Langmuir monolayers and proteoliposomes as models of matrix vesicles involved in biomineralization

Chondrocytes and osteoblasts are able to mineralize the extracellular matrix (ECM) by promoting the nucleation of apatite in the lumen of matrix vesicles (MVs) during endochondral bone formation (Bottini et al. 2018; Plaut et al. 2019). The lipids and proteins present in the membrane of the MVs mediate the interactions of these vesicles with the ECM and regulate the initial mineral deposition and further propagation. Among the proteins, ion transporters control the availability of phosphate and calcium needed for initial apatite precipitation. Phosphatases (orphan phosphatase 1 (PHOSPHO1), ectonucleotide pyrophosphatase/phosphodiesterase 1 (NPP1), and tissue-nonspecific alkaline phosphatase (TNAP) play a crucial role in controlling the inorganic pyrophosphate/inorganic phosphate ratio that allows MVs-mediated initiation of mineralization. The lipidic microenvironment can serve as binding sites for Ca²⁺ associated with enzymes and transporters (type III sodium-dependent phosphate transporters (PIT1/2), annexins (specifically AnxA5), and Na⁺,K⁺-ATPase (NKA) to form calcium phosphate complexes (Bolean et al. 2017a; Cruz et al. 2020). This exquisite, highly regulated multistep process can be studied with the aid of simplified membrane models such as Langmuir monolayers, liposomes, and proteoliposomes (Fig. 1).These models are useful for understanding the lipid-protein interactions with emphasis on physicochemical and biochemical processes that trigger mineralization (Andrade et al. 2019; Derradi et al. 2019; Bolean et al. 2015, 2017a, b, 2020; Simão et al. 2010, 2019; Favarin et al. 2017, 2020). Our group has made a series of advances, based on the use of biomimetic models that have increased the general understanding of the biomineralization process. Among these developments, we highlight the following:

1. Proteoliposomes composed by different lipids and TNAP propagate mineralization after 48 h of incubation with ATP as a source of Pi, at saturating conditions, and in the presence of a nucleator (Simão et al. 2019; Favarin et al. 2020). We also found that DMPC:SM proteoliposomes displayed the highest efficiency of substrate hydrolysis, apparent affinity for ATP, and mineral propagation, related to the highest degree of membrane organization (highest ΔH of phase transition), among the tested proteoliposomes. Taken together, these data indicated that, in addition to the essential components required for mineralization, such as enzymes, substrates, ions, and lipid bilayers, the degree of packing of lipid bilayers and the presence of sterol can affect both TNAP catalytic efficiency and the supersaturation conditions required to precipitate apatite crystals (Simão et al. 2019, Favarin et al. 2020).

2. The presence of TNAP in the proteoliposomes did not significantly affect AnxA5-mediated Ca²⁺ transports. On the other hand, one can observe that the presence of AnxA5 affects significantly the hydrolysis of substrates by TNAP. Admittedly, this proteoliposome system is still incomplete since other proteins are necessary to adequately mimic the MVs in the biomineralization process, such as the protein Pit-1 for example, which may be responsible for the Pi influx into the vesicles (Boelan et al., 2015, 2017a).

3. An AFM study using proteoliposomes harboring TNAP and AnxA5 provides basic yet crucial information about the structure of lipid-protein microdomains on the surface of MVs that other microscopy techniques could not have provided. Since our experimental approach can provide information on specific regions on more complex protein-containing lipid vesicles, it can be exploited to shed light on processes involving lateral heterogeneity within cellular membranes, including domain-induced budding and possibly MV formation (Bolean et al. 2017b).

4. AnxA5-proteoliposomes showed the highest affinities for type II collagen, deposited during chondrocyte mineralization in joint cartilage. TNAP in the lipid/protein microenvironment disturbs interactions between AnxA5 and collagen. These findings support the hypothesis that TNAP is cleaved from the MV membrane just before ECM binding, such facilitating MV anchoring to ECM via AnxA5 interaction (Bolean et al. 2020).

5. TNAP, incorporated in Langmuir monolayers and Langmuir–Blodgett films, keeps enough activity to hydrolyze PPi and ATP. Moreover, cholesterol enhanced the incorporation of the enzyme in a lipid-raft model. Taken together, these results showed that both TNAP incorporation and catalytic activity are modulated by membrane fluidity, driven by the presence of cholesterol, providing a better understanding of the interfacial behavior between cholesterol and TNAP in MV membrane and helping to clarify complex biochemical and biophysical processes that occur in the MV membrane during biomineralization (Derradi et al., 2019).

6. The mineralization induced on DPPS-enriched monolayers reproduces the mineralization of monolayers composed by lipid extract from native MVs. Therefore, Langmuir monolayers proved to be a promising approach to determine lipid-lipid and lipid-protein interactions occurring during the mineralization induced by MVs. Our findings suggest the possibility that PS plays a major role during ossification, motivating future studies in order to unveil the origin of the biomineral precursor phase [Cruz et al., 2020].

Fig. 1.

Langmuir monolayer (A) and proteoliposome (B) with TNAP incorporated mimic MVs during biomineralization process. Adapted by Andrade et al. (2019), Bolean et al. (2017a), and Simão et al. (2019)

Therefore, Langmuir monolayers and proteoliposomes are suitable as MV biomimetics. They are reliable models to investigate the mechanisms involved in the mineralization process (i.e., the effects of the lipid bilayer organization on apatite nucleation and propagation) and may be also applied for the development of novel therapeutic strategies to either prevent or inhibit ectopic mineralization.

Declarations

Conflict of interest

The authors declare no competing interests.