Abstract Pikachurin is a recently identified, highly conserved, extracellular matrix-like protein. Murine pikachurin has 1,017 amino acids (∼110 kDa), can bind to α-dystroglycan, and has been found to localize mainly in the synaptic cleft of photoreceptor ribbon synapses. Its knockout selectively disrupts synaptogenesis between photoreceptor and bipolar cells. To further characterize this synaptic protein, we used an antibody raised against the N-terminal of murine pikachurin on Western blots of mammalian and amphibian retinas and found, unexpectedly, that a low weight ∼60-kDa band was the predominant signal for endogenous pikachurin. This band was predicted to be an N-terminal product of post-translational cleavage of pikachurin. A similar sized protein was also detected in human Y79 retinoblastoma cells, a cell line with characteristics of photoreceptor cells. In Y79 cells, endogenous pikachurin immunofluorescence was found on the cell surface of living cells. The expression of the N-fragment was not significantly affected by dystroglycan overexpression in spite of the biochemical evidence for pikachurin-α-dystroglycan binding. The presence of a corresponding endogenous C-fragment was not determined because of the lack of a suitable antibody. However, a protein of ∼65 kDa was detected in Y79 cells expressing recombinant pikachurin with a C-terminal tag. In contrast, in QBI-HEK 293A cells, whose endogenous pikachurin protein level is negligible, recombinant pikachurin did not appear to be cleaved. Instead pikachurin was found either intact or as dimers. Finally, whole and N- and C-fragments of recombinant pikachurin were present in the conditioned media of Y79 cells indicating the secretion of pikachurin. The site of cleavage, however, was not conclusively determined. Our data suggest the existence of post-translational cleavage of pikachurin protein as well as the extracellular localization of cleaved protein specifically by retinal cells. The functions of the pikachurin N- and C-fragments in the photoreceptor ribbon synapse are unknown.

Citation: Han J, Townes-Anderson E (2012) Cell Specific Post-Translational Processing of Pikachurin, A Protein Involved in Retinal Synaptogenesis. PLoS ONE 7(12): e50552. https://doi.org/10.1371/journal.pone.0050552 Editor: Steven Barnes, Dalhousie University, Canada Received: July 25, 2012; Accepted: October 22, 2012; Published: December 4, 2012 Copyright: © 2012 Han, Townes-Anderson. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Institutes of Health grant EY012031 and the F.M. Kirby Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Understanding the formation and function of the first synapse in the visual pathway- the tripartite ribbon contact between photoreceptors and the second order horizontal and bipolar neurons- is critical to understanding visual processing. Moreover, the integrity of this first synapse is essential since without it almost all vision is lost even if the rest of the visual pathway remains intact. Pikachurin was recently identified as a highly conserved extracellular matrix (ECM)-like protein with a molecular weight around 110 kDa and high mRNA abundance in retina [1], [2]. As seen by immunocytochemistry, pikachurin is present in the synaptic cleft of photoreceptor ribbon synapses in adult murine retina and most importantly its absence specifically disrupts the apposition of bipolar cell dendrites to photoreceptor terminals [1]. Finally, pikachurin has been reported to bind to α-dystroglycan (α-DG) and this interaction has been suggested to contribute to its function in the retina [1]. The characterization of pikachurin, however, is incomplete. Notably, there are to date no Western blot analyses. Thus, although it has been suggested that pikachurin links bipolar cell dendrites to photoreceptors [1], many questions remain. In this study, we began by examining endogenous pikachurin from vertebrate retinas with Western blot analysis. The protein appears more complex than first suggested. We report that the majority of pikachurin in adult retina is post-translationally cleaved resulting in an N-terminal fragment of 60 kDa. In a human retinoblastoma cell line, Y79, the N-terminal fragments were produced from both endogenous and recombinant pikachurin, and were found on the extracellular surface; a C-terminal fragment was additionally demonstrated using recombinant protein. Moreover, when recombinant pikachurin was expressed in Y79 cells, the products of pikachurin cleavage, the N- and C-fragments, along with the whole protein were present in the conditioned medium and apparently highly glycosylated. Our findings suggest that there is post-translational modification of the pikachurin protein in the retina, which may have unique, but as yet unknown, importance to the function of the photoreceptor-bipolar synapse. These results have been previously reported in part at meetings of the Association for Researchers in Vision and Ophthalmology (ARVO).

Materials and Methods Ethics Statement Salamanders and mice were maintained in a central animal facility under pathogen-free conditions. All animal protocols were approved by the UMDNJ Institutional Animal Care and Use Committee. Antibodies The polyclonal antibody against pikachurin [1] was provided by Dr. T. Furukawa (Osaka Bioscience Institute, Japan) in early experiments and purchased from Wako Chemicals USA after it was commercialized. Other primary antibodies used were: c-Myc mAb (Sigma), His-tag pAb (Cell Signaling Technologies), His-tag mAb, α-DG mAb, GFP mAb, GFP pAb, GAPDH mAb (Santa Cruz Biotechnology, Inc.), α-DG mAb (Millipore) and β-DG mAb (Developmental Studies Hybridoma Bank, University of Iowa). Secondary antibodies were conjugated to either Alex Fluor (Invitrogen) or horseradish peroxidase (HRP; Jackson ImmunoResearch Laboratories). Retinas Human retinal tissue was from donor eyes and was provided by Dr. M. Zarbin (New Jersey Medical School- UMDNJ) [3]. Adult porcine eyes were purchased from a local slaughterhouse (Animal Parts, Scotch Plains, NJ), delivered on ice, and retinas were harvested as previously described [4] within 2 hours of death. Salamander retinal tissue was obtained from adult aquatic-phase tiger salamanders and isolated as previously described [5], [6]. Cells Y79 human retinoblastoma cells (ATCC) were grown in suspension in RPMI 1640 supplemented with 10–20% FBS at 37°C with 5% CO2. Before experiments, Y79 cells were plated on poly-L-lysine (Sigma, >300 kDa) at appropriate density to form a monolayer and maintained in DMEM with 2% FBS (D2 Media). In some cases, Y79 cells were cultured in serum free media (N2 media) consisting of DMEM, 10 µg/ml transferrin (Sigma), 5 µl/ml insulin (Sigma), 5 ng/ml sodium selenite (Sigma), 7.3 ng/ml progesterone (Sigma), and 16 µg/ml putrescine (Sigma). To block protease activity in the culture media, a protease inhibitor cocktail (Sigma, P1860, 1∶400 dilution) was included in the media until cell lysates and conditioned media were prepared. QBI-HEK 293A cells (Qbiogene) were cultured in DMEM supplemented with 10% FBS at 37°C with 5% CO2. RT-PCR The PureLink RNA mini kit (Invitrogen) was used to purify total RNA from cultured cells. Pikachurin mRNA expression was examined by RT-PCR (Qiagen OneStep RT-PCR kit). Primers used in the reactions were: forward primer (ACTCCATGGTTATCAAGGGCC), reverse primer (AGGCCAGCTGGTGTTACTTGGC). The PCR product was predicted to be 2472 kb, encompassing most of the encoding sequence of the longest human pikachurin variant (GenBank # NM_001205301). Plasmids Wild type murine pikachurin plasmid was kindly provided by Dr. T. Furukawa [1]. To make a double-tagged pikachurin expression vector, a polyhistidine (6xHis) tag was first introduced to the C-terminus of pikachurin by PCR using primers FP1 (CTCGAGATGCGAACGGCTCTGCGAAAATC) and RP1 (ATCGATTTAATGATGATGATGATGATGCTTAGCCCCACAAGTGTTG). The resulting cDNA, devoid of the signal sequence (SS), was digested with XhoI/ClaI and inserted into a pCIG expression vector to generate plasmid PK-HIS. Next, a cDNA fragment encompassing pikachurin’s SS was cloned into the BamHI/ClaI sites of plasmid pCS2+MT, in which SS is immediately upstream of six copies of c-Myc. Finally, the sequence of SS and 2 copies of c-Myc were amplified by PCR and directionally cloned into the XhoI site of plasmid PK-HIS. In some experiments, a FLAG tag was substituted for c-Myc. Three N-terminal mutants (amino acids 1–354, 1–498, and 1–600) were also amplified by PCR, digested with XhoI/ClaI, and inserted into pCIG vectors. They were designated P39, P55 and P66, respectively, based on their predicted molecular weights. Dystroglycan cDNA (NM_010017 in GenBank) was amplified from mouse eye tissue and cloned into a pCIG vector. Plasmid transfection in both Y79 and QBI-HEK 293A cells was done using Lipofectamine 2000 (Invitrogen) and lasted 48–72 hrs before cells were fixed for immunocytochemistry or lysed for Western blot. Western Blot and Analysis Retinal tissue and cultured cells were lysed on ice in 1xIP buffer (Sigma) supplemented with Complete Protease Inhibitor Cocktail (Roche Applied Science) and phosphatase inhibitors. Typically, 1200 µl of lysis buffer was used for one piece of human or pig retina, 150 µl for salamander retina, and 200 µl for cells cultured on a 35-mm petri dish with >85% confluence. Before lysis, cells were rinsed with PBS twice to remove possible contamination by conditioned media (CM). At the same time, CM was collected, centrifuged, and filtered through a 0.22 µm filter. To maximize the effect of protease inhibitors, tissue and cell lysates were incubated on ice with periodic vortexing for over 1 hr before proceeding to subsequent steps. Western blot was performed as previously described [7]. Briefly, protein samples were first heat-denatured in the presence of β-mercaptoethanol, separated on 3–8% Tris-acetate gel (Invitrogen) and transferred to nitrocellulose membrane. After incubation with blocking buffer (5% non-fat milk in Tris buffered saline) and primary antibodies, target proteins were detected by HRP-conjugated secondary antibodies and Luminata™ Western HRP substrate (Millipore). For quantitive analyses of pikachurin signals in the CM, Western blot results were first scanned to 16-bit Tiff files in grayscale at a resolution of 600 dpi, and the band densities were then determined using Image J software. Statistical analysis was performed with a one-way ANOVA test and a p value <0.05 was considered to be significant. Immunocytochemistry, Quantification of Fluorescence Intensity, and Live Cell Imaging Immunocytochemistry was conducted as previously described [7]. In some experiments, cells were not permeabilized with 0.5% Triton X-100/PBS in order to detect primarily extracellular immunolabeling. After labeling, cells were mounted on slides with Fluoromount-G (SouthernBiotech) and visualized with a 63×oil objective (N.A. 1.4) on a Zeiss Axiovert 135 microscope in ApoTome optical sectioning mode. Fluorescence images, taken using AxioVision 4.5 software (Zeiss), were transferred to ImageJ v1.43 software (NIH) for fluorescence intensity measurement and color coding. The settings for imaging and processing were fixed throughout the experiments. Quantification of the fluorescence intensity was done by first subtracting the background and then measuring the average intensity from the regions of interest that were hand-traced using ImageJ. Results of measurements from multiple cell groups were then compared. A confocal microscope (LSM510; Zeiss) was used to verify key findings obtained with the ApoTome technique. For live cell imaging of pikachurin, cells were cultured on custom made coverslip-bottomed petri dishes. All antibody incubation and rinse steps were performed on ice to minimize internalization of antibodies. Imaging and analysis procedures were as above.

Discussion In this report, we show that a recently identified retinal protein, pikachurin, is post-translationally cleaved in adult retina and human retinoblastoma Y79 cells. This modification is reminiscent of that for dystroglycan. Translated initially as a single protein with signal sequence at its N terminal and a trans-membrane domain at the C terminal, dystroglycan is trafficked into cellular membrane, where its extracellular portion is proteolytically cleaved generating two subunits (α and β). The α-dystroglycan is thus an extracellular protein which interacts with extracellular matrix [12]. Mammalian pikachurin contains an N-terminal signal sequence and has been routinely found in the culture medium of cells expressing the recombinant protein [1], [2], [8], [11]. The analogy with dystroglycan is however only partial. This current study with Y79 cells suggests that endogenous pikachurin mainly underwent cleavage instead of being secreted into the medium as a whole protein. In pikachurin-transfected cells, the cleavage generated two major fragments with a combined molecular weight approximating that of the whole protein. Our immunolabeling data indicate that endogenous N-fragments immobilized extracellularly in Y79 monolayer cell culture (Fig. 2), which is in agreement with previous findings that recombinant pikachurin is able to deposit in the extracellular matrix of differentiated myoblasts [2]. Moreover, the N-fragment of pikachurin very likely contains two type III fibronectin domains (FN3, Fig. 3A). FN3 is an evolutionary conserved protein domain that is often found in multiple repeats in the extracellular regions of various cell adhesion molecules [14]. At present, the function of the endogenous N-terminal fragments of pikachurin in the photoreceptor ribbon synapse is unknown. The presence of endogenous C-terminal fragments remain to be directly determined although cleavage of the exogenous pikachurin indicates that C-terminal fragments are possible. It is noteworthy that enzymatic digestion also took place for secreted pikachurin in conditions of overexpression (Fig. 4). The mechanisms for these cleavage processes are not clear and need further investigation. It has been proposed that pikachurin is an adaptor protein binding to α-DG on one side of the ribbon synapse and with another partner, yet to be identified, on the other side [1]. Despite biochemical evidence demonstrating that pikachurin binds to α-DG through LamG domains at its C-terminus [1], [8], [11], the interaction between these two proteins in a more physiological setting is elusive. First, although the localization of pikachurin in the outer plexiform layer (OPL) of the retina appears to be unambiguous [1], [8], [11], convincing evidence for the presence of α-DG in the OPL is lacking. Montanaro et al. (1995) did present immunofluorescence data showing faint α-DG staining in the OPL, but that labeling was later attributed by one of the authors to anti-α-DG antibody’s cross reactivity with β-DG [15]. Second, immunohistochemical studies demonstrated co-localization of pikachurin and β-DG within the ribbon synapses [1], [8], [11], however, the distribution of α-DG and β-DG is not always overlapping [16], [17]. In our study, we have not observed any correlation among the expression of pikachurin, pikachurin fragments, and dystroglycans. Pikachurin is a potentially useful molecule in synaptogenesis, and future studies on this protein may unavoidably involve upregulation of its expression. Thus, it should be noted that overexpressed pikachurin tends to form intracellular oligomers and accumulate as monomers in the culture medium. Both of these outcomes seem distinct from the endogenous situation in retina. Further, overexpression of double-tagged pikachurin in Y79 cells produced only a moderate increase in the signals of N-fragments on Western blot (Fig. 3B) and extracellular immunofluorescence of c-Myc (N-terminal tag, Fig. 3E), suggesting that the amount of extracellular N-fragments was dependent on the availability of as yet unidentified receptors as well as the competition between endogenous and exogenous pikachurin for these receptors. The phenomenon of pikachurin oligomerization, first reported by us at the ARVO meeting in 2009, was later confirmed by Kanagawa et al. (2010), who also showed that the first LamG domain was critical for oligomerization. In agreement with their observations, our results indicate that the amino acid sequence from 354 to 498, which includes most of the first LamG domain, is required for pikachurin oligomerization (Fig. 3D). It is likely that oligomerization induces some conformational change in pikachurin, e.g. the C-terminus becomes less exposed, as suggested by our observations with His antibody (Fig. 3B, C). It is intriguing that very little oligomerized recombinant pikachurin was found in the conditioned media of Y79 cells. Perhaps, concealment of oligomerization sites by glycosylation could be one contributing factor. Finally, oligomerization was observed in both Y79 and HEK293 cells, suggesting it is a general capability of pikachurin and that understanding of pikachurin function in retina may require use of specific cell types. In conclusion, our work underscores the unique role of pikachurin in retina. Not only is pikachurin highly expressed in retina, but it has tissue specific post-translational processing. The fact that most endogenous pikachurin is cleaved suggests intrinsic regulation within retinal tissue and Y79 cells. The mechanisms underlying the post-translational processing of pikachurin have yet to be determined.

Supporting Information Figure S1. Overexpression of murine dystroglycan in Y79 cells. (A, B) Simultaneous increase in the levels of α-DG (red) and β-DG (green) levels after overexpression of murine dystroglycan in Y79 cells. Two sets of representative immunofluorescent images are shown. (C) Overexpression of dystroglycan in Y79 cells, indicated by the nuclear GFP, had no significant influence on pikachurin staining. Bar = 10 µm. https://doi.org/10.1371/journal.pone.0050552.s001 (TIF)

Acknowledgments We are grateful to Dr. Takahisa Furukawa (Osaka Bioscience Institute, Japan) for pikachurin antibody and plasmid, Drs. Annie Beuve and Marco Zarbin (New Jersey Medical School, USA) for technical help with immunocytochemistry and human retinas, respectively.

Author Contributions Conceived and designed the experiments: JH ETA. Performed the experiments: JH. Analyzed the data: JH ETA. Contributed reagents/materials/analysis tools: JH ETA. Wrote the paper: JH ETA.