., at least 10 in liver) engage onecarboxylase, creating a potentially competitivestate. To understand how this process is normallyaccomplished with such remarkable fidelity, wehave begun analyzing the intracellular events thatoccur during VKD protein carboxylation andsecretion. Our recent in vivo studies show, forexample, that saturation of carboxylation is notdue to limiting amounts of carboxylase. Theysuggest that the limitation in expressing fullycarboxylated proteins is due to vitamin Kbioavailability, and we are currently investigatingthis possibility. We are also determining howVKD protein carboxylation is regulated in vivo.We discovered that the carboxylase itself is aVKD protein that undergoes autocarboxylation.Preliminary studies indicate that carboxylasecarboxylation may function to downregulate theenzyme when VKD substrate levels are low.120