Reconsidering the role of albumin towards amorphous calcium phosphate-based calciprotein particles formation and stability from a physico-chemical perspective

The formation of inorganic nanostructures in living organisms is a fascinating phenomenon in which proteins have a crucial role in modulating the functional properties of the obtained biominerals [1]. Among them, the liver-derived glycoprotein Fetuin-A (Fet-A), known in humans as α2-HS-glycoprotein, circulates in serum at a concentration of ∼ 0.8 g/L [2] and is able to bind to amorphous calcium phosphate (ACP) clusters which spontaneously form in plasma and inhibit their growth, thus preventing the surge of ectopic calcifications. The role of Fet-A and other mineral chaperones, such as acidic proteins, is thus fundamental to scavenge calcium ions and nascent calcium phosphate crystals, limiting the ionic supersaturation that would provoke extra-skeletal calcifications [3], [4]. Fet-A/ACP complexes are referred to as CPPs (calciprotein particles) and consist of inorganic particles stabilized by Fet-A molecules, that bind calcium phosphate through their acidic residues present in an extended β-sheet in the D1 domain of the protein [1], [5]. When in the amorphous state, they are called primary calciprotein particles (CPP1). In time, CPP1 may ripen and crystallize, forming hydroxyapatite-based needle-like structures called secondary CPP (CPP2). Both CPP1 and CPP2 may induce endothelial dysfunction, although primary CPPs cause less deleterious effects than secondary CPPs [6], [7], [8], [9], [10], [11]. This biological pathway is especially important for patients affected by kidney diseases, which often suffer from imbalanced homeostasis of phosphate and, in some cases, calcium ions [11], [12]. Fet-A has recently been defined as a “mineral chaperone” protecting tissues from calcification and inflammation-related damage [13]; in addition, this glycoprotein displays other biological functions such as the regulation of bone metabolism and the involvement in insulin signaling pathway, also acting as protease inhibitor and inflammatory mediator [14].

Despite Fet-A is well recognized as the major responsible for the stabilization of CPPs in serum, it has to be considered that hundreds of different types of proteins are present in our blood [15] and among them Albumin (Alb) is by far the most abundant one. Alb circulates in serum at a concentration of 35–50 g/L [16], which is about 50 times higher than that of Fet-A. This protein plays a multifunctional role in blood, as it binds to various endogenous and exogenous molecules and ions, being involved in their transport and homeostasis [17]. Among the ions, Alb is able to bind Ca2+ by means of its acidic residues, lowering the concentration of free ions and contributing to the serum calcification inhibition [18]. The efficacy of Alb in preventing serum calcifications is tough controversial: studies from the nineties on the inhibitory capacity of human serum attributed two-thirds of the inhibitory potential to proteins and other macromolecules, Alb accounting for half of the inhibitory effect of the serum proteins [19]. More recent investigations estimated that the contributory fraction of Alb could be as low as ∼10% [1]. A series of studies also compared the calcification inhibition potential of Fet-A and Alb: while in 1994 it was shown that, in bovine fetal serum, Alb displays a lower Ca-binding activity than Fet-A [20], Schinke et al. described the calcification propensity of several proteins (Fet-A from different sources, Alb, ovalbumin, calmodulin, lysozyme), and concluded that Fet-A accounts for about half of the serum ability to inhibit mineral precipitation, while Alb had no remarkable effect even at higher concentrations [21]. In terms of CPPs, some works demonstrated that both endogenous and synthetic CPPs contain a substantial fraction of Alb [2], [22], [23], [24], suggesting that this protein might play a role in their formation and stability. Heiss et al. prepared CPP-like nanoparticles at different Fet-A/Alb concentrations and found that the conversion from CPP1 to CPP2 is driven by Fet-A, whereas Alb only affects the sedimentation process of CPP2, which was monitored for 2 days [2]. They concluded that Fet-A is mainly involved in the stabilization of CPP1 while Alb (and other acidic proteins) stabilize CPP2. A successive paper by Wu et al. studied the effect of Fet-A and Alb towards calcium phosphate mineralization, focusing on the turbidity of cell culture media supplemented with Fet-A/Alb in different conditions, and concluded that both proteins, but in particular Fet-A, exert a strong inhibitory influence on mineral growth and precipitation [25]. Out of note, in their experiment they observed similar results using either human or bovine Fet-A and Alb. A few years later Pasch et al., while developing a nephelometric test to study the calcification propensity of serum [24], observed that Alb has no intrinsic inhibitory effect, but in combination with Fet-A a synergistic inhibitory effect could be observed. In the same work, they conclude that "proteins largely define primary CPP assembly and shape, whereas small molecules largely define primary CPP stability and timing of primary to secondary CPP conversion". The literature presented so far showcases the importance of Alb in the framework of CPPs. Nonetheless, most of the works address the topic from a clinical perspective, without focusing on the colloidal stability and on the nanoscale structure of these inorganic-organic hybrid particles. In particular, the effects of different Alb concentrations on the shape, size, and amount of formed CPPs are of interest, as well as their long-term colloidal stability.

In this work, we investigated the role of Alb on synthetic CPPs formation and stability at constant Fet-A concentration, using 5 µM of Fet-A (1/3 of the physiological one); this concentration was selected in order to evaluate if in a potential pathological situation of low Fet-A levels (associated with an increased risk of mortality in patients suffering from kidney diseases [26], [27]), Alb would compensate such deficiency by contributing to CPPs stabilization. Different concentrations of Alb were investigated, from 0 to 606 µM, and the system was analyzed in situ at 37 °C by means of turbidimetry, sedimentation and dynamic light scattering, to study the ripening process. The features of the CPP1 formed at different Alb concentrations were thoroughly investigated by combining different analytical approaches including scanning and transmission electron microscopy, infrared spectroscopy and elemental analysis. The obtained results demonstrate that Alb controls both the size and morphology of CPP1 and the stability towards sedimentation of CPP2 dispersions.

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