Accelerated Nucleation of Hydroxyapatite Using an Engineered Hydrophobin Fusion Protein

Abstract

Calcium phosphate mineralization is of particular interest in dental repair. A biomimetic approach using proteins or peptides is a highly promising way to reconstruct eroded teeth. In this study, the screening of several proteins is described for their binding and nucleating activities toward hydroxyapatite. Out of 27 tested candidates, only two hydrophobin fusion proteins showed binding abilities to hydroxyapatite in a mouthwash formulation and an increased nucleation in artificial saliva. Using a semirational approach, one of the two candidates (DEWA_5), a fusion protein consisting of a truncated section of the Bacillus subtilis synthase YaaD, the Aspergillus nidulans hydrophobin DEWA, and the rationally designed peptide P11-4 described in the literature, could be further engineered toward a faster mineral formation. The variants DEWA_5a (40aaYaaD-SDSDSD-DEWA) and DEWA_5b (40aaYaaD-RDRDRD-DEWA) were able to enhance the nucleation activity without losing the ability to form hydroxyapatite. In the case of variant DEWA_5b, an additional increase in the binding toward hydroxyapatite could be achieved. Especially with the variant DEWA_5a, the protein engineering of the rationally designed peptide sequence resulted in a resemblance of an amino acid motif that is found in nature. The engineered peptide resembles the amino acid motif in dentin phosphoprotein, one of the major proteins involved in dentinogenesis.

Matrix vesicles from chondrocytes and osteoblasts: Their biogenesis, properties, functions and biomimetic models

“…Apatite in a crystalline phase is induced by a nucleation step, even if it is generated from ACP. The nucleation can be induced without MVs in vitro by chemical agents such as citrate [110, 111], dimethyl sulfoxide [112] and other natural pro-nucleation agents such as positively charged amino acids [113], aspartic acid [113, 114], acidic carboxyproteins and phosphoproteins [115–117], bone sialioprotein [118], proteins of the enamel matrix (ameloblastin, amelogenin, dentin sialophosphoprotein, enamelin, tuftelin) [119] and hydrophobic fusion protein [120]. However, the contribution of these proteins in mineralization in vivo is highly hypothetical.…”

“…Taken together, these facts indicate that MVs are involved with the initiation of the mineralization process [10] in an efficient manner. At least in vitro , other types of vesicles [98], including unidentified types of vesicles [133], intracellular vesicles [98] or natural pro-nucleating agents [113–120] as well as necrotic cells and cellular remnants [98] may also contribute to the mineralization process. All these patterns of mineralization are the result of different activities of one cell type [98].…”

Applications of Functional Amyloids from Fungi: Surface Modification by Class I Hydrophobins

“…Moreover, two HFB proteins were exploited in dental repair applications. DewA_4 and DewA_5 showed binding abilities to hydroxyapatite in a mouthwash formulation and an increased nucleation in artificial saliva [ 57 ].…”

Morphology, Structure Evolution and Site‐Selective Occupancy of Eu3+ in Ca10(PO4)6(OH)2 Nanorods Synthesized via Subcritical Hydrothermal Method

“…Hydroxyapatite is the most important inorganic component in hard tissues that support the body skeleton and movement in our daily life . Nanostructures of hydroxyapatite have been reported to show excellent mechanical and biocompatible properties with potential biomedical applications in bone, and teeth regeneration, antibacterial and anticancer drug carrier,, and gene cellular information transporters . The unique hydroxyl group in the structure of hydroxyapatite could also endow the material as catalyst in activating small organic molecules, such as HCHO .…”