Ultrashort peptides induce biomineralization

Introduction

The process of physiological biomineralization is widespread in vertebrate bones and teeth, and it determines the shape and mechanical properties of the hard tissue [1,2]. Biomineralization is a natural process of mineral formation mediated by biological macromolecules [3]. In this process, mineral deposition is mainly regulated by proteins and then self-assembles into hierarchical structures with outstanding mechanical properties [4]. Despite this, there is a limited understanding of how biomineralization takes place. Therefore, research has focus on understanding the mechanisms by which organisms control the shape and size of crystals through proteins [5]. In the meantime, due to the complex structure of proteins and the potential risks associated with in vivo application of proteins [6], finding novel types of biomolecules to replace natural proteins to promote hydroxyapatite formation and hard tissue mineralization is the key challenge in the field of hard tissue development and regeneration [7].

In recent years, a number of peptides have been demonstrated to have important biological functions as drugs, with significant efficacy in immune defense, tumor therapy, metabolic disorders, and bone tissue engineering [8], and have gradually evolved into novel biomaterials development for a variety of applications over the decades [9,10]. It has recently been found that the difficulty and high cost of synthesizing recombinant protein can be overcome by using functional protein-derived short peptides in place of full-length proteins [11,12]. Therefore, the study of short peptides that can effectively promote mineralization is beneficial to hard tissue health. The classical theory of biomineralization holds that the primary structure of proteins provides crystal nucleation sites and the secondary structure controls crystal growth [13,14]. The reported biomineralization-promoting peptides generally contain more than 10 amino acids, which can form certain secondary structures and affect the spatial conformation of hydroxyapatite crystals [15]. It is important to note that because ultrashort peptides are less than 7 amino acids in length [16,17], they are simpler to synthesize and function primarily through the primary structure itself (i.e., the amino acid sequence). However, it is unclear whether ultrashort peptides can promote the formation of hydroxyapatite. If ultrashort peptides could be synthesized to perform the same functions in mineralization as full-length proteins, it would not only help to elucidate biomineralization mechanisms but would also provide new candidate molecules for the treatment of mineralization-deficient diseases. In addition, how these ultrashort peptides exert their biological effects and whether the underlying principles of their action are similar to their derived proteins need to be further explored.

To find suitable candidate proteins to screen for mineralization-promoting ultrashort peptides, we focused on various non-collagenous proteins (NCPs) in the extracellular matrix of hard tissues. NCPs contain a variety of proteins that could promote biomineralization [18]. The SIBLING (Small Integrin-Binding LIgand, N-linked Glycoprotein) family is a critical category of NCPs, which contains dentin matrix protein 1 (DMP1), osteopontin (OPN), matrix extracellular phosphoglycoprotein (MEPE), bone sialoprotein (BSP), and dentin sialophosphoprotein (DSPP) [19,20]. SIBLING members are mainly secreted by osteoblasts or odontoblasts during bone or tooth mineralization, and mainly exhibit the typical characteristics of negatively charged acidic proteins. On one hand, they regulate the nucleation and crystal growth of the mineral substance in hard tissue by directly binding calcium and phosphorus ions; on the other hand, they can promote the differentiation of mesenchymal stem cells and maturation of osteoblasts, which produce bone matrix and result in hard tissue formation [21]. For example, DMP1, a key member of the SIBLING family, thought to be a hard tissue-specific pro-mineralization protein. The Dmp1 knockout mice have a severe defect in the mineralization of bone and dentin [22,23], suggesting that it is important for biomineralization. However, there are technical and economic barriers to the application of bioengineered and biomimetic repair materials in hard tissue due to the large molecular weight, complex post-translational modification process, and high cost of synthesizing these proteins. Therefore, it is important to explore methods to study these osteogenic protein-derived functional ultrashort peptides and to assess whether they can promote biomineralization based on their sequence characteristics.

To analyze the functional fragments of the mineralization-related protein, we applied AlphaFold2, a neural network-based approach for protein structure prediction [24], and to determine the structural character of SIBLINGs. With the prediction analysis of protein structure by AlphaFold2, the bone and dentin matrix proteins SIBLING family members could be recognized as intrinsically disordered proteins (IDPs), whose structures are highly flexible, and do not assume typical secondary structures (α-helix, β-sheet, etc.) (Fig. 1a). Thus, based on their properties of rich acidic amino acids (Fig. S1) and lack of typical spatial structure, we believed that the way these SIBLING protein members act in biomineralization are highly related to the characteristics of their primary structure information (amino acid sequence). Interestingly, the function of ultrashort peptides is also dependent on specific and simple amino acid combinations, but not secondary structures in proteins [25]. In addition, only DMP1 is highly expressed and plays an important role in both bone and tooth mineralization among the five protein members [26]. Our analysis of the amino acid sequence characteristics of DMP1 revealed that its sequence is rich in S and E (serine and glutamic acid), with a ratio of S: E = 3: 2 (Fig. 1b). Therefore, in this study, we referenced the native protein of DMP1 and used a combination of S + E and a 3:2 amino acid ratio to synthesize ultrashort peptides. Using an inorganic mineralized system that mimics physiological conditions, we examined the effect of S + E ultrashort peptides on the formation and morphology of hydroxyapatite and the remineralization of demineralized dentin in vitro and preliminarily examined the role of the peptides during bone remodeling in vivo [13,27,28]. We showed that ultrashort peptides have good effects on hydroxyapatite formation and dentin remineralization in vitro, as well as bone formation in vivo.

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Section snippets

Ultrashort peptides design pattern

Spatial structure prediction images of SIBLING protein members (Fig. 1a) based on AlphaFold2 were shown. All the above SIBLING proteins were found to lack typical secondary structures such as α-helix and β-sheet, showing loose and disordered structural features. We focused on the amino acid sequence of DMP1 and found that S and E repeatedly appeared (Fig. 1b). Meanwhile, since the ratio of S and E in the DMP1 sequence is about 3: 2 (Fig. 1b), ultrashort peptide SESSE was synthesized (After

Discussion

The proteins involved in biomineralization are usually acidic intrinsically disordered proteins, suitable for shellfish [29] and vertebrates [30]. It has been reported that protein-derived peptides can effectively promote biomineralization and osteogenesis. For example, collagen-binding motif (CBM) peptide derived from osteopontin selectively stimulated osteogenic differentiation rather than adipogenesis, thus being an available therapeutic agent for osteoporosis [31]. As a matrix protein in

Conclusions

In conclusion, we designed ultrashort peptides based on the primary structure characteristics of intrinsically disordered matrix proteins and demonstrated that S + E based ultrashort peptides can induce biomineralization. It is evaluated that the different lengths and amino acid ratios of ultrashort peptides could affect the crystal size and cell adhesion. SESSE significantly promoted the formation of hydroxyapatite crystals and the deposition of demineralized dentin minerals in a cell-free

Protein structure prediction

The SIBLING proteins structure were predicted by AlphaFold [51] (https://alphafold.ebi.ac.uk), DMP1 (UniProt ID: O55188), BSP2 (UniProt ID: Q61711), DSPP (UniProt ID: P97399), MEPE (UniProt ID: Q8K4L6) and OPN (UniProt ID: P10923).

Ultrashort peptides synthesis

All peptides were synthesized by the Chinese Peptide Company (Shanghai, China) using the standard Fmoc solid-phase peptide synthesis method and were more than 95% pure. The sequences were as follows: SESSE, H–S–OH, H-E-OH, SE, SESSE-NH2, SRSSR, SNSSN, EEEEE, SSSSS,

Funding

This work was supported by grants from Shanghai Experimental Animal Research Project of Science and Technology Innovation Action Plan (201409006400), Shanghai Academic Leader of Science and Technology Innovation Action Plan (20XD1424000), National Natural Science Foundation Projects of China (81822012, 82061130222, 81771043), and a collaboration grant from State Key Laboratory of Molecular Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences.

Author contributions

Yanan Ding: Methodology, Validation, Investigation, Visualization, Writing - Original Draft. Mingxiang Cai: Validation, Investigation, Data Curation. Pingping Niu: Investigation, Writing - Review & Editing. Han Zhang: Investigation, Visualization. Shao-Qing Zhang and Yao Sun: Conceptualization, Methodology, Writing - Review & Editing, Supervision.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

The authors gratefully acknowledge Dandan Kong for the work at the early of the research and Zhi Liu and Dike Tao for their technical assistance.

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