Pigment epithelium-derived factor engineered to increase glycosaminoglycan affinity while maintaining bioactivity

Abstract

Pigment epithelium-derived factor (PEDF) is a secreted protein that is essential in tissue homeostasis and is involved in multiple functions in the eye, such as antiangiogenesis and neuroprotection. However, short retention in the retinal microenvironment can limit its therapeutic effects. In this study, we modified the amino acid sequence of PEDF to increase its affinity for heparin and hyaluronic acid (HA), which are negatively charged extracellular matrix (ECM) molecules. HA is the major component of the vitreous humor. We selectively converted neutral or anionic residues into cationic residues to obtain engineered PEDF (ePEDF). Using in vitro binding assays, we demonstrate that ePEDF had higher affinity for heparin and HA than wild-type PEDF (wtPEDF). ePEDF exhibited antiangiogenic and retinal survival bioactivities. It inhibited endothelial cell proliferation and tube formation in vitro. In an ex vivo model mimicking retinal degeneration, ePEDF protected photoreceptors from cell death. The findings suggest that protein engineering is an approach to develop active PEDF with higher ECM affinity to potentially improve its retention in the retina microenvironment and in turn make it a more efficient therapeutic drug for retinal diseases.

Keywords: Hyaluronic acid; Pigment epithelium-derived factor; Retinal disease.

Fig. 1.. Rational design of ePEDF based on the wtPEDF structure (PDB ID: 1IMV).

(A) Coulombic surface coloring describes positive (blue) and negative (red) charge distribution. One side of PEDF (top left) is negatively charged, while the opposite side is less charged or neutral (top right). Bottom: Two bioactive domains, D34-N77 (yellow) and V78-T121 (green), critical for the anti-angiogenic and neuroprotective properties, respectively, are located on the same side as most negative surface charges and are distal to the cationic residues (cyan) that bind HA and heparin. (B) Coulombic surface coloring showing charge distribution of ePEDFs. ePEDF is denoted by x.y. (x represents the number of cationic residues, and y distinguishes two analogs having the same x). (C) A plot of the calculated net charges of wt- and ePEDF (pH 5–8.5), using Protein Calculator v3.4 is shown.

Fig. 2.. Biochemical characterization of ePEDF.

(A) Heparin affinity of wt- and ePEDF. Photographs of selected slices of SDS-PAGE gels stained with Coomassie blue of eluted fractions of wt- or ePEDF with the indicated NaCl concentrations (top of each slice) of from heparin resin are shown. (B) HA affinity of wtPEDF, ePEDF_5.2 and ePEDF_6.1. Proteins at 1, 0.1, 0.01, and 0.001 μg/ml (x-axis) were added to the HA-conjugated surface, and then ELISAs were performed to detect bound PEDF (y-axis). (C) Collagen I affinity of wtPEDF and ePEDF_6.1. Proteins at 1, 0.1, 0.01, and 0.001 μg/ml (x-axis) were added to the surface coated with collagen I, and then, ELISAs were performed to measure PEDF bound to collagen (y-axis). Higher absorbance values indicate more PEDF bound to the HA or collagen I surface. a.u.: arbitrary unit.

Fig. 3.. In vitro bioactivity of ePEDF

(A) Inhibition of tube formation by PEDF. Representative images of HMEC-1 cells treated with (a) no FGF2 or PEDF, (b) FGF2, (c, d) FGF2 + wtPEDF, (e, f) FGF2 + ePEDF_5.2, (g, h) FGF2 + ePEDF_6.1. (B) Statistical comparison of tube lengths. Tube length was measured, and the values were normalized to that of (a), the group without FGF2 or PEDF. **p<0.05, (b) vs. (c) to (h). (C) Reduction in HMEC viability by PEDF. Viability of HMEC-1 was determined after incubation in the medium containing no VEGF₁₆₅, VEGF₁₆₅, VEGF₁₆₅ + wtPEDF, or VEGF₁₆₅ + ePEDF_6.1. All values were normalized to that of a group without VEGF or PEDF. **p<0.01, vs. wtPEDF or ePEDF_6.1 (100 ng/ml VEGF); *p<0.05, vs. ePEDF_6.1 (50 and 25 ng/ml VEGF). (D) Ex vivo protection of photoreceptor cells against death by wt- and ePEDF. Representative confocal images showing the photoreceptor layer treated with wtPEDF and ePEDF_6.1, with or without the induction of cell death by zaprinast. Each ROI shows total nuclei (DAPI) and dead cells (TUNEL) (top). (E) The plot shows the rate of photoreceptor cell death as calculated by the percentage of TUNEL label per DAPI in each ROI. Each group included assays with three biological replicates (n=3), and a total of nine ROIs were compared by one-way ANOVA (***p<0.001, zaprinast without PEDF vs. Control, zaprinast with wtPEDF and ePEDF_6.1).