Comparison of two neurotrophic serpins reveals a small fragment with cell survival activity

Amino acid sequence alignments

Amino acid sequence alignments between human PEDF (human PEDF: pigment epithelium-derived factor precursor [Homo sapiens] NCBI Reference Sequence: NP_002606.3), human PN-1 (glia-derived nexin isoform a precursor [Homo sapiens] NCBI Reference Sequence: NP_006207.1), mouse PEDF (pigment epithelium-derived factor precursor [Mus musculus] NCBI Reference Sequence: NP_035470.3), and mouse PN-1 (glia-derived nexin precursor [Mus musculus] NCBI Reference Sequence: NP_033281.1) were performed using the ClustalW2 Multiple Sequence Alignment EMBL-EBI program.

Tertiary structure comparison

Tertiary structures of human PEDF (PDB: 1IMV_A) and human PN-1 (PDB: 4DY7_C) were evaluated by comparing their crystal structures and corresponding sequence alignments on the Cn3D macromolecular structure viewer (Version 4.3).

cDNA synthesis

RNA was obtained from RPE and retinas dissected from eyes of C57BL/6N [Crb1^rd8] mice and human ARPE-19 cells and retinas (Clontech, Mountain View, CA). An oligo(dT) probe was used to reverse-transcribe mRNA from cell samples in a final volume of 20 µl using SuperScript first-strand synthesis system (Life Technologies, Frederick, MD) following the manufacturer’s instructions.

RT–PCR

Specific primers for screening the expression of human SERPINE2 were huPN-1-forward 5′-CCG CTG AAA GTT CTT GGC A-3′ and huPN-1-reverse 5′-CAG CAC CTG TAG GAT TAT GTC G-3′. Specific mouse Serpine2 primers were mPN-1-forward 5′-CAG TGT GAA GTG CAG AAT GTG A-3′ and mPN-1-reverse 5′-TTG GGG AAA GCA GAT TAT CAA-3′. Templates were cDNAs prepared from the cells and tissues in HotStarTaq DNA Polymerase reactions following the manufacturer’s instructions (Qiagen, Valencia, CA). Reverse-transcription PCR was performed in a final volume of 50 µl containing 2 µl of cDNA and 10 µM primers as listed above using a thermocycler (MJ Research PTC-200 DNA Engine; Bio-Rad, Hercules, CA) according to the following program: initial denaturation for 15 m at 95 °C, 60 cycles of denaturation for 1 m at 95 °C, annealing for 30 s at 58 °C, extension for 30 s at 72 °C, and a 5 m hold at 72 °C. PCR products were resolved by electrophoresis in E-Gel® Agarose Gels, 2%, containing ethidium bromide for visualization (Life Technologies). For quantitative RT–PCR, total RNA was purified using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. Total RNA (0.1–1 μg) was used for reverse transcription using the SuperScript III First-Strand Synthesis System (Invitrogen). Bcl2 mRNA levels were normalized to 18S levels with quantitative RT–PCR using SYBR Green Mix (Applied Biosystems, Foster City, CA) in the Bio-Rad Chromo4 Real-Time PCR System. The primers used were as follows: rat Bcl2, 5′-TGG ACA ACA TCG CTC TGT GGA TGA-3′ (forward) and 5′-GGG CCA TAT AGT TCC ACA AAG GCA-3′ (reverse); and 18S, 5′-GGT TGA TCC TGC CAG TAG-3′ (forward) and 5′-GCG ACC AAA GGA ACC ATA AC-3′ (reverse).

Proteins and peptides

Human recombinant wild-type (WT) PN-1 and human recombinant PN-1[R346A] rendered inactive by a mutation of the reactive site were prepared from Escherichia coli containing expression vectors as previously described [10,23]. Briefly, the cDNA coding sequence for human PN-1 was inserted into pGEX-6P-1 vector resulting in a fusion gene construct that encoded glutathione S-transferase (GST)-PN-1. PN-1 mutagenesis was performed to change the arginine (R346) residue of the reactive site to alanine. The constructs were expressed in BL21(DE3) E. coli strain. After induction of expression of the recombinant gene, GST-PN-1 was purified from cell lysate by glutathione-Sepharose affinity chromatography. On-column cleavage of the protein from GST was performed to yield PN-1 and PN-1[R346A] proteins. Human recombinant PEDF was prepared from stably transfected baby hamster kidney cells containing a PEDF expression vector as previously described [24]. The recombinant protein was purified from culturing media of the cells by ammonium sulfate precipitation, cation exchange chromatography followed by anion exchange chromatography. Peptides 44mer and 17mer derived from PEDF were synthesized as previously described [19,20]. These peptides were chemically synthesized by a commercial source (Biosynthesis, Lewisville, TX). Peptide PN-1-17mer, a peptide designed from the homologous neurotrophic region of PEDF, corresponds to the human PN-1 polypeptide sequence Gly⁷¹-Gly⁸⁷ (GRTKKQLAMVMRYGVNG) and was chemically synthesized (Biosynthesis, Lewisville, TX).

Cell culture

ARPE-19 cells, a spontaneously arising human RPE cell line (American Type Culture Collection (ATCC), Manassas, VA), were cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12, HEPES, catalog # 11330057, ThermoFisher, Waltman, MA) with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cell line STR Validation analysis was performed by BioSYNTHESIS (catalog # CL1003, Lewisville, TX; Appendix 1). ARPE-19 cells in passage numbers 30 to 35 were used. R28 cells, an immortalized retinal progenitor cell line derived from the neonatal rat retina [25] (Kerafast, Boston, MA), were validated by IDEXX BioResearch (Columbia, MO; Appendix 2). Cells were cultured in DMEM with 10% FBS, 3% sodium bicarbonate, 1% MEM nonessential amino acids (Catalog # 11140050, ThermoFisher Scientific), 1% minimum essential medium (MEM) vitamins (Catalog # 11120029, ThermoFisher Scientific), 1% L-glutamine (Catalog # 25030-024, ThermoFisher Scientific), and 0.125% gentamicin (Sigma-Aldrich, Catalogue # G1397) at 37 °C with 5% CO₂ and 95% humidity. Cells were revalidated at passage number 42, and all experiments were performed using cells in passage numbers 37 to 45. The cells were maintained in a T75 flask at 70–75% confluency.

Processing of ARPE-19 conditioned media and lysates

ARPE-19 cells were cultured in a six-well plate until confluent and serum-starved for 5 days. The media were collected and concentrated tenfold (from 1 ml to 100 µl) in Amicon Ultra Centrifugal Filters Ultracel-30K Membrane (Millipore, Temecula, CA) by centrifugation on the Beckman Allegra 6KR Kneewell Refrigerated Centrifuge (Analytical Instruments LLC, Minneapolis, MN) for 10 m at 4 °C. Wells containing the ARPE-19 cells were washed twice with cold PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, pH 7.4). Then 300 µl cold RIPA Lysis and Extraction buffer (Thermo Fisher Scientific, Catalogue # 89,900, Waltham, MA) was added to each well, and the plate was incubated on ice for 5 m. Cell lysates were collected in microcentrifuge tubes and sonicated for 20 s with a 50% pulse. Sonicated samples were centrifuged at maximum speed at 4 °C for 15 m to pellet any cell debris. Supernatants were concentrated as described above. All media and lysate samples were stored at −20 °C. Proteins were analyzed with western blotting against human serpin E2/PN1 antibody goat Ab-PN-1 (polyclonal goat IgG by R&D Systems, Catalogue # AF2980, Minneapolis, MN)) at 50 ng/ml (diluted in 1% bovine serum albumin/Tris-buffered saline/Tween 20 [BSA/TBST]) and anti-goat immunoglobulin (IgG) (H⁺L) antibody, human serum absorbed and peroxidase labeled secondary (KPL, Gaithersburg, MD, 14–13–06) diluted 1:200,000. Western blotting against anti-PEDF were incubated in rabbit AB-PEDF (BioProducts MD, Middletown, MD) at 1:100,000 in 1% BSA/TBST, followed by secondary antibodies peroxidase anti-rabbit IgG (KPL) diluted 1:200,000. Signaling was detected using West Dura Super Signal (ThermoFisher) and exposed to X-ray films.

Preparation of bovine IPM wash

All preparation procedures were performed at 4 °C. Adult bovine eyes were obtained from J. W. Trueth & Sons (Baltimore, MD). The soluble components of the IPM were obtained with the “no-cut” method described by Adler that ensures that the extracellular fluid is free of significant cellular contamination [26]. Each eye was dissected as follows: The anterior segment and the vitreous were removed from each eye, and a solution of PBS was gently introduced between the neural retina and the RPE with a needle at 0.5 ml per eye. The IPM wash was extracted and centrifuged at 1,000 xg for 10 min to remove cellular debris, and the supernatant was passed through a 0.45 µm syringe filter and stored at −80 °C. This filtrate constituted the IPM wash.

Bovine retinal extracts

All preparation procedures were performed at 4 °C as described by Aymerich et al. [27]. Fresh adult bovine eyes (J. W. Trueth & Sons) were dissected below the iris, the vitreous was removed from the inner retinal surface, and the neural retinas were gently separated from the pigment epithelium with forceps. Retinas were homogenized in a solution of cold 0.32 M sucrose in Tris-buffered saline (TBS; 20 mM Tris/HCL [pH 7.5] and 150 mM NaCl) containing protease inhibitors (1 mM aminoethyl-benzenesulfonyl fluoride hydrochloride [AEBSF], 5 µg/ml aprotinin, 1 µ/ml pepstatin, and 0.5 µg/ml leupeptin) at 7.5 ml per retina with a homogenizer (Polytron model 3000; Brinkman Instruments, Westbury, NY) set at 10,000 rpm for 20 s. The homogenized material was separated from tissue and cellular debris by centrifugation at 1,000 ×g for 10 m.

TUNEL assay

Rat retina R28 cells (1.5 × 10⁴/well) were cultured in an eight-well Nunc chamber slide for 16 h in media containing 5% FBS. The media were then replaced with serum-free media containing effectors and incubated at 37 °C for 48 h. Cell death was evaluated at the end point by performing the terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay using the ApopTag^® Fluorescein in situ Apoptosis Detection Kit (Millipore, Billerica, MA) following the manufacturer’s protocol. The nuclei were counterstained with Hoechst, and images were taken using a Zeiss (Peabody, MA) Imager Z1 fluorescence microscope. Five different fields per treatment were imaged from two independent wells in each experiment. Cells were counted with ImageJ version 1.42 using fixed threshold values within each experiment to quantify based on intensity.

Statistical analyses

Data are shown as the mean ± standard deviation (SD). The data were analyzed with the two-tailed unpaired Student t test. A p value of less than 0.05 was considered statistically significant.

Primary and tertiary structural comparison between PEDF and PN-1

We used the ClustalW2 Multiple Sequence Alignment EMBL-EBI program to compare the amino acid sequences of human and mouse PEDF and PN-1 (Figure 1). With 25% identity at the primary level between PN-1 and PEDF, the sequences feature a conserved set of residues that predominantly governs the integrity of the spatial structure of serpins and biologic activity [28]. The presence of this common set, comprised of approximately 51 invariable residues, ensures proper folding of the native, metastable state protein [29]. Human and mouse PN-1 sequences contain 36 and 37 residues identical to those in this set, respectively, of which six residues have strongly similar properties to those in the set (Figure 1). Both proteins were crystallized, and the 3D structures have extensive overlap between the alpha helical and beta sheet regions (Figure 2). They contain exposed RCLs of 15 residues, in which P1, the protease specificity-determining site, is an arginine in PN-1 and a leucine in PEDF [30,31]. There is a degree of sequence similarity in the 17mer ligand-binding region of PEDF to its receptor PEDF-R [22]. Recent alanine scanning studies revealed that R99 of human PEDF within the 17mer region is crucial for receptor binding and survival activity, and an alteration of H105A increases the affinity for PEDF-R tenfold [20]. Alignment and comparison show that both requirements are present in the corresponding positions of human and mouse PN-1 (R/K at 72 and A at 78). Other residues crucial for receptor binding in PEDF (S102, I104, and Y110; [22]) are also present in human PN-1 as their semiconservative and conserved residues K, L, and Y, respectively. Overall, the distributions of alpha helices and beta sheets are consistent between the structures and are typical of the serpin protein superfamily. The PEDF 17mer and the corresponding region in PN-1 are visible in alpha helix C of the crystal structures presented in Figure 2 [32,33].

PN-1 in ocular tissue

We assessed the presence of SERPINE2 transcripts in ocular tissues via RT–PCR. We used RNA from the mouse RPE and retina, human retina, and the spontaneously arising RPE cell line ARPE-19. Using specific primers for mouse Serpine2, the expected amplified fragment of 112 base pairs was detected with cDNA derived from murine RPE and retina in agarose gels (Figure 3). Similarly, the expected DNA fragments of 247 base pairs were amplified and detected from cDNAs of human ARPE-19 and from cDNAs of native human retina cDNA using specific primers for human SERPINE2 (Figure 3).

Selbonne et al. reported the presence of PN-1 in the mouse retina [34]. Western blots of protein extracts from the mouse retina with a specific antibody to PN-1 showed a main immunoreactive band for PN-1 protein (Figure 4) confirming what was previously reported [34]. An ARPE-19 cell culture was prepared with serum-free media conditioned for 5 days. After the conditioned media were removed, the cells were washed with 2 M NaCl in PBS to isolate proteins attached by ionic interactions to the cells and the ECM. The cells were also collected, and protein extracts were prepared. Western blots of conditioned media, the salt wash, and cell lysates showed PN-1-immunoreactive bands (50 kDa) only in the ARPE-19 cell lysates (Figure 4). The purified, bacterially derived, recombinant human PN-1 migrated slightly faster (40-kDa), as it corresponds to an unglycosylated form of the PN-1 polypeptide. The slower migration of ARPE-19 PN-1 is likely due to post-translational modification. PEDF was detected only in the conditioned media.

As murine eye tissue yielded insufficient sample material to further evaluate physiologic PN-1, we implemented a previously established protocol describing the purification of PEDF from the bovine IPM in an effort to investigate the presence of the native ocular PN-1 protein [26]. Western blots of native bovine RPE and retina extracts revealed the presence of PN-1 bands with the expected migration patterns (Figure 5A). An additional slower migrating PN-1-immunoreactive band (approximately 50 kDa) was detected in the bovine retina, which was present also in the mouse retina but at a lower level. Given that PN-1 associates with ECM components via ionic interactions [4], we used 1 M NaCl to remove proteins bound to the IPM. We found PN-1 in IPM lavage with PBS, which increased in lavages with 1 M NaCl in PBS. The bacterially derived recombinant human PN-1 served as a positive control. To compare the distribution of PN-1 in the bovine ocular samples with those of PEDF, western blots of the same samples were performed with an antibody to PEDF (Figure 5B). PEDF was similarly detected in the bovine IPM lavage extracted with 1 M NaCl in PBS but was detected less intensely in the RPE and retina tissue extracts.

Cytoprotective activity of PN-1 on retina cells

We have demonstrated that PEDF protects retina precursor R28 cells against serum deprivation-related injury [22]. To evaluate the retinoprotective activity of PN-1 on R28 cells, we set to determine the extent to which PN-1 can prevent cell death in serum-deprived R28 cells. Cells were treated with serpins upon serum removal, and death was followed by TUNEL labeling of the nuclei in the dying R28 cells, as previously described [24]. The treatment conditions employed were 2.5 nM and 10 nM of PN-1, as well as PEDF at concentrations known to exert a cytoprotective effect in the cells. Given that the cytoprotective action of PEDF is independent of its inhibitory potential against proteases, we tested PN-1 versions without inhibitory activity against serine proteases, such as PN-1[R346A] and PN-1-17mer. PN-1[R346A] is a recombinant PN-1 with a single residue alteration that abolishes the serine protease inhibitory capacity [10]. PN-1-17mer is a peptide designed from the homologous neurotrophic region of PEDF and distant from the RCL [20]. Figure 6A shows representative images of TUNEL-positive nuclei of R28 cells treated with PN-1 versions and PEDF at 2.5 nM and the cultures that remained untreated without serpins (None), as well as 4',6-diamidino-2-phenylindole (DAPI)-stained nuclei for the total number of cells. A lower number of TUNEL-positive nuclei was observed when the cells were treated with the PN-1 versions relative to the untreated cells, mirroring the protective effects of PEDF. Quantitation of the fluorescent images revealed a significant decrease in TUNEL-positive nuclei/total number of cells in cultures treated with PN-1 when compared to the cultures that were without serpins (None). Given that death by serum deprivation-mediated injury ranged between 4% and 10% from cell batch to cell batch, the number of TUNEL-positive nuclei/total cells was normalized to the cell death of those in untreated cultures (None) as 100% within each of three experiments, and the averages are plotted in Figure 6B. Wild-type PN-1 and PN-1[R346A], each at 2.5 nM or 10 nM, decreased the percentage of TUNEL-positive cells to 46–57% from those without effectors (100%). The cells treated with the 2.5 nM PN-1-17mer peptide exhibited an even further decrease in the percentage of dead cells (37%) and then less effectively with 10 nM peptide (62%). Cell death was also diminished to 32% in the presence of 10 nM PEDF (Figure 6C), a bit more effective than those with PN-1. We conclude that PN-1 with and without protease inhibitory capacity and even the 17mer of PN-1 protected serum-deprived R28 cells.

We have shown previously that atglistatin, a selective and competitive inhibitor of the lipase enzymatic activity of PEDF-R, attenuates the PEDF-mediated cytoprotective activity in R28 cells [20]. We used atglistatin to investigate its effects on PN-1-mediated cytoprotection. R28 cells were incubated in media with atglistatin before the effectors were added. Cell death among cell batches in three experiments ranged between 9% and 12%. As shown in Figure 6C, atglistatin completely abolished the survival activity of PEDF, but no effect on PN-1 and PN-1-17mer was observed. Atglistatin treatment alone did not affect the number of TUNEL-positive cells. Altogether, although PN-1 was similar to PEDF in that the protease inhibitory activity was dispensable for cytoprotection, PN-1 differed from PEDF as PN-1 promoted cell survival by a mechanism of action likely independent of the enzymatic activity of PEDF-R.

Antiapoptotic Bcl2 expression in retinal R28 cells

We and others have shown that PEDF protects retina R28 cells from serum-starvation injury by induction of the antiapoptotic Bcl2 gene [22,35]. We examined the Bcl2 expression of serum-starved R28 cells treated with PN-1. As shown in Figure 7, antiapoptotic Bcl2 expression increased upon treatment with PN-1, PN-1[R346A], and PN-1-17mer relative to untreated cells (None). FBS, PEDF, and the neurotrophic PEDF-derived peptides 44mer (a fragment spanning positions 78 and 121 of the human PEDF polypeptide) and 17mer were positive controls that induced Bcl2 by more than twofold. We conclude that similar to PEDF, PEDF-44mer, and PEDF-17mer, protection from serum-starvation injury was accompanied by induction of the antiapoptotic Bcl2 gene expression in R28 cells treated with PN-1 and PN-1-17mer.