Brain-derived and glial cell line-derived neurotrophic factor fusion protein immobilization to laminin

Abstract

Damage to the recurrent laryngeal nerve often causes hoarseness, dyspnea, dysphagia, and sometimes asphyxia due to vocal cord paralysis which result in a reduction of quality of life. Brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) play critical roles in peripheral nerve regeneration. However, methods for efficiently delivering these molecules are lacking, which limits their use in clinical applications. The present study reports an effective strategy for targeting BDNF and GDNF to laminin by fusing the N-terminal domains of these molecules with agrin (NtA). More specifically, laminin-binding efficacy was assessed and sustained release assays of the delivery of BDNF or GDNF fused with NtA (LBD-BDNF or LBD-GDNF) to laminin were conducted in vitro. In addition, the bioactivity of LBD-BDNF and LBD-GDNF on laminin in vitro was investigated. LBD-BDNF and LBD-GDNF were each able to specifically bind to laminin and maintain their activity in vitro. Moreover, neurotrophic factors with NtA retained higher concentrations and bioactivity levels compared with those without NtA. The ratio of LBD-BDNF and LBD-GDNF that produced optimal effects was 4:6. BDNF and GDNF fused with NtA were effective in specifically binding to laminin. As laminin is a major component of the extracellular matrix, LBD-BDNF and LBD-GDNF may prove useful in the repair of peripheral nerve injuries.

Keywords: PC12 cell; brain-derived neurotrophic factor; glial cell line-derived neurotrophic factor; immobilization; laminin.

Figure 1.

Schematic drawing of the engineered (A) NAT-BDNF, NAT-GDNF, (B) LBD-BDNF and LBD-GDNF proteins. His₆, six histidine (an affinity tag used to purify proteins); LBD, laminin-binding domain; BDNF, brain-derived neurotrophic factor (functional domain); GDNF, glial cell line-derived neurotrophic factor (functional domain); NAT, native.

Figure 2.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis of purified proteins. Coomassie blue staining was used to detect the purification of (A) LBD-BDNF and (B) LBD-GDNF. Western blotting with an antibody to polyhistidine was then used to evaluate (C) LBD-BDNF and (D) LBD-GDNF. LBD, laminin-binding domain; BDNF, brain-derived neurotrophic factor; GDNF, glial cell line-derived neurotrophic factor.

Figure 3.

Laminin binding assay in vitro. Binding curves of (A) NAT-BDNF and LBD-BDNF and (B) NAT-GDNF and LBD-GDNF to laminin measured by enzyme-linked immunosorbent assay. n=6. *P<0.05 vs. NAT control. NAT, native; LBD, laminin-binding domain; BDNF, brain-derived neurotrophic factor; GDNF, glial cell line-derived neurotrophic factor; OD, optical density.

Figure 4.

Sustained release assay from laminin in vitro. Release curves of (A) NAT-BDNF and LBD-BDN and (B) NAT-GDNF and LBD-GDNF from laminin in vitro. n=6. *P<0.05 vs. NAT control. NAT, native; LBD, laminin-binding domain; BDNF, brain-derived neurotrophic factor; GDNF, glial cell line-derived neurotrophic factor.

Figure 5.

Characterization of PC12 cells was conducted by phase contrast microscopy for cells that were cultured under conditions (A) without recombinant proteins or (B) with LBD-BDNF. Black arrows indicate the neurites. Magnification, ×200. LBD, laminin-binding domain; BDNF, brain-derived neurotrophic factor.

Figure 6.

Bioactivity comparison of recombinant proteins in vitro. Effect of (A) LBD-BDNF, NAT-BDNF, (B) LBD-GDNF and NAT-GDNF on neurite outgrowth from PC12 cells. Effect of (C) LBD-BDNF, NAT-BDNF, (D) LBD-GDNF and NAT-GDNF on the ratio of neurite length/cell diameter in PC12 cells. Effect of (E) LBD-BDNF, NAT-BDNF, (F) LBD-GDNF and NAT-GDNF on PC12 cell survival as determined by Cell Counting kit-8 assay. Data are presented as mean ± standard deviation (n=6). NAT, native; LBD, laminin-binding domain; BDNF, brain-derived neurotrophic factor; GDNF, glial cell line-derived neurotrophic factor; OD, optical density.

Figure 7.

Bioactivity comparison of recombinant proteins on laminin in vitro. Effect of (A) LBD-BDNF, NAT-BDNF, (B) LBD-GDNF and NAT-GDNF on neurite outgrowth from PC12 cells. Effect of (C) LBD-BDNF, NAT-BDNF, (D) LBD-GDNF and NAT-GDNF on the ratio of neurite length/cell diameter in PC12 cells. Effect of (E) LBD-BDNF, NAT-BDNF, (F) LBD-GDNF and NAT-GDNF on PC12 cell survival as determined by Cell Counting kit-8 assay. Data are presented as mean ± standard deviation (n=6). *P<0.05 the NAT control. NAT, native; LBD, laminin-binding domain; BDNF, brain-derived neurotrophic factor; GDNF, glial cell line-derived neurotrophic factor; OD, optical density.

Figure 8.

Bioactivity comparison of different porportions of recombinant proteins on laminin in vitro. Effect of different porportions of recombinant proteins on (A) neurite outgrowth and (B) the the ratio of neurite length/cell diameter in PC12 cells. (C) Effect of different proportions of recombinant proteins on cell survival in PC12 cells by Cell Counting kit-8 assay. Blank controls (ratio 0 or 10) indicates cells were cultured under similar conditions with the exception that LBD-BDNF or LBD-GDNF was omitted. Data are presented as mean ± standard deviation (n=6). *P<0.05 vs. NAT control. The asterisk within a square indicates the optimum ratio of mixing to promote growth. NAT, native; LBD, laminin-binding domain; BDNF, brain-derived neurotrophic factor; GDNF, glial cell line-derived neurotrophic factor; OD, optical density.

Figure 9.

Images of PC12 cell (Aa) cytoskeletons; (Ab) nucleim, or (Ac) overlays, viewed by confocal fluorescent microscopy following treatment with different proportions of LBD-BDNF and LBD-GDNF. (A) Only LBD-BDNF, LBD-GDNF and LBD-BDNF in a (B) 1:4 ratio, (C) 2:3 ratio, (D) 3:2 ratio and (E) 4:1 ratio, and (F) only LBD-GDNF. NAT, native; LBD, laminin-binding domain; BDNF, brain-derived neurotrophic factor; GDNF, glial cell line-derived neurotrophic factor.