Calcium and hydroxyapatite binding site of human vitronectin provides insights to abnormal deposit formation

Abstract

The human blood protein vitronectin (Vn) is a major component of the abnormal deposits associated with age-related macular degeneration, Alzheimer’s disease, and many other age-related disorders. Its accumulation with lipids and hydroxyapatite (HAP) has been demonstrated, but the precise mechanism for deposit formation remains unknown. Using a combination of solution and solid-state NMR experiments, cosedimentation assays, differential scanning fluorimetry (DSF), and binding energy calculations, we demonstrate that Vn is capable of binding both soluble ionic calcium and crystalline HAP, with high affinity and chemical specificity. Calcium ions bind preferentially at an external site, at the top of the hemopexin-like (HX) domain, with a group of four Asp carboxylate groups. The same external site is also implicated in HAP binding. Moreover, Vn acquires thermal stability upon association with either calcium ions or crystalline HAP. The data point to a mechanism whereby Vn plays an active role in orchestrating calcified deposit formation. They provide a platform for understanding the pathogenesis of macular degeneration and other related degenerative disorders, and the normal functions of Vn, especially those related to bone resorption.

Calcium-induced environmental adaptability of the blood protein vitronectin

“…As predicted by NMR (24), the m1-bound Ca 2+ is not deeply buried within the channel, and locates 0.4-0.8 Å further away from Cl − than m1-bound Na + (Fig. 1C-F; Fig.…”

“…Previously, we showed that Ca 2+ binds Vn HX with a dissociation constant in the range of 27 μM (24) and, since this value is approximately ten times higher than the blood concentration of Vn (2.5-5 μM) (1-3) and thirty times lower than the total blood concentration of Ca 2+ (1.3-1.5 mM) (26), we concluded that Vn is Ca 2+ -associated in vivo . Using NMR, we showed that Ca 2+ enhances conformational exchange of Vn on the ms time scale, and by monitoring protein unfolding with DSF and an environment-sensitive fluorescent dye, we showed that Ca 2+ markedly increases the unfolding transition temperature of Vn and, thus, protects the structure from thermal denaturation.…”

“…Previously, we determined the three-dimensional structure of the hemopexin-like (HX) domain of Vn (23). We showed that it specifically binds both soluble Ca 2+ and mineralized hydroxyapatite [Ca 10 (PO 4 ) 6 (OH) 2 ] – the major component of pathological calcified protein-lipid deposits – acquiring appreciable thermodynamic stability in the process (24). Here we describe the structural mechanism for this Ca 2+ -dependent effect, and show that it also confers Vn HX with mechanical stability against high shear stress, such as that of the vasculature.…”

Vitronectin and Its Interaction with PAI-1 Suggests a Functional Link to Vascular Changes in AMD Pathobiology

“…The delineation of this pathway could facilitate the development of diagnostic, preventive, or therapeutic treatment strategies. Vitronectin has been recognized previously as a promising therapeutic target to regulate complement activation and inflammation [ 111 ] and to prevent ectopic deposits associated with AMD [ 19 ]. Addressing the need to regulate and not simply inhibit pathological angiogenic events in NV-AMD [ 112 ], therapeutic modulation of vitronectin and PAI-1 protein expression, either through genetic or pharmacological approaches, could contribute to the restoration of vascular homeostasis and, thus, help to prevent or treat vascular abnormalities in late stage AMD.…”

“…Interactions with lipids, glycosaminoglycans, collagens, plasminogen activator inhibitor-1 (PAI-1) and adhesion receptors make vitronectin a multifunctional protein involved in the regulation of a variety of cellular events that are also known to be affected by AMD, such as ECM deposition, cell adhesion and migration, immune response, angiogenesis and fibrinolysis [ 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 ]. Notably, vitronectin has repeatedly been identified as the main component and coordinating factor in the formation of AMD-associated retinal deposits [ 16 , 17 , 18 , 19 ].…”