pigment epithelium - derived factor ( pedf ) is a 50  kda secreted glycoprotein and a member of the serpin superfamily with no demonstrable protease inhibitory activity .
pedf is associated with several biological processes due to its antiangiogenic , anti - inflammatory , anti - oxidative , neurotrophic , and neuroprotective properties .
moreover , it has been implicated in another interesting role , as an antitumor and antimetastatic agent with applications in multiple malignancies such as retinoblastoma , lung , breast , prostate , ovarian and pancreatic carcinomas , uveal melanoma , glioma , and osteosarcoma [ 38 ] .
pedf levels are decreased in tumor cells relative to normal cells , and pedf addition inhibits tumor formation and metastasis , blocks angiogenesis , and induces apoptosis in tumor and endothelial cells .
in contrast , pedf promotes retinal cell survival and neuronal differentiation , protects retinal pigment epithelial cells against oxidative stress [ 2 , 3 , 9 ] and plays a role in expansion of neural stem cells .
previous structure - function studies revealed that two peptide regions toward the amino end of the pedf polypeptide have distinct biological activities [ 2 , 11 , 12 ] .
the peptide region termed 34-mer ( amino acid residues asp - asn of the human pedf sequence ) forms alpha helix a of the 3d structure of human pedf   and confers antiangiogenic and antitumorigenic properties to the pedf polypeptide . the 44-mer peptide ( val - thr ) derived from the region that forms alpha helices b , c , and part of d exhibits neurite - outgrowth activity and protects spinal cord motor neurons against chronic toxicity [ 11 , 14 ] . a smaller peptide derived from amino acid positions 82121 exhibits
interestingly , the native pedf has several isoforms differing in isoelectric point ( pi ) values [ 16 , 17 ] implying differences in posttranslational modifications of the polypeptide backbone .
reported that the secreted human recombinant pedf from human embryonic kidney ( hek ) cells has at least two species varying in their carbohydrate composition of the n - glycosylation site and efficacy of suppressing vascular endothelial growth factor - induced proliferation and migration of retinal microvascular endothelial cells .
maik - rachline and seger showed that the human plasma pedf is a phosphoprotein and that extracellular phosphorylation converts the recombinant protein from a neurotrophic to an antiangiogenic factor .
konson et al . demonstrated that a triple phosphomimetic - altered pedf is more efficient than wild - type pedf in inhibiting neovascularization and tumor growth in vivo and suppresses cultured endothelial cell proliferation and cell migration much better than the wild - type pedf .
given the above , the aim of this study was to identify and characterize pedf isoforms that could contribute to the complexity of pedf action and its multifunctional modality .
human bladder carcinoma t24 cells ( cell line collection , lombardi comprehensive cancer center , passage 33 ) , human breast cancer 231-br cells   ( passage 13 ) , mouse breast cancer 4t1-br5 cells   ( passage 11 ) , human breast tumor mda - mb-231 cells ( cell line collection , lombardi comprehensive cancer center , passage 6 ) , mouse breast cancer 4t1 cells ( cell line collection , lombardi comprehensive cancer center , passage 9 ) , and rat retinal r28 ( generous gift of gail seigel , passages 4755 ) , were cultured in dmem medium . human retinal pigment epithelial arpe-19 cells ( american type culture collection , passages
media were supplemented with 10% of fetal bovine serum ( fbs ) and 1% penicillin / streptomycin and cultures were incubated at 37c with 5% co2 .
recombinant human pedf was purified from the culture media of bhk cells harboring an expression plasmid containing full - length pedf cdna .
the culture media was concentrated by ammonium sulfate precipitation and subjected sequentially to cation- and anion - exchange column chromatography as described before with the following modifications .
cation - exchange column chromatography was performed using a poros s resin connected to a biocad 700e perfusion chromatography system , with buffer s ( 20  mm na phosphate , ph 6.5 , 50  mm nacl ) and elutions were with a linear gradient of 50500  mm nacl in buffer s. pedf - containing fractions were pooled , dialyzed against buffer q ( 50  mm tris - hcl , ph 8) and subjected to poros q column chromatography in buffer q and elutions were done with a linear gradient of 100300  mm nacl in buffer q. the pedf - containing fractions were pooled , concentrated , and the buffer exchanged to pbs using ultrafiltration devices ( centricon-30 or amicon-30 , millipore ) .
proteins were analyzed by sds - page using 1020% polyacrylamide in sds - tricine gels ( invitrogen ) or nupage 10% polyacrylamide gel in bis - tris buffer with nupage mops - sds as running buffer ( invitrogen ) under reducing conditions , and isoelectric focusing ( ief ) .
after separation by sds - page electrophoresis , proteins were transferred to a nitrocellulose membrane , blocked for 1  h at room temperature , and incubated with polyclonal antibody to pedf ( bioproducts md , inc . ) in blocking solution at 1  :  5,000 or 1  :  10,000 dilution , followed by secondary antibody anti - rabbit igg ( h + l ) in a 1  :  1000 dilution and the vectastatin abc kit ( vector laboratories ) with colorimetric detection reagent 4-chloro-1-naphthol ( biorad laboratories ) as substrate .
alternatively , the secondary antibody was affinity - purified peroxidase - labeled goat anti - rabbit igg ( h + l ) in a 1  :  200,000 dilution , and chemiluminescence detection with super signal west dura extended duration substrate ( thermo scientific ) on x - ray films .
protein concentrations were determined using the protein assay kit ( biorad ) and beckman du 640 spectrophotometer .
protein concentration was calculated from absorbance values using the formula : 
 ( 1)[absorbance595  nm3.5  g  bsa ]  [sample  volume  ( l)0.2 ]  = protein  concentration  ( mg / ml ) . 
 
isoelectric focusing was performed using ph 37 or ph 310 ief gels ( invitrogen ) , following manufacturer 's instructions .
two micrograms of protein was treated with n - glycosidase f ( new england biolabs , ipswich , ma ) following a previously described method .
briefly , the protein sample was denatured by boiling in a solution containing 0.5% sds , 40  mm dtt . to avoid inactivation of the enzyme by sds , a total of 1% np-40 in 50  mm sodium phosphate ,
ph 7.5 was added to the denatured sample before adding 1000 units of n - glycosidase f in 20  l final reaction volume .
enzymatic reactions were incubated at 37c for 1  h followed by addition of sds - page sample buffer and samples were boiled for 10  min .
reaction mixtures contained chymotrypsin ( worthington , lakewood , nj ) and pedf substrate : at a w / w ratio of 1  :  10 in 80  mm tris - hcl , ph 7.5 and 100  mm cacl2 .
the reactions were stopped by freezing in dry ice and the addition of sds - page sample buffer .
the cells were plated on a microplate biosensor platform and real - time electrical impedance of cells was followed with rt - ces system from acea biosciences ( san diego , ca ) ( http://www.aceabio.com ) , a system described previously [ 25 , 26 ] .
t24 , 231-br , and 4t1-br5 cells were seeded at 6,000  cells per well ( a 96-well format ) and allowed to attach for 18  h. then the cells were serum - starved for 8  h followed by addition of serum - free media containing pedf .
r28 cells were seeded at a density of 3000 cells / well on 16-well strips of 96-well format with microelectrodes .
the cells were allowed to attach for 8  h with media containing 5% fbs .
this was followed by changing to media with or without pedf at the desired concentration .
real - time electrical cell impendence was monitored every hour in each well for several hours .
data from two replicates was averaged . at the end point of the rt - ces assay , cell viability was measured by determining the relative levels of intracellular atp as a biomarker for live cells using a celltiter - glo kit ( promega , madison , wi ) and following instructions by manufacturer . after 30  min of incubation at room temperature ,
the incubation solution in each well was transferred into wells in a 96-well microtiter plate .
the luminescence intensity was measured using an automated plate reader ( envision , perkin elmer , ma ) .
in parallel experiments , cells cultured in 24-well plates in triplicates were imaged and at the end point , cell viability was measured by determining the relative levels of mitochondrial dehydrogenase activity as live cell biomarker using the cell counting kit-8 ( dojindo ) following instructions by manufacturer .
cells in each well were incubated with 50  l of cck-8 solution diluted 1  :  25 and incubated for 4  h at 37c .
absorbance of each well was measured at 450  nm using an automated plate reader ( envision ) . in all the cases ,
data from replicates were averaged and statistical analysis was performed by a t - test . a p value of < 0.05 was taken as significant .
confluent cultures of arpe-19 cells on 24-well culture plates and of mda - mb-231 cells in 6-well plates were scratched with 10-l pipette tips to create fixed - width linear 
wounds  in the cell monolayers , followed by incubation with medium containing indicated concentrations of pedf .
cell migration was monitored by capturing the images of linear wound closure using a 2x brightfield objective in an olympus 1  70 inverted microscope coupled with a sony camera .
images were taken immediately after wounding and later at defined time intervals until complete closure in the control cultures .
matrix assisted laser desorption ionization time of flight ( maldi - tof ) mass spectrometry was performed to determine the average molecular mass of the two pedf protein forms .
purified samples of pedf-1 ( 0.8  g/l ) and pedf-2 ( 2.5 or 1  g/l ) in pbs were mixed 1  :  3 ( v / v ) with a saturated solution of sinapinic acid in 50% acetonitrile , 0.3% trifluoroacetic acid , and 0.5  l of each sample was spotted onto a stainless steel maldi plate .
mass spectra were manually acquired with 100 laser shots / spectrum using a maldi voyager de - str ( ab sciex ) in positive ion , linear mode .
the spectra were calibrated using a mixture of bsa and igg calibration standards ( ab sciex ) .
additionally , some samples were mixed 1  :  1 ( v / v ) with the standard and calibrated internally , postacquisition .
molecular masses of both forms of pedf were determined by averaging the means of 5 spectra collected from each of three sample spots .
human recombinant pedf was purified from culture media by a two - step procedure involving sequential cation- and anion - exchange column chromatography .
the chromatogram from the cation - exchange column showed one broad peak of pedf eluting with 200250  mm nacl , which was pooled and prepared for anion - exchange column chromatography .
the anion - exchange column chromatogram showed two main protein peaks ( figure 1 ) . using conductivity values for the fractions ( 18.3  ms and 21.96  ms )
we determined that protein in the first and second peaks eluted with about 200  mm nacl and 253  mm nacl , respectively .
isoelectric focusing of the proteins revealed a higher pi value for the protein in the first peak ( peak 1 ) ( pi = 7 ) than in the second one ( peak 2 ) ( pi = 6.1 ) . proteins in fractions from the two peaks contained pedf - immunoreactive bands migrating as ~50,000-mr by sds - page ( see figure 3 ) .
in addition to the ief gel , coomassie blue stained sds - page gels demonstrated that the pedf forms were highly pure , and the proteins from the first peak migrated slightly faster than those in the second peak ( see figure 3 ) .
the protein eluting with lower ionic strength was termed pedf-1 , and the version eluting with higher ionic strength was labeled pedf-2 . as determined by maldi - tof mass spectrometry ,
the average molecular mass ( mean  standard error ) of pedf-1 was 46,063  13  da and the molecular mass of pedf-2 was 47,176  87  da , in agreement with the differences in migration by sds - page . to find out how surface exposed charges affect fractionation of pedf
, alterations at positively charged residues k146/k147/r149 and k189/k191/r194/k197 of pedf to neutral side chain residue alanine were prepared .
fractionation by anion - exchange column chromatography showed that in both cases two peaks of pedf were eluted with conductivity values similar to those of wild - type pedf ( figure 2 ) .
recombinant pedf fused with two tandem repeats of flag peptide at the carboxy - end produced by hek cells   was also subjected to anion - exchange column chromatography under identical conditions as above . flag peptide ( dykddddk ) with several acidic amino acid residues would increase the overall negative charge of the pedf protein .
we found that flag - pedf eluted in two peaks with higher ionic strength than the untagged pedf ( with 316  mm nacl and 367  mm nacl , resp . )
these results indicate that recombinant human pedf contains at least two isoforms varying in charge and in apparent molecular weight , and that this characteristic is general . to determine whether the glycosylation attachments on the pedf-1 and pedf-2 polypeptide backbones are involved in their differences , the proteins were treated with n - glycosidase f to release asn - linked oligosaccharides from glycoproteins .
figure 3 shows that n - glycosidase f digested both pedf-1 and pedf-2 causing them to migrate faster by sds - page .
it indicates that the enzymatic treatment decreased their apparent molecular weight by identical values of ~8000 .
both pedf forms were glycosylated , and the deglycosylated pedf-1 still migrated slightly faster than pedf-2 by sds - page .
these observations demonstrate that each pedf-1 and pedf-2 molecule had similar carbohydrate content of about 15% attached to an internal asparagine residue and that it does not account for the differences between the two forms pedf-1 and pedf-2 .
treatment with limiting amounts of chymotrypsin revealed that both pedf forms had identical patterns after digestion .
by incubation with chymotrypsin at a protease  :  substrate ratio of 1  :  10 ( w / w ) , both pedf forms of about 50  kda were digested in a one - step fashion into a product of about 46  kda , that is , a decrease of ~4  kda .
a band corresponding to the released low - molecular - weight peptide was not readily identified by coomassie blue staining of the gel . given that pedf has affinity for heparin , we examined binding to heparin by both forms .
heparin affinity column chromatography showed that both forms bound to heparin - conjugated beads with similar affinity ( figure 4(b ) ) .
hyaluronan binding assays were performed with pedf-1 and pedf-2 ( data not shown ) and showed similar results .
altogether , the results indicate that there are no differences in the overall protein conformation and in binding affinity to glycosaminoglycans between the two pedf forms . to compare the antitumorigenic effects of pedf-1 and pedf-2 , t24 , 231-br , and 4t1-br5 cell cultures were incubated with serum - free medium containing increasing concentrations of each pedf form , and monitored , in parallel , in real time , for cell impedance using a microelectronic system and under the microscope .
cells treated without pedf increased the electrical impedance represented as cell index value with time up to 7296  h. however , the cell index decreased in those treated with pedf-2 in a dose - dependent fashion , while this was not observed with pedf-1 ( figure 5 ) , suggesting a differential effect on lowering the cell number .
examination under the microscope revealed a decline in cell number with increasing pedf-2 additions and not with pedf-1 with the three types of cells ( figure 6 ) .
the numbers of cells with 100  nm pedf-2 in each case were significantly decreased in each field relative to those without or with pedf-1 .
quantification of relative cell numbers using two different biomarkers for live cells , intracellular atp content , and mitochondrial dehydrogenase activity at end point corroborated the observation that pedf-2 decreased the viability of each cell type , while pedf-1 did not have a significant effect ( figure 7 ) .
altogether , treatment with pedf-2 resulted in a dramatic direct negative effect in tumor cell viability in contrast to pedf-1 .
it has been reported that pedf inhibits the migration of breast tumor mda - mb-231 cells and retinal pigment epithelial ( rpe ) cells [ 20 , 27 ] .
thus , the effect of the pedf-1 and pedf-2 proteins on cell migration was examined in mda - mb-231 cells and arpe-19 cells . for this wound healing assay , a fixed - width scratch in a cell monolayer was created and the advancement of the migrating front was followed in the presence of either pedf-1 or pedf-2 .
pedf-2 affected negatively the migration of the mda - mb-231 cells , while pedf-1 was not significant ( figure 8(a ) ) . wound healing assays performed with arpe-19 cells showed similar results as with the cancer cells ( figure 8(b ) ) .
these observations indicate that pedf-2 was more efficient than pedf-1 in inhibiting cancer and rpe cell migration .
the above results suggest that pedf-2 is more efficient than pedf-1 , implying that pedf-1 may have lost its activity .
it has been reported that pedf also protects retina cells against trophic factor depletion [ 28 , 29 ] .
thus the activity of pedf-1 was also examined using a method in which pedf protects retina cells against death induced by serum starvation .  
figure 9 shows cell viability of retina r28 cells treated with increasing amounts of pedf in serum - free media for 48 hours .  
figure 9(a ) shows that real - time cell index of r28 cells decreased almost linearly between the 20th hour and the 48th hour after plating .
however treatments with increasing concentrations of pedf-1 prevented the cell index decrease , which with the highest dose of pedf ( 100  nm ) even increased from the 20th to the 35th hour after plating .
quantification of relative cell numbers at end point using intracellular atp content as biomarker of live cells corroborated the observation that pedf-1 increased viability of r28 cells in a dose - dependent fashion ( figure 9(b ) ) .
the effects on the retina survival activity of pedf-2 were similar to those of pedf-1 ( figures 9(c ) and 9(d ) ) .
altogether , the results show that both pedf forms pedf-1 and pedf-2 behaved as active survival factor for retina cells in culture .
we have identified two distinct biochemical forms of recombinant pedf , which we termed pedf-1 and pedf-2 .
the distinction is based on ionic potential , pi values , and apparent molecular weight and demonstrates that pedf-2 has higher overall negative charge than pedf-1 .
pedf-2 elution required higher ionic potential from anion - exchange column chromatography ; the protein has a lower pi and migrated slower in sds - page than pedf-1 .
this characteristic is shared with pedf from different sources and even when it is altered or fused to tags . altered and flag - tagged
pedf yielded two peaks similar to the wild - type pedf ( figure 2 ) , confirming that the existence as two forms is a general feature of pedf . because the protein derives from one pedf cdna sequence transfected into bhk cells , the sequence of the polypeptide backbone is identical in both forms . upon maturation and secretion ,
the pedf versions may have adopted modifications posttranslationally that confer charge differences , and in turn grant diversity in biological activities to pedf .
recombinant pedf isoforms were reported to have differences in carbohydrate composition at its glycosylation site .
pedf isoforms have also been reported in peripheral neuropathies , with fucose residues on the oligosaccharide chain found only in the isoforms downregulated in neuropathic patients without pain .
although both pedf-1 and pedf-2 are glycosylated , they differ in apparent molecular weight even after deglycosylation , suggesting that this is not the main difference between them .
more recently , it was reported that the triple phosphomimetic pedf , eee - pedf , containing three negatively charged glutamic acids instead of serines , possesses significantly increased antiangiogenic , and anticancer activities than wild - type pedf [ 20 , 31 ] . however , our pedf forms were not phosphorylated ( data not shown ) .
the significance of these findings is that pedf isoforms have been identified in ocular , tumor , and nontumor tissues [ 16 , 17 , 32 ] , and interestingly their distribution varies between normal and tumor tissues .
several reports on posttranslational modifications of pedf offer explanations for the multifunctional modality of pedf .
purified two forms of pedf , a and b , using cation - exchange column .
form a eluted at a lower nacl concentration , that is , less positively charged and had a slightly larger molecular weight than from b ; and pedf a was a more efficient antiangiogenic form than pedf b. this implies a match of pedf-1 to pedf b and pedf-2 to pedf a. petersen et al .   reported that a complete map of all post - translational modifications revealed that authentic plasma pedf carries an n - terminal pyroglutamate blocking group and an n - linked glycan at position asn266 .
they proposed that the pyroglutamate residue may regulate the activity of pedf analogously to the manner in which it regulates thyrotropin - releasing hormone .
maik - rachline et al .   demonstrated that extracellular phosphorylation converts pedf from a neurotrophic to an antiangiogenic factor .
later they proposed that differential phosphorylation induces variable effects of pedf , and therefore contributes to the complexity of pedf action .
although the observations with pedf phosphomimetics match our results , our pedf forms were not found to be phosphorylated and therefore , another chemical difference is sought for pedf-1 and pedf-2 that is yet to be determined .
these observations are in agreement with the idea that the multifunctional modality of pedf may be explained by differences in posttranslational modifications of the pedf polypeptide , which may regulate their biological activities . given that synthetic peptides 34-mer and 44-mer derived from pedf are biologically active , these regions may require to be properly exposed in the folded protein to interact with cell surface receptors and trigger the necessary signals for activity .
posttranslational modifications may be tools to open up or expose the active regions in the folded pedf protein .
it is envisioned that in pedf-2 the active region for antitumorigenic activity is more exposed than in pedf-1 .
other factors that enhance efficacy in pedf-2 may include post - translational modification(s ) that confers an increase in negative charge to the protein .
further studies to identify the chemical differences between the pedf forms will be of great interest for the development of second generation pedf molecules .
 
 purpose . 
 pigment epithelium - derived factor ( pedf ) is a multifunctional serpin . 
 the purpose of this study is to identify pedf protein forms and investigate their biological activities on tumor cell lines . 
 methods . 
 recombinant human pedf proteins were purified by cation- and anion - exchange column chromatography . 
 they were subjected to sds - page , ief , deglycosylation , heparin affinity chromatography , and limited proteolysis . 
 cell viability , real - time electrical impedance of cells , and wound healing assays were performed using bladder and breast cancer cell lines , rat retinal r28 , and human arpe-19 cells . 
 results . 
 two pedf protein peaks were identified after anion - exchange column chromatography : pedf-1 eluting with lower ionic strength than pedf-2 . 
 pedf-1 had higher pi value and lower apparent molecular weight than pedf-2 . 
 both pedf forms were glycosylated , bound to heparin , and had identical patterns by limited proteolysis . 
 however , pedf-2 emerged as being highly potent in lowering cell viability in all tumor cell lines tested , and in inhibiting tumor and arpe-19 cell migration . in contrast , pedf-1 minimally affected tumor cell viability and cell migration but protected r28 cells against death caused by serum starvation . 
 conclusion . 
 two distinct biochemical forms of pedf varying in overall charge have distinct biological effects on tumor cell viability and migration . 
 the existence of pedf forms may explain the multifunctional modality of pedf . 
1. Introduction
2. Methods
3. Results
4. Discussion