DBP Functions
Vitamin D Metabolite Transport
The main role of DBP is the transport of vitamin D metabolites [17]. Steroid hormones are lipophilic and need to be carried by a protein to become soluble in the bloodstream. Therefore, after cutaneous synthesis, vitamin D is transported bound to DBP. In the liver, it is converted into 25(OH)D by the action of vitamin D 25-hydroxylase (CYP21R) and re-enters the bloodstream where it circulates once more, mainly bound to DBP. This is the metabolite measured to establish vitamin D status; however, it is not the active form. To be converted into 1,25(OH)2D in the kidney, the DBP-bound 25(OH)D needs to undergo endocytosis by the proximal tubular cells. The process is mediated by megalin, a large transmembrane protein, and facilitated by two others, cubilin and disabled-2. In the kidney, 1,25(OH)2D is synthesized by the action of CYP27B1. The active form is transported bound to DBP.
Fig. 1. Bioavailability of 25(OH)D. About 85% of Vitamin D metabolites circulate bound to DPB, and less than 1% of 25(OH)D is free. Albumin concentrations in blood are 130 times higher than DBP, and a significant amount of Vitamin D (10 to 15%) circulates bound to it, but because this binding is not so tight, both free and Albumin-bound Vitamin D are considered bioavailable.
Other tissues such as the placenta, and the mammary and parathyroid glands also express megalin; however, the role of megalin in DBP transport outside the kidney is less clear [17]. DBP-bound vitamin D may also be internalized by other tissues in a process that is megalin-independent, as is observed, for example, in lymphocytes [18]. Besides the bound forms, small amounts of free vitamin D metabolites also circulate and may enter the cells via diffusion.
Both 25(OH)D and 1,25(OH)2D circulate bound to DBP (85–90%) or to albumin (10–15%) or in the free form (<1%). DBP’s affinity for vitamin D metabolites is much greater than that of albumin [16]. DBP’s measured affinity constant for 25(OH)D is 7 × 108M–1 and for 1,25(OH)2D it is 4 × 107M–1, while ALB is 6 × 105 and 5.4 × 105M–1, respectively [17]. DBP binding reduces hepatic degradation of vitamin D metabolites, increasing the circulating half-life, and it limits the access of target cells to them [2].
Although the affinity for DBP is much greater than for ALB, ALB is much more abundant in serum (approximately 130-fold) and a significant amount of vitamin D metabolites (10–15%) binds to this protein. However, as the binding is not so tight, ALB-bound vitamin D is considered bioavailable. Therefore, although less than 1% of vitamin D metabolites are found free in the serum, 10–15% is considered bioavailable once it is not bound to DBP (Fig. 1) [17].
Absence of DBP has never been described in humans [1], suggesting that the protein is essential for human viability. Surprisingly, DBP knockout mice are healthy and fertile despite lower circulating levels of 25(OH)D and 1,25(OH)2D while on a vitamin D-replete diet [19]. However, on vitamin D-deficient diets, the mice briefly developed low phosphate, high PTH, and high alkaline phosphatase levels, signs of vitamin D deficiency [19]. In the animal model, vitamin D metabolites are more likely to bind to albumin but the mice are less effective in preventing urinary loss of vitamin D and more sensitive to vitamin D deficiency [19]. Although the absence of DBP in knockout mice decreases the circulating 1,25(OH)2D levels, this does not seem to influence the ability of the hormone to enter the cells and the biological actions in vivo [3]. Animal studies have also shown that DBP slows vitamin D actions in the intestines and other target tissues and protects vitamin D from degradation [16]. Therefore, DBP acts as a vitamin D modulator, protecting it from degradation, facilitating renal uptake, and reducing renal loss, and also limiting tissue bioavailability.
DBP circulates in the bloodstream at higher levels than vitamin D metabolites and less than 5% of the protein’s sterol binding sites are occupied [1, 12]. The molar excess suggests that DBP could act as a protective mechanism against vitamin D toxicity, but also that the protein is involved in other physiological functions.
Other DBP Functions
Actin Scavenger System
Actin is an important cytoskeletal protein, highly conserved in eukaryotic cells. Two main forms of the protein are described, a monomeric globular form (G-actin) and a linear polymeric form called F-actin. Once in the extracellular compartment, actin polymerizes in the filamentary form [2]. The polymer may cause coagulation cascade activation, vascular obstruction, and cellular dysfunction. Actin is released by tissue injury and cell death, and DBP is able to bind to it with high affinity and to prevent filament formation [18]. In conjunction with gelsolin, another serum protein, DBP forms an actin scavenger system. It is able to rapidly sequester free actin from circulation, and the major DBP polymorphisms have equal binding affinity. It is possible that the molar excess of the protein is related to this function [2].
Fatty Acid Transport
All the members of the albumin superfamily of binding proteins are able to transport free fatty acids (FFA). ALB has several low- and high-affinity binding sites for FFA, while DBP has a single high-affinity binding site. ALB is also much more abundant in the plasma, which results in DBP having only a contributory role. The binding of unsaturated FFA reduces DBP affinity for vitamin D metabolites, but this effect is not observed for saturated FFA [17]. These variations are a result of the specific conformational changes induced for each FFA in DBP [2].
DBP-Macrophage Activation Factor
DBP can be deglycosylated by T- and B-cell glycosidases and act as a macrophage activation factor (MAF), which is called DBP-MAF [17]. This activity was discovered when mouse peritoneal cells were stimulated with lysophosphatidylcholine, and it resulted in an increase in phagocytic activity [20]. Analysis revealed that this increase was associated with the deglycosylation of DBP by β-galactosidase and sialidase of the B and T cells, resulting in DBP-MAF. The different isoforms of DBP show variable susceptibility to deglycosylation. This feature could explain the association of the isoforms with diseases like chronic obstructive pulmonary disease, bronchiectasis, and tuberculosis [17].