Development of Proteoliposome-Based Matrix Vesicle Models for Investigating the Protein Corona Role in Biomineralization

Matrix vesicles (MVs) play a fundamental role in the process of bone biomineralization. They are extracellular structures released by cells such as osteoblasts and chondrocytes. These vesicles carry lipids, including phospholipids, cholesterol, sphingomyelin, and ceramide, as well as enzymes, among which the most important is tissue non-specific alkaline phosphatase (TNAP). TNAP is an ectoenzyme anchored to the membrane by GPI anchors and is essential for the hydrolysis of pyrophosphate, an inhibitor of biomineralization. Additionally, proteins can associate with the surface of MVs, forming a protein corona (PC), which is capable of modulating the biomineralization function of MVs by altering TNAP activity.

To test this hypothesis, we prepared a simplified model of a dimyristoyl phosphatidylcholine (DMPC)-containing vesicle in which TNAP was reconstituted, producing a proteoliposome. Subsequently, TNAP activity was studied in the absence and presence of PC isolated from MC3T3-E1 osteoblast cell line MVs, as previously standardized. TNAP activity was determined by measuring the hydrolysis of p-nitrophenyl phosphate (PNPP).

DMPC liposomes were prepared using the extrusion technique and characterized by Dynamic Light Scattering (DLS), zeta potential (ζ-potential), and Nanoparticle Tracking Analysis (NTA), resulting in an average diameter of approximately 101.3 nm, a ζ-potential of -14.5 mV, and low polydispersity indices (PDI) of less than 0.1. Incorporation of TNAP into the liposomes produced proteoliposomes with an average diameter of 131.4 nm, a ζ-potential of -19.8 mV, protein 55 µg/mL, and a 2.19-fold increase in TNAP specific activity compared to the solubilized enzyme. Incubation of these proteoliposomes with PC (66 µg/mL total protein) resulted in average diameter of 139.2 nm, a ζ-potential of -16.5 mV, with 1.45-fold increase in the specific activity of TNAP compared to proteoliposomes without PC.

These data suggest that PC modulates enzyme activity, while also increasing the surface charge and homogeneity of the vesicles. Therefore, the use of proteoliposomes is an excellent tool for studying interactions between membranes and proteins, contributing to a better understanding of the mechanisms of biomineralization.

This work was supported by CNPq (124587/2024-0 and 305426/2021-2) and by FAPESP (2019/08568-2).