Gamma-Carboxylation (γ-carboxylation)
Bone formation and vitamin K are closely connected biochemically. To understand this, we first need to explore the core concepts of Gla proteins and γ-carboxylation. Simply put, Gla proteins are specialized proteins whose purpose is to bind calcium ions (Ca²⁺). In order to bind calcium effectively, these proteins must undergo a specific chemical modification called γ-carboxylation, which absolutely requires vitamin K as a cofactor. γ-carboxylation, which converts proteins into Gla proteins, plays roles not only in bone formation but also in blood coagulation and regulation of vascular calcification. The mechanism of the widely known anticoagulant drug warfarin also involves inhibiting this very γ-carboxylation reaction. Given its pivotal role in diverse biological processes, it is important to take a closer look at this mechanism.
Let us revisit the basic structure of amino acids, the building blocks of proteins. In simplified terms, amino acids are linked like beads through peptide bonds to form polypeptide chains. These peptides then fold and twist into complex three-dimensional structures to become functional proteins. Every amino acid has a central alpha (α) carbon bonded to four substituents: an amino group (-NH₂), a carboxyl group (-COOH), a hydrogen atom (H), and a side chain (R-group). The amino group, carboxyl group, and hydrogen constitute what is called the backbone. Each amino acid has a unique side chain that determines its characteristics. Peptide bonds form through a dehydration condensation reaction between the amino group of one amino acid and the carboxyl group of the next, releasing a water molecule (H₂O). Thus, the carboxyl group in the backbone is not available for other reactions.
Now, let us look at glutamic acid—the only amino acid that undergoes γ-carboxylation. This reaction removes a hydrogen from the γ-carbon (the third carbon from the alpha carbon) and adds CO₂ to form an additional carboxyl group. Only glutamic acid has the right structure for this, including the presence of the γ-carbon and the requisite hydrogen. Glutamic acid originally has two carboxyl groups, but one is involved in peptide bonding, while the other ionizes into COO⁻ under physiological pH. This single COO⁻ is insufficient for binding tightly to divalent calcium ions (Ca²⁺). Two carboxyl groups are necessary to firmly grasp calcium—like claws. The post-translational modification that creates this second carboxyl group is γ-carboxylation.
In the structure of the amino acid glutamic acid, the carbon atoms extending from the central alpha (α) carbon of the backbone are referred to sequentially as the beta (β) and gamma (γ) carbons. The second carboxyl group is added at the γ-position. The enzyme that catalyzes this reaction is gamma-glutamyl carboxylase, and it requires vitamin K. Through this γ-carboxylation process, glutamic acid (Glu) is converted into gamma-carboxyglutamic acid (Gla). The resulting Gla structure chelates calcium ions using its two negative charges, enabling the protein to carry out its physiological function.
Roles of Gla Proteins (γ-carboxyglutamate proteins)
Gla proteins are those that contain Gla residues formed through the γ-carboxylation of glutamic acid. But what functions do these Gla proteins serve? They are involved in three major physiological processes: blood coagulation, prevention of soft tissue calcification (e.g., in blood vessels), and bone formation—all of which are critically important.
In Blood Coagulation
As explained in detail in previous discussions on hemostasis, both anticoagulant proteins (C, S, and Z) and procoagulant factors (Factors II, VII, IX, and X) require binding to Ca²⁺ in order to function properly. For example, coagulation factors must adhere to the phospholipid membranes of platelets at injury sites to perform their role. This binding is facilitated by the Gla residues in these factors, which use their double negative charges to strongly chelate Ca²⁺. This enables them to localize precisely at the injury site and execute the coagulation process effectively.
In Bone Formation
What does the presence of Gla proteins mean for bone formation? After osteoclasts resorb bone, osteoblasts secrete new bone matrix. One crucial organic molecule secreted by osteoblasts is osteocalcin, composed of 49 amino acids, three of which are glutamic acid. These three residues must undergo γ-carboxylation to become carboxylated osteocalcin (cOC), which can then bind strongly to calcium. This calcium is subsequently transferred into the bone matrix, aiding in hydroxyapatite crystal formation. Only γ-carboxylated osteocalcin can attract calcium ions effectively to form mineralized bone matrix.
In Inhibition of Vascular Calcification
While osteocalcin naturally helps trap calcium in the bone matrix, abnormal calcium deposition in soft tissues like blood vessels, heart valves, kidneys, or lungs can be dangerous. Vascular calcification is a pathological process in which hydroxyapatite precipitates in the vessel walls (especially in the tunica media and intima), reducing elasticity and leading to hypertension, myocardial infarction, heart failure, arrhythmias, sudden cardiac death, and stroke. It can occur in arteries of all sizes and is most prominent in the intima and media.Two important Gla proteins—MGP (matrix Gla protein) and GRP (Gla-rich protein)—help inhibit such calcification.
Vascular smooth muscle cells, which control the contraction and growth of vessel walls, are capable of phenotypic switching in response to environmental changes. Under pathological conditions like inflammation, hyperphosphatemia, or hypercalcemia, these cells can transform into osteoblast-like cells and begin depositing calcium in vessels. Fortunately, they also secrete MGP and GRP, which act as intrinsic defense mechanisms against calcification. These Gla proteins must undergo γ-carboxylation to bind calcium or hydroxyapatite crystals and prevent their growth. GRP, as its name suggests, can contain up to 16 glutamic acid residues, making its calcification-inhibiting effect very powerful when fully carboxylated. Cartilage also secretes GRP to prevent abnormal calcification.
Vascular calcification can severely impair blood vessel elasticity, disrupt blood pressure regulation, and compromise heart function and systemic circulation. Therefore, the roles of MGP and GRP in maintaining vascular health cannot be overstated.
Having understood the mechanism and significance of γ-carboxylation, it is now essential to focus on vitamin K. Without vitamin K, γ-carboxylation cannot occur. For this reason, all Gla proteins are classified as vitamin K-dependent proteins. The next step is to examine vitamin K in detail and explore the γ-carboxylation process itself.
Vitamin K
Let’s begin with some general facts about vitamin K. It was first discovered for its role in preventing bleeding, hence the name vitamin K, derived from the Danish word “Koagulation,” meaning coagulation. Vitamin K is a fat-soluble vitamin and an essential nutrient that the human body cannot synthesize on its own. Although gut microbiota produce some vitamin K (though strictly speaking, they are not “us”), the majority must be obtained from food or supplements. All forms of vitamin K share a central ring structure called naphthoquinone. Based on the carbon length, saturation level, and number of repeating isoprene units on the side chain attached to the 3rd carbon, vitamin K can be classified into about 11 types. However, the three most commonly referenced types are K1, K2, and K3.
Structure and Common Types: K1, K2, K3
These all share a core naphthoquinone (2-methyl-1,4-naphthoquinone) structure. That is, the naphthalene ring, formed by the fusion of two benzene rings, carries ketone groups (C=O) at the 1st and 4th carbon, and a methyl group (CH₃) at the 2nd carbon. The redox reactions involving electron transfer in this common structure are key to the function of vitamin K as a coenzyme in the γ-carboxylation reaction. This portion forms the physiologically active backbone of all vitamin K types.
Vitamin K1 (phylloquinone) is found abundantly in plants, particularly green leafy vegetables. Its side chain includes a phytol group, a lipid produced by plants that is also a component of chlorophyll, thereby closely related to photosynthesis. It is primarily utilized in the liver for blood clotting.
Vitamin K2 (menaquinone) is derived from vitamin K1 by replacing the phytol group with isoprene units. Depending on the number of isoprene units, it is further categorized into MK-4, MK-7, etc. It is found in animal products (eggs, meat), cheese, and fermented foods such as natto and cheonggukjang.
Vitamin K3 (menadione) is a synthetic precursor of vitamin K that lacks a side chain and is water-soluble. Although it was previously used to treat vitamin K deficiency, concerns about its toxicity have led to its discontinuation in humans; it is now primarily used in animal feed.
Vitamin K and γ-Carboxylation
γ-Carboxylation of proteins is essential for physiological processes like hemostasis, coagulation, bone formation, and inhibition of vascular calcification. Vitamin K serves as a crucial coenzyme in this process. Although only a small amount of vitamin K exists in the body, it is efficiently recycled after functioning as a coenzyme. This recycling mechanism allows vitamin K to be reused repeatedly. Warfarin, a medication, inhibits this recycling process, thereby suppressing γ-carboxylation and inhibiting the function of blood clotting factors. Let’s look in more detail at how dietary vitamin K is converted into its active form in the liver and participates in γ-carboxylation before being recycled.
Both K1 and K2 obtained from food exist in the quinone form (phylloquinone, menaquinone), which must be converted to the quinol form to be biologically active. This reduction reaction converts the ketone (which forms double bonds due to electron deficiency) to a hydroxyl group (OH), resulting in a hydroquinone structure. Only the vitamin K in this active form can participate in γ-carboxylation.
This reduction is facilitated by enzymes such as vitamin K reductase (VKORC1) or NAD(P)H-dependent reductases, which provide two electrons and two protons (2e⁻ + 2H⁺). Once converted to quinol, vitamin K acts as an electron donor in γ-carboxylation, providing electrons to the γ-position carbon so that a proton is displaced and replaced with CO₂, forming another carboxyl group.
During this process, the hydroquinone form is oxidized and converted to a three-membered epoxide ring with oxygen atoms at carbon positions 2 and 3. Then, vitamin K epoxide reductase (VKOR) reduces this back to the quinone form. This form is again reduced to hydroquinone, completing the vitamin K cycle.
Vitamin K1 constitutes about 90% of dietary vitamin K intake. However, it has low biological activity, only about 20% absorption, and a short half-life of 1–2 hours, leading to rapid clearance from the liver and circulation—thus requiring high intake. In contrast, MK-7 has a half-life of 2–3 days and is effective throughout the body even in small amounts.[1]
Being fat-soluble, vitamin K is absorbed in the intestine, transported to the liver, and circulated systemically. While K1 has a short half-life and remains mostly in the liver, K2, with a longer half-life, is more effective in systemic processes such as inhibiting vascular calcification and aiding in bone formation through γ-carboxylation. However, since clotting factors are produced in the liver, vitamin K1 is sufficient for their γ-carboxylation.
Warfarin (a type of coumarin), a widely used anticoagulant, blocks the recycling of vitamin K in the liver, preventing γ-carboxylation of clotting factors. This leads to vitamin K deficiency, affecting not only coagulation but also other vitamin K-dependent proteins. In patients who took coumarin for over 10 years, higher levels of uncarboxylated MGP (matrix Gla protein) were observed, suggesting systemic vitamin K deficiency and progression of vascular calcification. [2] Some studies suggest that sufficient intake of vitamin K may help prevent arterial calcification and coronary heart disease in the elderly. [3]
Low Blood Levels of ucOC, Vitamin K, and Osteoporosis
From the perspective of osteoporosis, γ-carboxylation of osteocalcin—a bone-forming protein secreted by osteoblasts—is crucial. When osteocalcin is not sufficiently carboxylated due to low vitamin K intake, the proportion of undercarboxylated osteocalcin (ucOC) increases, reducing calcium-binding capacity in bone. High blood ucOC levels are predictive of low bone mineral density and fracture risk.
Elevated ucOC levels may serve as a biochemical marker of vitamin K deficiency or impaired bone formation. While this may also result from increased bone resorption by osteoclasts, both scenarios indicate negative implications for bone health—either increased bone resorption or decreased bone formation, leading to bone loss.
Other Functions of Undercarboxylated Osteocalcin (ucOC)
Interestingly, ucOC also performs several functions when not carboxylated. In addition to vitamin K deficiency, carboxylated osteocalcin (cOC) can be decarboxylated in the acidic, low-pH environment created by osteoclasts during bone resorption, reverting back to ucOC. In this form, it loses its bone affinity and is released into the bloodstream.
While cOC binds tightly to calcium and plays a central role in bone formation and mineralization, ucOC acts like a hormone, traveling through circulation and influencing various tissues. Recent studies suggest ucOC may function as a regulatory protein in glucose metabolism, testosterone synthesis, muscle mass, and cognitive function.
The hypothesis that ucOC improves insulin resistance and glucose metabolism has gained attention. It proposes that ucOC acts hormonally to stimulate β-cell proliferation and insulin secretion in the pancreas, while also inducing adiponectin production in adipocytes, thereby enhancing insulin sensitivity. [4] Insulin, in turn, increases osteoblast differentiation and osteocalcin secretion, forming a loop between bones (ucOC) and the pancreas (insulin) to regulate glucose metabolism and improve insulin resistance. Though this remains a hypothesis requiring further research and validation, these effects are specific to osteocalcin that is secreted from bone in its undercarboxylated form.
Additionally, ucOC has been reported to influence brain development and function. In experiments with mice, those deficient in ucOC exhibited increased anxiety, reduced memory, and underdeveloped hippocampi. [5] Osteocalcin appears to regulate the synthesis of neurotransmitters like dopamine, serotonin, and norepinephrine, and may improve memory loss and neurodevelopmental impairments. Some studies in older women have shown a correlation between high osteocalcin levels and better cognitive function, though others have not, indicating the need for more research.
There is also interest in the relationship between ucOC and sex hormones. ucOC binds to receptors (Gprc6a) on Leydig cells in the testes, activating genes involved in testosterone biosynthesis. [6] Low ucOC levels are associated with reduced testosterone, smaller reproductive organs, and decreased sperm count, but supplementation with ucOC appears to mitigate these effects. ucOC may also be linked to exercise adaptability. With aging, osteocalcin levels decline, leading to reduced muscle mass and exercise capacity. However, injection of osteocalcin (ucOC) has shown potential to improve these outcomes.
[References]
[1] Vitamin K2 for bone health
[2] Vitamin K Dependent Proteins and the Role of Vitamin K2 in the Modulation of Vascular Calcification: A Review
DOI 10.5001/omj.2014.44
[3] Dietary Intake of Menaquinone Is Associated with a Reduced Risk of Coronary Heart Disease: The Rotterdam Study1
https://doi.org/10.1093/jn/134.11.3100
[4] Osteocalcin: The extra-skeletal role of a vitamin K-dependent protein in glucose metabolism
https://doi.org/10.1016/j.jnim.2017.01.001
[5] Osteocalcin—A Versatile Bone-Derived Hormone
https://doi.org/10.3389/fendo.2018.00794
[6] The “soft” side of the bone: unveiling its endocrine functions
https://doi.org/10.1515/hmbci-2016-0009
