Comparative Analysis of the Tear Protein Expression in Blepharitis

The aim of this study was to screen prognostic or diagnostic marker proteins for blepharitis and investigate the pathogenesis of this disease using a ...
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Comparative Analysis of the Tear Protein Expression in Blepharitis Patients Using Two-Dimensional Electrophoresis Bon-Suk Koo†, Do-Yeon Lee†, Hyo-Shin Ha,‡ Jae-Chan Kim,‡ and Chan-Wha Kim*,† Graduate School of Life Sciences and Biotechnology, Korea University, Seoul, Korea and Department of Ophthalmology, College of Medicine, Chung-Ang University, Seoul, Korea Received October 21, 2004

Change in the expression of body fluid proteins is caused by many diseases or environmental disturbances. The changes in tear proteins are also associated with various pathological eye conditions. Especially, chronic blepharitis is one of the most common conditions seen in the ophthalmologist’s office. However, there are no specific clinical diagnostic tests for blepharitis, and it is difficult to treat effectively. Therefore, the aim of this study was to screen prognostic or diagnostic marker tear proteins for blepharitis and investigate pathogenesis of this disease using proteomics techniques. The tear proteins expressed in patients suffering from blepharitis (patient, n ) 19) and healthy volunteers (control, n ) 27) were analyzed using the two-dimensional electrophoresis (2-DE) technique. The differentially expressed proteins in patients were identified with ESI-Q-TOF (electrospray-quadrupole-time-of-flight) mass spectrometry and confirmed with western blotting. Nine proteins in patient were down regulated about 50% compared to those of the control: serum albumin precursor, R-1 antitrypsin, lacritin precursor, lysozyme, Ig-κ chain VIII, prolactin inducible protein (PIP/GCDFP-15), cystatin-SA III, pyruvate kinase, and an unnamed protein. The use of the two-dimensional eletrophoretic technique could give more insight into the disease-related protein expression changes in tear fluids. Our findings reveal that the composition of tear proteins in blepharitis patients is different from that of healthy subjects and may provide further insights into the pathogenesis of blepharitis. Keywords: tear protein • blepharitis • two-dimensional electrophoresis • ESI-Q-TOF mass spectrometry

Introduction The ocular surface is covered with a tear film (between 7 µm to 40 µm in thickness) which plays on important role in the defense mechanism of the external ocular surface.1,2 In general, tears are composed of mucins, glycoprotein, unglycosylated protein, lipid, and salt. The major tear proteins are lysozyme, lactoferrin, secretory immunoglobulin A (sIgA), lipocalin, albumin, and lipophilin.3,4 Especially, some levels of tear proteins are associated with various pathological conditions.5-7 Tear proteins have been investigated extensively in the past, but only with regard to revealing the relationship between the concentrations of single tear proteins and ocular surface disorders.8-10 Recently, the tear proteins were analyzed by two-dimensional gel electrophoresis (2-DE) providing more dedicated information on the composition of proteins in tears.11,12 However, this method has limitations for the large volume of tears required, as well as needing additional sample preparation for analysis. In this study, our procedure has the * To whom correspondence should be addressed. Graduate School of Life Sciences and Biotechnology, Korea University, 5-1 Anam-dong, Sungbukku, Seoul 136-701, Korea. Tel: +82-2-3290-3439. Fax: +82-2-3290-3957. E-mail: [email protected]. † Graduate School of Life Sciences and Biotechnology, Korea University. ‡ Department of Ophthalmology, College of Medicine, Chung-Ang University. 10.1021/pr0498133 CCC: $30.25

 2005 American Chemical Society

advantage of requiring only 2∼3 µL of tears (equivalent to 20 µg of protein) to carry out one analysis and has enabled analysis of proteins directly from crude mixtures without labor intensive processing. In addition, the combination with ESI-MS/MS analysis can identify proteins which are differentially expressed in patient compare with those of control. Chronic blepharitis is one of the most common diseases seen in the ophthalmologist’s office. The disease is an extremely complex condition that manifests several different and overlapping arrays of signs and symptoms.13 Symptoms can include crusting of the lid margins and itching and burning eyelids, as well as signs of lid inflammation and associated conjunctival and corneal change. From previous studies, blepharitis is a complex condition that would not only be difficult to investigate but also difficult to understand adequately enough to treat effectively.13,14 Proteomic analysis can provide more insight about protein expression patterns which are associated with various pathological conditions, and identification of tear proteins whose expression is altered in patients has the potential to reveal important mechanisms of eye disease. The objective of this study was to use a proteomic approach to compare tear protein patterns of control subjects with blepharitis patients. An electrophoretic procedure was demonstrated which allows the two-dimensional quantitative analysis of the tear proteins. Blepharitis patient tear proteins were Journal of Proteome Research 2005, 4, 719-724

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research articles compared with those of control group, and differentially expressed proteins were identified by ESI-MS/MS analysis and confirmed by Western blotting.

Material and Methods Human Subjects. This research followed the tenets of the Declaration of Helsinki. The nineteen patients (14 male and 5 female with a mean age 58 ( 7.5 years) with blepharitis that were evaluated by the principal investigator in the Department of Ophthalmology, College of Medicine, Chung-Ang University were enrolled. After the nature of the procedure had been fully explained, the complete informed consent was obtained from each subject prior to participating in this study. Chronic blepharitis was diagnosed on the basis of the findings of slitlamp examination (e.g., lid margin telangiectasia, oily capping or obstruction of meibomian gland orifices, crusting of lashes) among the patients complain of ocular irritation or dryness. We excluded from the study any patients with eyes with disorders that could have affected the tear composition (e.g., patient undertaking systemic antibiotics or hormones, and users of any eyedrops). Twenty-seven normal controls (22 male and female 5 with a mean age of 27 ( 2.2 years) and eight agematched controls (4 male and 4 female with a mean age of 62 ( 5.5 years) with no abnormalities of the cornea, conjunctiva, lacrimal system, or meibomian glands as assessed by slit-lamp examination, and showing normal values for the tear breakup time (TBUT, over 10 s) were recruited. Briefly, the tear fluid was collected from the interior tear meniscus, causing the lease irritation possible, using a preweighed polyester wick (Transorb rods; American Filtrona, Richmond, VA) to obtain the sample as previously decribed.15 Wicks were then placed into the end of a micropippet tip located within a 1.5 mL tube and centrifuged at 10 000 rpm for 5 min. The concentration of protein in the sample was measured by the modified Bradford methods.16 The tear samples were then stored at -70 °C, and thawed only once prior to analysis. 2-DE. Ready-to-use Immobiline DryStrips (24 cm, pH 3-10 NL) were used for IEF. DryStrips were rehydrated with the sample (20 µg of protein) in 450 µL of the rehydration solution containing 8 M urea, 2% CHAPS, 1% IPG buffer (pH 3-10 NL), 65 mM DTT and a trace of BPB for 5 h without current and 5 h with current fo 80 V. IEF was carried out for a total of 100.000 Vhr using the IPGphor IEF system (AP Biotech, Sweden). Following IEF separation, the IEF gel strips were equilibrated in two equilibration solutions for 15 min with gentle shaking. The first equilibration solution contains 50 mM Tris-HCl (pH 8.8) with 6 M urea, 20% glycerol, 2% SDS, and 1% DTT. In second equilibration solution, DTT of the first equilibration solution was replaced with 2.5% idoacetaminde.17-19 The IEF strip was applied onto 11-16% gradient SDS-PAGE gel. The second-dimensional SDS-PAGE was performed with Ettan DALT II (AP Biotech) at current a constant voltage of 70 V for 1 h, 140 V 2 h and 320 V for 5 h. Image Analysis. Proteins were visualized by silver staining method with some modification.20 The gels were fixed in 50% methanol, 12% acetic acid and 0.05% formaldehyde (37%) at least for 2 h. Then, the fixed gels were sensitized by incubating in 0.02% sodium thiosulfate, and subsequently, were immersed in 0.1% silver nitrate for 20 min. The development stage was carried out with 6% sodium carbonate and 0.05% formaldehyde (8 min, 4 °C). Finally, the reaction was terminated with fixing by 50% methanol and 12% acetic acid. All staining procedures 720

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were done at precise time and temperature. Digitalized images of the silver stained gels were analyzed using the ImageMaster 2D Elite Software (AP Biotech). Expression levels of the spots were determined by the relative spot volume of proteins compared to the volume of a single spot in the gel, and expressed as relative volume intensity; this technique is called single-spot normalization. For each spot, the normalization volume was averaged and expressed as a mean ( standard error of mean (SEM) in each group. The proteins differently expressed with statistical difference were selected and identified. Mass Spectrometric Identification. Proteins were subjected to in-gel trypsin digestion. Excised gel spots were destained with 100 µL of destain solution with shaking for 5 min. After removal of the solution, gel spots were incubated with 200 mM ammonium bicarbonate for 20 min. The gel pieces were dehydrated with acetonitrile and dried in a vacuum centrifuge. The dried gel pieces were rehydrated with 50 mM ammonium bicarbonate containing 0.2 µg modified trypsin (Promega, Medison, WI) for 45 min on ice. After removal of solution, 50 mM ammonium bicarbonate was added. A column consisting of 100-300 nL of Poros reverse phase R2 material (20-30 µm bead size, PerSeptive Biosystems) was packed in a constricted GELoader tip (Eppendorf, Hamburg, Germany). Thirty microliters of the peptide mixture from the digestion supernatant was diluted in 5% formic acid, loaded onto the column, and washed with 5% formic acid. For analyses by MS/MS, peptides were eluted with 50% methanol, 49% H2O, 1% formic acid directly into a precoated borosilicate nanoelectrospray needle (Micromass, Manchester, UK). MS/MS of peptides generated by in-gel digestion was performed by nano-ESI on a Q-TOF mass spectrometer (Micromass). Product ions were analyzed using an orthogonal TOF analyzer, fitted with a reflector, a microchannel plate detector and a time-to-digital converter. The data were processed using a Mass Lynx Windows NT PC system (Micromass). For identification of proteins, all MS/MS spectra recorded on tryptic peptides derived from spot were searched against protein sequences from NCBInr databases using the MASCOT search program. Search parameters were as follows; MS/MS Ion Search in type of search, trypsin in enzyme, carbamidomethyl (c) in fixed modifications, oxidation (M) in variable modifications, monoisotopic in mass values, ( 1 Da in peptide mass tolerance, ( 0.8 Da in fragment mass tolerance and 1 in max missed cleavage. The highest score identification in mascot search results were selected, considering species. Western Blot Analysis. For Western blot of cystatin and PIP, 40-50 µg of the tear protein were separated by 15% (w/v) SDSPAGE, and then blotted onto nitrocellulose membranes. The membrane was incubated with blocking solution containing 1:500 dilution of anti-cystatin SA antibody (R&D systems, MN) and anti-PIP antibody (Novocastra Laboratories Ltd., UK). These subsequently incubated with blocking solution containing 1:2500 dilution of horseradish peroxidase-conjugated antimouse IgG secondary antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA). Detection was performed with Enhanced chemiluminescence (ECL) system (Pierce Biotechnology, Inc., IL). Western blot was scanned with flat-bed scanner and digitalized using Scion image analysis software (Scion Corporation, MD). Values are mean ( standard error of mean (SEM) and statistical significance was evaluated by unpaired student’s t test.

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Tear Protein Expression in Blepharitis Patients

Figure 1. Representative 2-DE gel map of normal and patient tear proteins. Twenty microgram proteins were separated by 2-DE using 24 cm pH 3-10 NL IPG strip and 11-16% gradient SDS-PAGE. The map was analyzed by the Image Master 2D Elite Software (AP Biotech, Sweden). (A) The 2-DE gel map of normal subject. (B) The 2-DE gel map of patient subject.

Result and Discussion Tear Protein Map of Normal Control and Blepharitis Patients. Like most techniques, the quality of the 2-DE analysis also depends on how to prepare the source material. Since the volume of tear sample is usually limited to few microliters, many researchers pool the sample or mix with physiological water for 2-DE analysis. In our study, we did not pool the sample nor add any buffer except a rehydration solution for IEF. Moreover, there were no additional sample preparation steps for 2-DE analysis. Sufficient resolving power and reproducibility also need for a comparative proteome analysis for 2-DE. Our electrophoretic procedure and staining method which were described in the section of Material and Methods are suitable for the analysis of tear proteome using 2-DE. Finally, we could get high-resolution 2-DE gel image from this methodology without streaking and high background. This could be a very useful tool to compare tear protein profiles which associated with various pathological eye conditions. Figure 1 shows 2-DE map of the tear proteins in patients and control group and about five hundred tear proteins were visualized (Figure 1). Comparison of the spots in 2-DE gels in control and blepharitis patients, through ImageMaster 2D Elite software analysis (AP Biotech) revealed that twelve spots were differently expressed (Figure 2a). The spots in patient were down regulated about 50% (p < 0.05) compared to those of control (Figure 2b). Identification of Differentially Expressed Proteins and Confirmed by Western Blotting. Recent advances in mass spectrometric technology, together with progress in genomic sequencing and bioinformatics, have dramatically changed the status of mass spectrometric methods as a general strategy of molecular and cell biology. Matrix-assisted laser desorption/ ionization time-of flight (MALDI-TOF) peptide mass mapping is the most commonly employed approach for protein identification. It is suitable for automation, which is an important issue for high-through-put application. Generally speaking,

Table 1. Identification of the down-regulated Proteins in Patient Using ESI-Q-TOF MS/MS spot

protein identification

Mw (Da)

pI

10 18 34 43 47 48 116 148 350 396 406 434

serum albumin precursor Ig κ chain-VIII pyruvate kinase unnamed protein R-1-antitrypsin R-1-antitrypsin prolactin-inducible protein serum albumin precursor cystatin SA-III lacritin precursor lysozyme serum albumin precursor

69832 23037 58447 71246 46787 46787 16847 70550 14409 14237 15269 70550

5.85 5.75 7.15 5.92 5.42 5.42 8.26 5.95 4.74 5.43 9.28 5.95

scorea accession no.

88 44 62 26 32 136 214 68 62 74 106 36

Q28522 A23746 P11979 CAA23753 AAA51546 AAA51546 NP_002643 P35747 AAB19889 NP_150593 BAA00314 P35747

a Score is -10*log(P), where P is the probability that the observed match is a random event, it is based on NCBInr database using MASCOT searching program as MS/MS data.

MALDI peptide mapping may operate efficiently when a statistically significant number of peptide peaks can be measured, but it possesses virtually no power to characterize an individual peak in the MALDI mass spectrum. Protein identification by electrospray tandem mass spectrometric analysis (ESI-MS/MS), developed by Mann and co-worker,21 is a more sophisticated method for protein identification. We use ESIQ-TOF MS/MS analyzer to obtain more accurate protein identification data. Table 1 shows identified proteins which are down-regulated in patient group. Spot Nos. 10, 148, and 434 were identically identified as serum albumin precursor and Spot 47 and 48 were identified as R-1-antitrypsin. The identity of Cystatin SA-III was confirmed by Western blotting (Figure 3a). The age-matched control which age is similar to patient group is shown in the figure. The 15 kDa of cystatin-SA was detected in the tear of control, patient and age-matched control group. Its expression in patient group (41 ( 1.26) was decreased more than 60% compared to control (104.86 ( 0.13, p < 0.001) and about 40% decreased compared to age-matched control (73.17 ( 1.37, p < 0.001) (Figure 3b). The identity of prolactin Journal of Proteome Research • Vol. 4, No. 3, 2005 721

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Figure 2. Magnified 2-DE map of spots which were down-regulated in belpharitis patient tear. (A) Twelve spots were down-regulated (about 50%) in patients. (B) The average volume intensity in control and patient. Each bar represents the mean ( standard error of the mean (SEM) of each spots. Significant difference from normal tear based on a two-tailed student’s T-test (*p < 0.05, **p < 0.01, ***p < 0.001).

inducible protein (PIP) was also confirmed (Figure 4a). The 16 KDa of PIP was detected in all groups and its expression of patient group (58.16 ( 3.75) was decreased more than 50% compared to control and age-matched control (116.83 ( 5.79/ 109.5 ( 3.27, p < 0.001) (Figure 4b). The age-matched control might explain that down-regulation of some proteins in patient group were not aging problem but associated with disease state. Functional Implication of Differentially Expressed Proteins. Since the levels of all identified proteins decreased in patient group, the down-regulation of these proteins may affect the function of tear film (Table 2). Among them, cystatin SAIII and R-1-antytrypsin (R-1-protease inhibitor, R1-PI) are protease inhibitors which are found in body fluid and tissues of animal. The balance between protease and protease inhibitor is believed to play a significant role in maintaining normal ocular surface condition.22 Cystatins are generally tight-binding inhibitors of cysteine proteases and have a protective function by regulating the activities of endogenous cysteine proteases causing uncontrolled proteolysis and tissue damages.23-25 In 722

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tears of patients with blepharitis, significant lower levels of cystatins have been found and some pathologic conditions of the eye are related with a lower level of cysteine proteases inhibitor are demonstrated.26,27 Furthermore, some researchers showed cystatin upregulated IL-6 production which played a central role in host defense mechanism.28 The R-1-antitrypsin (R1-PI) is a major protease inhibitor in human serum.29 The importance of this protein was proposed in the 1960s based on observations that genetically R1-PI-deficient patients developed an early-onset degenerative lung disease30 or a liver disease.31 Recently, the synthesis of R1-PI by human corneal cells has been reported.22 The cornea, located in the anterior portion of the eye, is a transparent connective tissue made up of epithelial, stromal, and endothelial layers. A proper level of R1-PI may protect the cornea from degradation by neutrophil elastase during inflammation. Additionally, R1-PI may function as a backup inhibitor for other serine proteinases such as plasmin and cathepsin G in the cornea. Therefore, in this study, a reduction of cystatin and R1-PI levels in blepharitis patients

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Tear Protein Expression in Blepharitis Patients Table 2. Enzymatic Function and Their Catalytic Activity of Identified Protein spot

accession no.

protein identification

10

Q28522

serum albumin precursor

34

P11979

pyruvate kinase

47 116 350 396

AAA51546 NP_002643 AAB19889 NP_150593

R-1-antitypsin prolactin-inducible protein cystatin SA-III lacritin precursor

406

BAA00314

lysozyme

could not inhibit the protease activity and the degration processes in blepharitis conditions would be increased. Prolactin inducible protein (PIP), which has been found in saliva, tears, sweat, seminal plasma, submucosal gland of the lung, and amniotic fluid.32 Some researcher found that blepharitis could be induced in rabbits by sensitization to Staphylococcus aureus cell wall components.33 Enhanced cellmediated immunity to S. aureus had been detected in 40% of patients with chronic blepharitis but not among normal subjects.34 Interestingly, it is suggested that the PIP in human saliva binding to some bacterial strains commonly found in human and mice35 and PIP inhibited the growth of Streptococcus in culture by interaction with microorganisms.36 These evidences suggest that PIP involved local immune systems as a function. Furthermore, PIP is capable of suppressing T-cell apoptosis in the reproductive system via high-affinity interaction with CD4.37 Though the exact function of PIP remains undetermined, the down-regulation of this protein in blepharitis patient provide the insight into a possible involvement for maintaining healthy eye condition. Especially, PIP level in tear could be important for bacteriostasis in the eye, and downregulation of this protein would promote blepharitis conditions.

function

regulation of the colloidal osmotic pressure of blood ATP + pyruvate ) ADP + phosphoenolpyruvate serine protease inhibitor suppressing T- cell apoptosis cysteine protease inhibitor stimulate cell secretion and signaling in lacrimal gland and cornea anti-bacterial action in body fluids

Another down-regulated protein, human lacritin is highly expressed in human lacrimal gland, but no lacritin protein and mRNA detected elsewhere among more than 50 human tissues.38 This protein enhanced unstimulated secretion and rapid tyrosine phosphorylation in larcrimal acinar cell, and it also stimulated corneal epithelial cell calcium signaling. Therefore,

Figure 4. Expression of prolactin inducible protein (PIP) by Western blotting. (A) Western blot analysis of tear protein (40 µg of tear protein) from control and patient was performed. (B) Quantitative analysis of gel band was performed using Scion image analysis software (Scion Corporation, MD). Each data point represents the mean ( standard error of mean (SEM). The unpaired student’s t test was carried out and showed a significant difference (*p < 0.001) between the two control groups and patient group (CON; control, PAT; patient, AMC; age-matched control).

Figure 3. Expression of cystatin-SA (CYS) by Western blotting. (A) Western blot analysis of tear protein (50 µg of tear protein) from control and patient was performed. (B) Quantitative analysis of gel band was performed using Scion image analysis software (Scion corporation, MD). Each data point represents the mean ( standard error of mean (SEM). The unpaired student’s t test was carried out and showed a significant difference (*p < 0.001) between the two control groups and patient group (CON; control, PAT; patient, AMC; age-matched control).

lacritin may play a key role in the function of the lacrimal gland-cornel axis and their deficiency may cause some problems in the eye. Since many blepharitis patients have tear deficiency and abnormal tear conditions, lacritin might important for tear secretion and maintaining normal tear conditions.

Conclusion The 2-DE in combination with mass spectrometry (MS) is very powerful approach to proteome research. In this study, the 2-DE patterns of blepharitis patients were compared to those of healthy volunteers, and found down regulated proteins which are associated with disease eye state. Probably, many Journal of Proteome Research • Vol. 4, No. 3, 2005 723

research articles other eye diseases could influence the tear protein composition in a similar way to that which blepharitis does, and the proteomic analysis could be applied to understand the eye disease mechanism and find new diagnostic or therapeutic targets.

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