Adhesive Growth Factors Inspired by Underwater Adhesion Proteins

Chapter 5

Adhesive Growth Factors Inspired by Underwater Adhesion Proteins Downloaded by UNIV OF FLORIDA on October 26, 2017 | http://pubs.acs.org Publication Date (Web): October 25, 2017 | doi: 10.1021/bk-2017-1252.ch005

Chen Zhang,1,2,3 Hideyuki Miyatake,1 and Yoshihiro Ito1,3,4,* 1Nano

Medical Engineering Laboratory, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan 2School of Pharmaceutical Sciences, Jilin University, No. 1266 Fujin Road, Changchun, Jilin 130021, P. R. China 3Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, P. R. China 4Emergent Bioengineering Materials Research Team, RIKEN Center for Emergent Matter Science, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan *E-mail: [email protected]

The generation of material surfaces with biological properties such as cell growth-enhancement and differentiation-inducing abilities could be useful for the development of functional materials for medical applications. Here, by the extension of conventional protein engineering into bio-orthogonal protein engineering using the specific incorporation of non-natural amino acids, we have developed new binding growth factors for the surface modification of materials, to impart biological activity on them.

Introduction A wide variety of different biomaterials are currently employed for medical applications. Research towards the development of new biomaterials has traditionally focused on the preparation of functional materials capable of the simple adhesion of cells or the connection of tissues to metals and ceramics. However, there is a growing interest in the development of biomaterials involving the immobilization of growth factors, which would allow these artificial materials to regulate specific cellular functions, including the gene expression processes © 2017 American Chemical Society Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Downloaded by UNIV OF FLORIDA on October 26, 2017 | http://pubs.acs.org Publication Date (Web): October 25, 2017 | doi: 10.1021/bk-2017-1252.ch005

associated with cell growth and differentiation (1, 2). Binding growth factors have been designed and applied for two purposes, as shown in Figure 1. One is delivery to specific sites or enhancement of local concentrations at specific sites using direct injection. Another involves surface modification of scaffolds for implantation.

Figure 1. Applications of binding growth factors. Binding growth factors have been applied using two methods. One involves delivery to specific sites or enhancement of local concentrations at specific sites using direct injection. The other involves surface modification of scaffolds for implantation of bio-functional materials. Reproduced with permission from ref. (4). Copyright [2013] (4) [Elsevier Ltd.] In terms of the latter application, various types of materials have been modified. However, although numerous studies have reported the use of metallic materials in medical devices such as artificial joints, dental implants and stents, there have been very few reports concerning the surface modification of metal or ceramic materials with biological agents (3). In recent years, an increasing number of reports have been published on natural adhesives (5–7). To design such proteins as a strategy for the surface modification of metals and ceramics, biomimetic approaches inspired by underwater adhesive proteins have been employed with biological materials. Figure 2 shows the use of motifs of two underwater adhesive proteins. One of the proteins is the salivary stathelin protein which is a multifunctional molecule that possesses a high affinity for calcium phosphate minerals such as hydroxyapatite (HA), maintains the appropriate mineral solution dynamics of enamel, promotes selective initial bacterial colonization, and functions as a boundary lubricant on the enamel surface (8). Another is mussel foot protein which is involved in a sticky pad at the end of threads which stick firmly to rock, or any other hard surface (9). It is known that the active sites for the adhesion of both proteins consist of post-translationally modified amino acids. 84 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 2. Combination of growth factors with the active sites of underwater adhesion proteins such as salivary stathelin and the mussel foot protein (Mfp). One is a phosphorylated serine of salivary statherin and another is the 3,4dihyroxyphenylalanine (DOPA) of mussel foot proteins. Both cannot be directly incorporated into a protein using conventional protein engineering (recombinant DNA ) techniques. Therefore, bio-orthogonal approaches are required to prepare proteins with site-specific incorporation of these amino acids. Here, two approaches were employed. The first is chemical synthesis using a solid phase method. The second is a combination of conventional protein (recombinant gene) engineering and enzymatic modification. We have prepared phosphoserine-incorporated epidermal growth factor (EGF) and bone morphogenetic protein-4 (BMP-4), and DOPA-incorporated insulin-like growth factor-1 (IGF-1) using these bio-orthogonal approaches.

Adhesive EGF To prepare phosphoserine-incorporated EGF, we employed the solid phase synthesis method because EGF is a small protein with 53 amino acids. The length of peptides that can be synthesized using a solid-phase method is generally limited to around 50 amino acids (10). The schematic design of the phosphoserine-incorporated EGF peptide (EGFN8P) is shown in Figure 3A. The minimum portion of bio-active EGF was conjugated with the short peptide with the binding sequence in statherin. Figure 4A shows that the modified EGF bound to HA and titanium (Ti), whereas unmodified EGF did not. This shows that the binding affinity of the stathelin fragment was maintained after conjugation with the EGF fragment. The mitogenic activity of EGF derivatives on HA and Ti is shown in Figure 4B. Smaller amounts of bound EGF had higher effects than soluble EGF. As a conclusion, the bound EGF has higher activity than the soluble form because of its immobilization. 85 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 3. Design of binding growth factors. (A) Adhesive EGF using a solid phase method. (B) Adhesive BMP-4 and IGF-1 synthesized using a combination of recombinant DNA technology and enzyme treatments.

Adhesive BMP-4 In the case of human BMP-4 (hBMP4), because its length is 117 amino acids, it is difficult to synthesize it using the solid phase method. Therefore, we combine conventional protein engineering with an enzyme treatment. Here, the sortase A enzyme was used for ligation of modified human BMP (hBMP) with a phosphoserine-containing peptide (11). Figure 3B shows the preparation of the protein derivative. The growth factor portion and the phosphorylated peptide portion were prepared using a gene engineering and the solid phase method, respectively, and they were ligated using the sortase enzyme. Figure 5 shows that the prepared phosphorylated peptide-carrying human BMP-4 (hBMP4-pSpS) has a higher binding affinity to HA than the non-phosphorylated one (hBMP4-SS). In addition, the phosphorylated BMP-4 significantly induced bone formation activity on HA. Considering these results, this novel protein incorporated with phosphoserine is expected to contribute to the preparation of bioactive bone regeneration materials in combination with HA.

86 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 4. (A) Binding of EGF and modified EGF (EGFN8P) onto HA (hydroxyapatite) and Ti (titanium). (B) Mitogenic activity of modified EGF (EGFN8P) bound to hydroxyapatite and titanium. Reproduced with permission from ref. (10) Copyright [2013] (10) [Elsevier Ltd.].

Adhesive IGF-1 The enzymatic treatment using tyrosinase can incorporate DOPA into IGF by converting the tyrosine residues to DOPA (Figure 3B) (12). In the procedure, a tyrosine-lysine-tyrosine-lysine-tyrosine sequence was added to the C-terminal of IGF-1 using conventional genetic recombinant technology. The resultant protein is referred to as IGF-Y. Subsequently, IGF-Y was treated with tyrosinase and the tyrosine residues were converted to DOPA residues. The final product is referred to as IGF-X. The binding affinities of the different IGF-1 derivatives towards Ti were investigated using quartz crystal microbalance (QCM) with dissipation monitoring, as shown in Figure 6. The binding affinity of IGF-X was significantly higher than that of IGF-Y at pH 8.5. Furthermore, the bound IGF-X did not dissociate even after it had been washed with phosphate buffered saline. Figure 7A shows the cell growth assay results for NIH 3T3 cells in the presence of soluble IGF-Y, commercial IGF-1 and bound IGF-X on Ti. Compared with soluble IGF proteins, the bound IGF-X produced a significant enhancement of cell growth.

87 Ito et al.; Advances in Bioinspired and Biomedical Materials Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 5. (A) Binding of engineered hBMP4 proteins to HA (hydroxyapatite). hBMP4-SS (non-phosphorylated serine-carrying human BMP4) or hBMP4-pSpS (phosphorylated serine-carrying BMP4) was incubated with HA beads. The amount of bound proteins was measured using an anti-BMP4 antibody. Mean (n = 3) ± SD is plotted. * p

Adhesive Growth Factors Inspired by Underwater Adhesion Proteins

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