Table 1.
List of potential tyrosine-sulfated proteins in cow RPE.
Table 2.
List of potential tyrosines that may be sulfated on human and cow vitronectin, opticin, and CFH, as identified by Sulfinator.
Figure 1.
Immunoblot analysis of neurosensory (NS) retinal and RPE extracts to identify tyrosine-sulfated proteins.
Immunoblot analysis of 50 µg of RPE (lanes 1–4) and neurosensory retinal extracts (lanes 5–8) from human, cow, pig, and mouse RPE probed with the anti-sulfotyrosine antibody PSG2. Blots were repeated 3 independent times using biologically different samples.
Figure 2.
PSG2-immunoaffinity column purification of tyrosine-sulfated proteins from cow RPE.
(A). The elution profile was monitored by following absorbance at 280 nm. Following loading, the column was washed with buffers W1, W2, and W3. Elution was performed in buffer W3 containing 4 mM sulfated pentapeptide (EB). (B). Twenty-six microliter aliquots from input (IN), flow-through (FT), wash 1 (W1), and wash 2 (W2) were fractionated by SDS-PAGE, and proteins were visualized by staining with Coomassie blue dye. (C). Left, SDS-PAGE of 26 µL of wash 3 (W3) and eluted samples (EB) from the immunoaffinity column stained with Coomassie blue (CB) and right, immunoblotted with PSG2. Asterisks indicate the bands that were prominent on Coomassie blue-stained gel (CB) and were also recognized by PSG2 as tyrosine-sulfated.
Figure 3.
Native human RPE vitronectin is tyrosine-sulfated.
Vitronectin was immunoprecipitated from 500 µg human RPE lysates using anti-VTN antibody (lane 2) or mouse IgG (lane 3). Immunoprecipitants were fractionated by SDS-PAGE, transferred, and immunoblotted using anti-VTN antibody or anti-sulfotyrosine PSG2 antibody. Immunoprecipitation and western blots were repeated 3 independent times using biologically different human RPE samples. (B). Ectopically-expressed vitronectin is tyrosine-sulfated. Recombinant VTN or empty vector (pcDNA3.1) were transfected into HEK 293T cells and immunoprecipitated from conditioned media using anti-VTN antibody (lane 4) or mouse IgG (lane 3). Immunoprecipitants were electrophoresed and immunoblotted using anti-VTN antibody or anti-sulfotyrosine PSG2 antibody. Immunoprecipitation and western blots were repeated 3 independent times after independent VTN transfections. (C). 35S-metabolic labeling of recombinant vitronectin in vitro. Vitronectin-transfectants were radiolabelled with 35Sulfate. Following radiolabeling, vitronectin was immunoprecipitated and blots were either subjected to autoradiography (AR) or immunoblotted with anti-VTN antibody. (D). Radiolabeled vitronectin bands were excised from the membrane along with equivalent areas from mouse IgG immunoprecipitants, and alkaline hydrolysis was performed. The samples were then spiked with sulfo-amino standards tyrosine sulfate, threonine sulfate, and serine sulfate, and subjected to thin layer electrophoresis (TLE) on cellulose plates. Following TLE analysis, sulfo-amino standards (NHD) were visualized either by spraying with Ninhydrin or autoradiography (AR). TLE experiments were repeated at least three independent times.
Figure 4.
Tyrosine sulfation of ectopically expressed opticin.
(A). Opticin was immunoprecipitated from 500 µg human RPE lysates using anti-OPT antibody (lane 3) or mouse IgG (lane 2). Immunoprecipitants were electrophoresed and immunoblotted using anti-OPT antibody (upper panel) or PSG2 (lower panel). Immunoprecipitation and Western blots were repeated 3 independent times using biologically different human RPE samples. (B). Metabolic labeling of recombinant opticin. Recombinant opticin or empty vectors were transfected into HEK293T cells and cells grown in presence of radioactive sulfate. Following radiolabeling, opticin isoforms were immunoprecipitated using anti-OPT antibody (lane 4) or mouse IgG (lane 3). The blots were then subjected to autoradiography (AR) and immunoblotted for opticin. The 65 kD and 55 kD isoforms are depicted as ‘H’ and ‘L’, respectively. Immunoprecipitation and western blots were repeated 3 independent times after independent OPT transfections. (C). Both the 65 kD ‘H’ and 55 kD ‘L’ isoforms were excised from the blot, along with equivalent areas around 55 kD from mouse IgG immunoprecipitants, and analyzed by barium hydroxide hydrolysis. The samples were then spiked with a mixture of tyrosine sulfate, threonine sulfate, and serine sulfate as standards, and subjected to thin layer electrophoresis (TLE) on cellulose plates. Following TLE analysis, the plate was sprayed with ninhydrin to visualize the sulfo-amino standards (NHD) and was autoradiographed (AR). TLE experiments were repeated at least three independent times.
Figure 5.
Native human plasma and RPE CFH is sulfated.
(A) About 4 µg of purified human plasma CFH was either untreated (lane 1) or treated with PNGase F (lane 2), and then immunoblotted with anti-CFH antibody or with PSG2. CFH was also immunoprecipitated from human RPE, and was either untreated (lane 1) or treated with PNGase F (lane 2), and then immunoblotted with anti-CFH antibody or with PSG2. Western blots were repeated 3 independent times from human plasma and RPE samples. (B) A recombinant human CFH clone was transfected into HEK293T cells, immunoprecipitated with anti-CFH antibody, and was either directly electrophoresed (lane 1) or first treated with PNGase F (lane 2). The immunoprecipitants were immunoblotted with anti-CFH antibody or with PSG2. Immunoprecipitation and Western blots were repeated 3 independent times after CFH transfections. (C). Recombinant CFH was transfected into HEK293T cells and radiolabelled with radioactive sulfate, then immunoprecipitated and subjected to PNGase treatment (lane 3) or left untreated (lane 2). The blots were autoradiographed (AR), then immunoblotted with anti-CFH antibody. (D). Radiolabelled CFH was analyzed by barium hydroxide hydrolysis and autoradiographed (AR). TLE experiments were repeated at least three independent times.