Keywords

1 Introduction

Tyrosine sulfation is a post-translational modification of proteins that is utilized in all ocular tissues (Kanan et al. 2009, 2012) and plays a very important role in vision (Sherry et al. 2010, 2012) . Eliminating tyrosine sulfation reduces scotopic electroretinographic responses to 25 % of normal and photopic responses to 15 % of normal (Sherry et al. 2010). Besides these functional deficits, ultrastructural examination reveals rod outer segments abnormalities (Sherry et al. 2010). To identify the tyrosine sulfated proteins that may be responsible for these effects, we immunoaffinity purified tyrosine sulfated proteins from neural retina using the anti-sulfotyrosine antibody PSG2 (Hoffhines et al. 2006) . Column elution using a sulfated pentapeptide was followed by mass spectrometry. Since all sulfated proteins are secreted, some of the tyrosine sulfated proteins produced by the cell line 661W (Tan et al. 2004) were identified by fractionating conditioned media followed by western blotting with PSG2 . Proteins that immuno-reacted were identified by in-gel digestion of excised bands followed by mass spectrometry.

2 Materials and Methods

2.1 Preparation of Bovine Retinal Lysates

Bovine eyes were obtained from Country Home Meat Slaughter House (Edmond, OK). Neural retinas were isolated and lysates were prepared in buffer A (25 mM MOPS, 100 mM NaCl, pH 7.5). Bradford assay was performed and lysate concentrations were adjusted to 4 mg/ml in buffer A prior to loading onto the column.

2.2 PSG2 Affinity Purification of Tyrosine O Sulfated Proteins

Ten mg of extracts were filtered using a 0.45 µm syringe filter (Millipore, Billerica, MA) and loaded onto the PSG2-Affi-Gel-10 HPLC column (Hoffhines et al. 2009) . Column was washed with buffer A, wash buffer 1 (25 mM MOPS, 200 mM NaCl), wash buffer 2 (25 mM MOPS, 400 mM NaCl) and eluted with elution buffer (25 mM MOPS, 400 mM NaCl, 4 mM sulfated pentapeptide). Eluted samples were concentrated with acetone precipitation. The tyrosine-sulfated pentapeptide LDYSDF was synthesized by Bio-Synthesis Inc. (Lewisville, TX).

2.3 Mass Spectrometry

Column fractions were separated by SDS-PAGE to remove the sulfated pentapeptide and gel lane was cut into 1 mm slices and subjected to in-gel trypsin digestion, reduction and alkylation followed by LC MS/MS analysis (ABI MDS Sciex Qstar Elite, (Life Technologies, Grand Island, NY). MS/MS data were collected using ABI Analyst QS 2.0 software and submitted to MASCOT (Matrix Science) server for protein identification against the NCBInr protein database.

3 Results

3.1 Tyrosine O Sulfated Proteins Identified by Immunoaffinity Purification

About 10 % of eluted proteins were run on a gel and immunoblotted with PSG2 (Hoffhines et al. 2006) . Identified proteins ranged from 250 kD to 37 kDa in size (Fig. 86.1a). The remaining eluate was acetone precipitated, fractionated by SDS-PAGE and subjected to mass spectrometry. Since tyrosine sulfated proteins transit the secretory pathway (Moore et al. 2003) , only membrane or secreted proteins were included in Table 86.1. Identified targets belonged to multiple protein families such as serpins, extracellular matrix proteins and complement proteins. Since the major function of tyrosine sulfation is protein-protein interaction (Zhu et al. 2011; Costagliola et al. 2002; Ramachandran et al. 1999) , immunoaffinity may have pulled down non-tyrosine sulfated proteins that co-purified due to their direct or indirect interaction with tyrosine-sulfated proteins. Therefore, each of the identified proteins was subjected to prediction of sulfated tyrosines using the software ‘Sulfinator’ (Monigatti et al. 2002). Seven of the proteins were predicted to be tyrosine sulfated (Table 86.2). Fibronectin and fibrinogen have previously been shown to be tyrosine sulfated (Liu and Suiko 1987; Hortin et al. 1986) and we have recently shown fibulin 2 and vitronectin to be tyrosine sulfated (Kanan et al. 2014a, 2014b) . Complement component 3 and fibrinogen beta have not been previously shown to be tyrosine sulfated. Sulfinator did not predict tyrosine sulfated sites on retinol binding protein 3 (IRBP), pigment epithelium-derived factor (PEDF) precursor, collagen (type I, alpha 1), neuronal membrane glycoprotein M6-b, isoform 2 and fibrinogen alpha. The presence of these proteins suggests that they may be interacting partners to some of the identified tyrosine sulfated proteins.

Fig. 86.1
figure 1

Tyrosine sulfated proteins in cow retinal extracts and 661W cells. a Immunoaffinity column purification of tyrosine O sulfated proteins from cow retinal lysates. SDS-PAGE and coomassie blue staining of cow retinal lysates eluted from the column and western blotted with PSG2. b Tyrosine O sulfated proteins from cone derived cell line 661W. SDS-PAGE and coomassie blue staining of 661W conditioned media and western blotting with PSG2

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Table 86.1 List of proteins identified in cow retinal lysates after immunoaffinity purification and mass spectrometric analysis. Eleven proteins were identified in cow retinal lysates by MALDI-MS/MS analysis of PSG2 immunoaffinity column eluent
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Table 86.2 List of potential sulfated tyrosines residues in cow retina as identified by Sulfinator. Citations are provided for the tyrosine sulfation sites that were experimentally identified
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3.2 Tyrosine Sulfated Proteins in 661W-Conditioned Media

661W-conditioned media was SDS-PAGE fractionated and immunoblotted with PSG2 revealing two major proteins between 150–250 kDa (Fig. 86.1b). These proteins were excised and subjected to MALDI MS/MS analysis. The top band (marked by an asterisk, Table 86.3) was a mixture of three proteins and the bottom band (denoted by #, Table 86.3) was identified as collagen, type V, alpha 2. Sulfinator predicted all the four proteins to be tyrosine sulfated (Table 86.4), which has been experimentally confirmed (Liu and Suiko 1987; Kanan et al. 2014a; Paulsson et al. 1985; Fessler et al. 1986) .

Table 86.3 List of proteins identified in 661W-conditioned media. Four proteins were identified from conditioned media of 661W cells following SDS-PAGE and MALDI-MS/ MS analysis
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Table 86.4 List of sulfated tyrosines residues in cone derived cell line, 661W. All four proteins have been experimentally proven to contain sulfated tyrosines and citations provided
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4 Discussion

We have previously shown the importance of tyrosine sulfation for vision (Sherry et al. 2010; Sherry et al. 2012) and here we identify some of the tyrosine sulfated proteins in ocular tissues. We subjected cow retinal extracts to immunoaffinity purification with PSG2. Eleven proteins were identified and seven of those were predicted to be tyrosine sulfated. The rest of the proteins may have been co-purified with their tyrosine sulfated interacting partners since tyrosine sulfation enhances protein-protein interactions (Zhu et al. 2011; Costagliola et al. 2002; Ramachandran et al. 1999) . Of the identified proteins, fibulin 2 and vitronectin were also detected in cow RPE to be tyrosine O sulfated (Kanan et al. 2014a, b) .

Since the retina is composed of six different classes of cells, identified proteins may belong to any and/or all cell types. Therefore, we used the cone derived cell line 661W to identify tyrosine sulfated proteins that may be cone specific. We identified fibronectin, fibulin 2, nidogen 2 and collagen V proteins from this cell line. These proteins were experimentally shown to be tyrosine sulfated (Liu and Suiko 1987; Kanan et al. 2014a; Paulsson et al. 1985; Fessler et al. 1986) .

This is the first report of the identification of tyrosine sulfated protein in the retina and in 661W cells. Further experiments will identify the cell type that produces these proteins in the retina. The function of tyrosine sulfation in these proteins and how it affects vision will only be revealed using ‘In-Vivo knock-in’ mutants that will have the tyrosine sulfated residues mutated to phenylalanines.