Impact of gamma-glutamyl carboxylase processivity on vitamin K-dependent protein modification and function in human health and disease

Dietary vitamin K is used by the gamma-glutamyl carboxylase to convert clusters of Glus to gamma-
carboxylated Glus (Glas) in vitamin K-dependent (VKD) proteins in virtually all tissues of the body. The first
VKD proteins identified were coagulation factors; however, the identification of nonhemostatic VKD proteins
has revealed additional roles, e.g. the regulation of calcification. Carboxylation activates VKD proteins by
generating a calcium-binding module required for their function, and a single gamma-glutamyl carboxylase
modifies all VKD proteins. Naturally occurring mutations in the carboxylase cause two diseases: vitamin K
clotting factor deficiency 1 that is associated with severe bleeding defects, and pseudoxanthoma elasticum-like
(PXE-like) that is associated with mild bleeding but excessive soft tissue calcification. How these carboxylase
mutations cause PXE-like was previously unknown. We studied two carboxylase mutations present in a PXE-
like patient. Analysis of a VKD clotting factor (factor IX) and a VKD protein that inhibits calcification (Matrix Gla
Protein) revealed partial carboxylation due to a defect in carboxylase processivity.
Processivity refers to the carboxylase remaining bound to a VKD protein until the multiple Glu residues are
carboxylated. We developed a novel assay to monitor processive carboxylation, and found that the wild type
carboxylase shields the VKD protein, i.e. limiting access of other VKD proteins into the active site until the VKD
protein is extensively carboxylated. In contrast, the PXE-like mutants allowed promiscuous access of VKD
protein substrates into the active site, resulting in the production of partially carboxylated VKD proteins. Our
studies also revealed that a single wild type carboxylase binds two VKD proteins at the same time. As tissues
express multiple VKD proteins thought to have widely different affinities, how full carboxylation of all VKD
proteins is achieved is an open question.
Our long-term goal is to understand how partial VKD protein carboxylation impacts human physiology.
Central questions are whether treatment with the anticoagulant warfarin, which limits VKD protein
carboxylation, generates partially carboxylated proteins, and whether warfarin evokes PXE-like phenotypes.
We will approach these questions using a combination of protein mapping and activity assays to determine
how partial carboxylation by PXE-like carboxylases impacts VKD protein function (Aim 1), determine whether
the carboxylation of a VKD protein is impacted by the presence of a different VKD protein (Aim 2), and
examine the consequence of warfarin therapy and a PXE-like mutant on VKD protein carboxylation and
function in vivo (Aim 3). Results from these studies will provide the first insights that link the extent of protein
carboxylation to different phenotypic outcomes.

Grant Number: 5R01HL152678-04
NIH Institute/Center: NIH
Principal Investigator: KATHLEEN BERKNER