PEDF association to hyaluronan secreted by human retinoblastoma Y-79 cells. A, human retinoblastoma Y-79 cells (1.25 × 10⁵ cells/ml) were cultured in serum-free medium for 16 h, and then the culture conditioned media were collected and concentrated 10-fold before being subjected to fluorophore-assisted carbohydrate electrophoresis. The fluorogram shows the separation of saccharides recovered from Y-79-conditioned media from two different batches of Y-79 cell cultures (M1 and M2). The image shown depicts oversaturated pixel intensity for the major derivatized components to allow visualization of less abundant components. The lane marked MW shows the resolution of a standard mixture of 13 AMAC-derivatized saccharides. The Control lane shows a derivatized medium sample not conditioned by human retinoblastoma Y-79 cells for background corrections. ΔDi2S standards at 62.5 and 31.5 pmol (as determined by hexuronic acid analysis) were added to lanes marked M1 and M2, respectively, for the quantification of resolved disaccharides. B, PEDF coprecipitates with glycosaminoglycans secreted into the Y-79-conditioned media. Serum-free medium from human retinoblastoma cell cultures (1.25 × 10⁶ cells/ml) maintained at 37 °C for 16 h was concentrated (25-fold), and 500-μl aliquots were either left untreated or treated with Streptomyces hyluronidase (10 turbidity reducing units/ml) for 10 min at 37 °C. Recombinant human PEDF (20 μg/ml) and BSA (100 μg/ml) were added, and the mixtures were incubated at 4 °C for 120 min. Glycosaminoglycans were precipitated with CPC. A Western blot of the precipitates was immunostained with Ab-rPEDF, and is shown. Treatments are indicated at the top of each lane.

Direct binding of PEDF to HA. Purified recombinant human PEDF and highly purified hyaluronan (Healon®) were incubated at 4 °C for 1 h. HA was precipitated with CPC, and the pellets were resuspended in SDS-PAGE sample buffer and applied to 10–20% polyacrylamide gels for detection of PEDF. A, binding reactions were performed with recombinant human PEDF and increasing concentrations of HA (indicated at the top of each lane) in 200 μl of buffer H (20 mm sodium phosphate, pH 6.5, 20 mm NaCl, and 10% glycerol). BSA was the negative control. Proteins in the precipitates were resolved by SDS-PAGE and visualized with Silver Stain. A photograph of a stained gel is shown. B, binding reactions were performed with 100 μg/ml HA and increasing concentrations of PEDF (indicated at the top of each lane) in 500 μl of Dulbecco's modified Eagle's medium plus 100 μg/ml BSA. PEDF in the precipitates was detected by Western blotting with antiserum Ab-rPEDF. C, concentration curve of PEDF binding to HA. Binding reactions were performed with 200 μg/ml HA and increasing PEDF concentrations in 200 μl of 150 mm NaCl in Buffer S (20 mm sodium phosphate, pH 6.4, 10% glycerol, 1 mm dithiothreitol) plus 100 μg/ml BSA (carrier protein). One-third of each reaction mixture was resolved by SDS-PAGE followed by silver staining, and two remaining thirds were analyzed by Western blotting with Ab-rPEDF. The density of each PEDF band was determined from scanned images using Stratagene Eagle Eye II. Data were analyzed by non-linear regression and one-site binding using a GraphPad Prism version 3.0 software program (plot shown). The best-fit values obtained from data combined from silver-stained gels and Western transfers were B_max = 1.723 bound PEDF, and K_D = 7.027 μg/ml PEDF (= 140.54 nm), with an R₂ = 0.9715.

Effect of increasing NaCl concentrations on the binding of PEDF to HA (A) and heparin (B). A, binding reactions were performed with recombinant human PEDF (14.5 μg/ml) and HA (200 μg/ml) in buffer S containing increasing concentrations of NaCl. After precipitation with CPC, proteins were resolved by SDS-PAGE and visualized with Coomassie Blue. A photograph of a gel is shown. B, heparin-affinity column chromatography was performed with heparin affinity beads in buffer H. Recombinant human PEDF (1.75 μg) was added to 0.5 ml of buffer H containing 17.5 μg of BSA and loaded onto a column with heparin-affinity beads (0.5 ml). The flow-through was collected and reloaded, repeating these steps three times. The column was washed with 20 column-volumes of buffer H. The bound proteins eluted with a NaCl step-gradient at 4 column-volume/fraction (numbers at top of each lane correspond to NaCl concentration). Fractions were concentrated to 40 μl, and an aliquot of 15 μl from each was resolved by SDS-PAGE followed by Western blotting. PEDF was detected by immunostaining with anti-PEDF. C, HA-affinity column chromatography was performed with HA affinity Sepharose in buffer H as in panel B, except that then the load was 10 μg of PEDF, and elutions were with one column-volume. Aliquots of the fractions (12 μl) were resolved by SDS-PAGE and Western blotting followed by immunostaining with Anti-PEDF.

Binding of chemically modified PEDF to HA and heparin. Exposed lysines of recombinant human PEDF were modified with fluorescein (Fl) using Sulfo-NHS-LC-fluorescein. PEDF (0.4 mg/ml) was preincubated without or with HA (1.6 mg/ml) in buffer H at pH 7.7 or 8.6 at 25 °C for 15 min followed by addition of Sulfo-NHS-LC-fluorescein (87.5 μg/ml) and incubation at 25 °C for 1 h. Ethanolamine was added to the reactions at (150 mm) and incubated for 2 h at 25 °C. Reaction mixtures without the glycosaminoglycan were supplemented with HA to match those with HA. Proteins were separated from unbound fluorescein by ultrafiltration using centricon-30 devices and concentrated in phosphate-buffered saline. A, CPC coprecipitation of HA and chemically modified PEDF. Proteins (2 μg) were mixed with HA (6 μg) in 20 μl of buffer H, pH 7.7, containing 5 μg of BSA, and incubated at 37 °C for 1 h. PEDF was coprecipitated with HA using CPC and resolved by SDS-PAGE. Fluoresceinated PEDF was detected by Typhoon scanning. Reactions were applied to lanes as follows: lane 1, Fl-PEDF preincubated without HA, pH 7.7; lane 2, Fl-PEDF preincubated with HA, pH 7.7; lane 3, Fl-PEDF preincubated without HA, pH 8.6; lane 4, Fl-PEDF preincubated with HA, pH 8.6. B, heparin-affinity column chromatography of chemically modified PEDF. Proteins (2 μg) were mixed with BSA (15 μg) in 500 μl of buffer H, pH 6.4, and applied to a heparin-affinity column (0.5-ml bead-volume). The flow-through (FT) was collected and reloaded three times, before washing with 12 column volumes of buffer H, pH 6.4. Bound material was eluted with 500 mm NaCl in buffer H, pH 6.4. Load, FT, and eluates were concentrated and applied to a gel at equivalent volumes and resolved by SDS-PAGE. Fluoresceinated PEDF was detected by Typhoon scanning. Lanes 1–3, Fl-PEDF preincubated without HA, pH 7.7; lanes 4–6, Fl-PEDF preincubated with HA, pH 7.7; lanes 7–9, Fl-PEDF preincubated without HA, pH 8.6; lanes 10–12, Fl-PEDF preincubated with HA, pH 8.6. Load was applied in lanes 1, 4, 7, and 10;FTin lanes 2, 5, 8, and 11; and eluate in lanes 3, 6, 9, and 12. C, heparin-affinity column chromatography of PEDF and HA mixtures. Unmodified PEDF (30 μg) was added to 2 ml of buffer H containing 0, 50, or 200 μg/ml HA and preincubated at 4 °C before mixing with heparin-affinity beads (1 ml) with gentle rotation in a column at 4 °C for 1 h. PEDF conjugated to fluorescein (Fl-PEDF) in bicarbonate buffer, pH 9, was also subjected to heparin-affinity chromatography. Unbound material was washed with 10 column-volumes of buffer H. Bound proteins were eluted with 500 mm NaCl, concentrated, and equivalent volumes were resolved by SDS-PAGE followed by Coomassie Blue staining. Photographs of the lanes with eluates are shown with components of each reaction mixture indicated to the top. D, PEDF-HA binding and pH. PEDF (17.5 μg/ml) was mixed with HA (60 μg/ml) in 100 μl of buffer H at pH ranging from 7.4 to 8.6, containing BSA (50 μg/ml), and incubated at 25 °C for 1 h. PEDF was coprecipitated with HA using CPC and resolved by SDS-PAGE followed by Western blot. PEDF was immunodetected with anti-PEDF antibodies. The pH of each reaction is indicated at the top of each lane. E, HA-affinity column chromatography was performed with HA affinity Sepharose in buffer H as in Fig. 3C, except that then the load was 5 μg of Fl-PEDF. Aliquots of the fractions (18 μl) were resolved by SDS-PAGE, and the fluoresceinated PEDF was detected by Typhoon scanning. The numbers at top of each lane correspond to NaCl concentration for each fraction.

Binding of genetically modified PEDF proteins to HA. BHK cells were transfected with mutated PEDF cDNA expression plasmids. A, culture media (100 μl) of stably transfected cells was concentrated and used in PEDF binding assays with HA (30 μg) and collagen I (2 μg) in 500 μl and 200 μl of phosphate-buffered saline, pH 7.5, respectively. HA-PEDF was isolated by CPC precipitation, and collagen I-PEDF by size exclusion ultrafiltration using centricon-100. PEDF was detected by immunoblotting versus anti-PEDF. HA-PEDF corresponds to PEDF bound to HA; Collagen I-PEDF to PEDF bound to collagen I; and Total PEDF to PEDF in media (20 μl). B, proteins in culture media of stably transfected cells were concentrated to ∼350 μg/ml protein, exchanged to buffer H (0.5 ml) and loaded onto heparin-affinity beads column (0.5 ml bed-volume). The flow-through was reloaded, and the unbound material was washed with 20 column-volumes of buffer H. Bound proteins were eluted with 2 column volumes of 1 m NaCl in buffer H, concentrated by ultrafiltration using centricon-30 devices, and resolved by SDS-PAGE. PEDF was detected by immunoblotting versus anti-PEDF. Heparin-PEDF corresponds to PEDF bound to heparin, and Total PEDF to PEDF in load (6 μg of protein per lane). C, quantification of PEDF bound to HA, collagen I, and heparin. PEDF-immunoreactive bands, as from above, were scanned using UNSCAN-IT software. Values from bound were divided by values from Total protein, plotted as a function of the genetically modified PEDF variant using EXCEL, Microsoft, and are shown.

Binding to HA of PEDF modified on putative exposed lysines. Altered PEDF proteins were purified and concentrated. Purified altered PEDF proteins and HA were mixed in buffer H and incubated at 4 °C for 1 h. Bound protein was coprecipitated with CPC. Western blot of CPC precipitates immunostained with anti-PEDF is shown at the top (HA-PEDF). SDS-PAGE of purified PEDF altered proteins stained with Coomassie Brilliant Blue is shown at the bottom (Total PEDF). A plot of bound PEDF quantification was performed as in Fig. 5C.

Three-dimensional structure of human PEDF (from Protein Data Bank ID 1IMV) to illustrate the location of the HA-binding site. The two structures are rotated about 180° from each other with highlighted positions of single alterations made in this study. In blue are basic amino acids Lys¹⁴⁶, Lys¹⁴⁷, and Arg¹⁴⁹ residues located in a turn between β-strand s2A and α-helix E, and Lys¹⁸⁹, Lys¹⁹¹, Lys¹⁹⁴, and Arg¹⁹⁷ located in another turn between α-helix F and β-strand s3A, both within BX₇B HA-binding sites; in red are acidic amino acids Asp²⁵⁶, Asp²⁵⁸, and Asp³⁰⁰ corresponding to the collagen-binding site. P2 corresponds to the residue next to the homologous serpin reactive site, P1; and N– in yellow indicates the position of the amino-end terminus of the polypeptide in the three-dimensional structure corresponding to position 26. Structures were visualized and reproduced using Cn3D (NCBI).