Journal of Biomedicine and BiotechnologyVolume 2011 (2011), Article ID 901329, 28 pagesdoi:10.1155/2011/901329

Research Article

Protein Profiling of Human Nonpigmented Ciliary Epithelium Cell Secretome: The Differentiation Factors Characterization for Retinal Ganglion Cell line

Ming-Hui Yang,¹ Raghu R. Krishnamoorthy,² Shiang-Bin Jong,³ Pei-Yu Chu,⁴  Yuan-Han Yang,⁵ Wen-Cheng Chen,⁶ Sharon Chia-Ju Chen,³ Adnan Dibas,² Thomas Yorio,² Tze-Wen Chung,¹ and Yu-Chang Tyan3,7,8,9

¹Department of Chemical and Material Engineering, National Yunlin University of Science and Technology, 123 University Road, Section 3, Douliou, Yunlin 64002, Taiwan²Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, USA³Department of Medical Imaging and Radiological Sciences, Kaohsiung Medical University, 100 Shi-Chuan 1st Road, Kaohsiung 80708, Taiwan⁴Department of Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, Kaohsiung 80708, Taiwan⁵Department of Neurology, Kaohsiung Medical University Chung-Ho Memorial Hospital, Kaohsiung 80708, Taiwan⁶Department of Fiber and Composite Materials, Feng Chia University, Taichung 40724, Taiwan⁷National Sun Yat-Sen University and Kaohsiung Medical University Joint Research Center, Kaohsiung 80708, Taiwan⁸Center for Research Resources and Development, Kaohsiung Medical University, Kaohsiung 80708, Taiwan⁹Center of Excellence for Environmental Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan

Received 5 April 2011; Revised 10 June 2011; Accepted 13 June 2011

Academic Editor: Daniel T. Monaghan

Copyright © 2011 Ming-Hui Yang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

The purpose of this paper was to characterize proteins secreted from the human nonpigmented ciliary epithelial (HNPE) cells, which have differentiated a rat retinal ganglion cell line, RGC-5. Undifferentiated RGC-5 cells have been shown to express several marker proteins characteristic of retinal ganglion cells. However, RGC-5 cells do not respond to N-methyl-D aspartate (NMDA), or glutamate. HNPE cells have been shown to secrete numbers of neuropeptides or neuroproteins also found in the aqueous humor, many of which have the ability to influence the activity of neuronal cells. This paper details the profile of HNPE cell-secreted proteins by proteomic approaches. The experimental results revealed the identification of 132 unique proteins from the HNPE cell-conditioned SF-medium. The biological functions of a portion of these identified proteins are involved in cell differentiation. We hypothesized that a differentiation system of HNPE cell-conditioned SF-medium with RGC-5 cells can induce a differentiated phenotype in RGC-5 cells, with functional characteristics that more closely resemble primary cultures of rat retinal ganglion cells. These proteins may replace harsh chemicals, which are currently used to induce cell differentiation.

1. Introduction

Primary open angle glaucoma (POAG), a leading cause of irreversible blindness worldwide, is an optic neuropathy characterized by the gradual and progressive loss of retinal ganglion cells (RGCs), optic nerve degeneration, and excavation of the optic disks [1–4]. The hypothesis has been that larger RGCs were selectively lost in the early stage of glaucoma [5]. Although the mechanisms of optic nerve damage in glaucoma have not been completely determined, it appears that the optic nerve head is a major site of damage [6].

RGCs can generate action potentials that travel along the optic fibers [7]. In general, RGCs are a mixture of more than 20 cell subtypes. They have energy-dependent axonal transport functions—orthograde and retrograde transports [8]. These terminal projection areas are in the lateral geniculate body. RGCs can be subdivided by their morphology and physiology, but they are usually discussed without classifications.

The in vitro study of the physiology and pathophysiology of RGCs has been limited to primary cultures. Previous studies have characterized a transformed rat retinal ganglion cell-line (RGC-5), which expresses many neuronal cell markers, including Thy-1, a cell surface glycoprotein found predominantly in the retinal ganglion cells [6, 9, 10], and Brn-3C, a POU domain transcription factor expressed exclusively in the retinal ganglion cells [11]. RGC-5 cells also express receptors of N-methyl-D aspartate (NMDA), GABA-B, and neurotrophin [6]. However, unlike primary RGCs, these cells were not sensitive to glutamate excitotoxicity in their undifferentiated state. RGC-5 cells pretreated with succinyl concanavalin-A (sCon A) were sensitive to 500 μM glutamate [12]. Lacking glutamate sensitivity causes the difficulties of using the RGC-5 cells in experiments involving glutamate.

Ocular ciliary epithelium cells have been shown to be involved in the synthesis and secretion of various proteins found in aqueous humor [13]. Several proteins, including neuropeptides and their processing enzymes, synthesized and secreted by a human nonpigmented ciliary epithelial (HNPE) cell-line, have been evaluated [14], and it is suggested that these secreted proteins can act in an autocrine or paracrine manner to affect ciliary epithelial functions and other target ocular cells, such as the trabecular meshwork [13]. Because of the neuroendocrine properties of the ciliary epithelium cells, the ability to confer differentiated neuroendocrine phenotypes and the physical locations of these ciliary epithelium cells and RGCs [15], we hypothesized that factors secreted by these HNPE cells may induce the RGC-5 cells to differentiate, and possibly induce glutamate and NMDA sensitivities.

Proteomic analysis, including identification and characterization, is a powerful tool for determination of biological roles and functions of individual proteins. In the present report, we have utilized a system involving HNPE and RGC-5 cells, and this system may result in the morphological and functional differentiation of RGC-5 cells. Although the origin of RGC-5 has been still in question, the expression of neuronal markers was validated [16]. Proteomic approaches have been applied to establish a map of expressed proteins for the characteristics of HNPE cells.

2. Materials and Methods

2.1. Cell Culture

The human non-pigmented ciliary epithelium cells (HNPE) were SV-40 transformed and were a gift from Dr. Miguel Coca-Prados (Yale University). HNPE were maintained at 37°C and 5% CO₂ in Dulbecco's modified Eagle's medium (DMEM, Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone Laboratories, Logan, UT), 1% penicillin/streptomycin (Gibco, Grand Island, NY, USA) and 44 mM NaHCO₃. After three days, the cells were washed with phosphate buffered saline (PBS) and the medium was replaced by serum-free (SF) DMEM for 12 h.

The HNPE cell conditioned SF-medium was filtered by 0.22 μm filter and diluted 25 times with autoclaved Milli-Q grade water (Millipore Co., Inc.). For each 5 kD cutoff centrifugal tube (Amicon Ultra-15, Millipore Co., Inc.), a 15 mL diluted sample was loaded. Following centrifugation at 5000 ×g for 20 min, the sample in the filter unit was collected. The protein concentration of the HNPE cell conditioned SF-medium was measured by the Bio-Rad Bradford total protein assay kit (Bio-Rad Laboratories, Inc.).

RGC-5 cells, a secondary cell culture, were transformed rat retinal ganglion cells developed and obtained from Dr. Agarwal (University of North Texas Health Science Center). RGC-5 cells were maintained in low glucose DMEM in T-150 culture flasks supplemented with 44 mM NaHCO₃, 10% FBS, and 1% penicillin/streptomycin (Gibco). Differentiated RGC-5 cells were obtained by using 50% HNPE cell conditioned SF-medium and 50% fresh DMEM (containing 10% FBS). HNPE conditioned medium, which consisted of low glucose DMEM, was incubated with human non-pigmented ciliary epithelial cells (HNPE).

2.2. Immunocytochemistry

RGCs were grown on glass coverslips for 1-2 days prior to experimentation. Coverslips were rinsed with PBS three times and then were fixed in 4% paraformaldehyde for 30 min. These cells were washed with PBS before being permeabilized in 0.1% Triton X-100 for 15 min, washed with PBS, and blocked with 5% bovine serum albumin for 60 min. After rinsing with PBS, the cells were incubated with a mixture of Thy-1 (monoclonal antibodies, Chemicon, Temecula, CA, 1 : 200) and Brn-3C (polyclonal antibodies, Convance Inc, Princeton, NJ, 1 : 1000) for 1.5 h at room temperature and subsequently incubated with a combination of secondary antibodies. After PBS rinses, these cells were incubated for 10 min in the dark with 300 nM DAPI to stain nuclear regions. Cover-slides were mounted on glass slides in antifade medium (FluorSave; Calbiochem, La Jolla, CA) and allowed to dry for 20 min in the dark. Cells were visualized and images were taken using a Zeiss LSM-410 Confocal Scanning Laser Microscope System. Controls were performed by omitting primary antibodies.

2.3. 1D SDS-PAGE

HNPE cell-secreted proteins were separated under denaturing conditions in a 4–12% polyacrylamide gel. The HNPE cell conditioned SF-medium was resuspended in the sample buffer (Invitrogen NuPAGE SDS sample buffer), heated at 80°C for 10 min and then stored on ice. Each well was loaded with 5 μg of sample solution. The SDS-PAGE gel was run in a Bio-Rad protean II xi cell (Richmond CA, USA) at 200 V for 1 h. After completion of electrophoresis, the protein bands in the gel were visualized by silver staining and image acquired using an image scanner (Amersham Biosciences, Uppsala, Sweden), which is operated by the software LabScan 5.00 (Amersham Biosciences).

2.4. Silver Staining

The gels were fixed in an aqueous solution having 40% ethanol and 10% acetic acid overnight, and then incubated in a buffer solution containing 30% ethanol, 6.8% w/v sodium acetate, and 0.312% w/v sodium thiosulfate for 30 min. After rinsing three times for 5 min each, the gels were stained in a 0.25% w/v silver nitrate solution containing 0.02% formaldehyde for 30 min. The development was performed for 10 min in a solution consisting of 2.5% sodium carbonate and 0.01% formaldehyde. An acetic acid solution (5% v/v) was used to stop the development, and the stained gels were then rinsed three times for 5 min each.

2.5. Protein Identification by Nano-HPLC-ESI-MS/MS

The protein bands were excised manually and digested using sequence grade trypsin (V511A, Promega, USA). The protein samples were reduced, alkylated, and then digested with trypsin using standard protocols [17, 18].

Reverse phase nano-high performance liquid chromatography electrospray ionization tandem mass spectrometry (RP-nano-HPLC-ESI-MS/MS) was used to identify the selected protein bands separated on the SDS-PAGE. The peptides obtained from the tryptic in-gel digestion were analyzed using a nano-HPLC system (LC Packings, Netherlands) coupled to an ion trap mass spectrometer (LCQ Deca XP Plus, ThermoFinnigan, San Jose, CA, USA) equipped with an electrospray ionization source. A linear acetonitrile gradient from 100% buffer A (5% acetonitrile/0.1% formic acid) to 60% buffer B (80% acetonitrile/0.1% formic acid) was used at a flow rate of approximately 200 nL/min for 70 min. The separation was performed on a C18 microcapillary column (Zorbax 300SB-C18, 3.5 μm, 75 μm I.D. ×150 mm, Agilent, Germany). Peptides eluted from the microcapillary column were electrosprayed into the nano-HPLC-ESI-MS/MS with the application of a distal 1.3 kV with heated capillary at the temperature of 200°C. Each cycle of one full scan mass spectrum (m/z 450–2000) was followed by three data-dependent tandem mass spectra with the collision energy was set at 35%.

2.6. Database Search

For protein identification, Mascot software (Version 2.2.1, Matrix Science, London, UK) was used to search the human protein sequence database (Swiss-Prot, Release 52.0 of 22-Feb-08). For proteolytic cleavages, only tryptic cleavage was allowed, and the number of maximal internal (missed) cleavage sites was set to 2. Variable modifications of cysteine with carboxyamidomethylation, methionine with oxidation, and asparagine/glutamine with deamidation were allowed. The mass tolerances of the precursor peptide ion and fragment ion were set to 1 Da. Positive protein identifications were defined if the Mowse scores of greater than 50 were considered significant (). Proteins were initially annotated by similar searches using UniProtKB/Swiss-Prot databases (Last modified September 22, 2009) [19–21].

3. Results and Discussion

Cell secretome (cell-conditional medium) studies can make major contributions in understand biomarker discovery and cell pathophysiological mechanisms. It is composed of proteins that are found in the extracellular growth medium. The cell secretome consists of proteins that are secreted, shed from the cell surface and intracellular proteins released into the supernatant due to cell lysis, apoptosis, and necrosis [22, 23]. The secretome which consists of proteins or peptides secreted from cells into the extracellular medium represents the major class of molecules involved in the intercellular communication in multicellular organisms. It constitutes an important class of proteins that control and regulate a multitude of biological and physiological processes and indicates a clinically relevant source for biomarker and therapeutic target discoveries [24].

Thus, secreted proteins constitute an important category of active molecules that play crucial roles in a number of physiological and pathological processes and may reflect a broad variety of pathological conditions and thus represent a rich source of biomarkers. Proteomic characterization of proteins for identification of specific biomarkers provides a powerful tool to gain deep insights into disease mechanisms in which proteins play major roles. In this study, we have used gel electrophoresis associated with mass spectrometry for identification of the proteome and secretome of HNPE cell conditioned SF-medium samples.

3.1. RGC-5 Cell Differentiation

The differentiation system consisted of RGC-5 cells on coverslips inside 6-well plates, which were exposed to the conditioned medium from HNPE cells. RGC-5 cells proliferated rapidly with a doubling time of less than a day. Decreasing the percentage of serum in the medium may slow down proliferation. The control RGC-5 cells were heterogeneous in shape. Morphological changes of RGC-5 cells were induced by HNPE cell conditioned SF-medium (Figure 1) and caused the shrinkage of the cell body with elongated neurite outgrowth (Figure 1(b)), which allows comparison with undifferentiated RGC-5 cells (Figure 1(a)). The overall morphology of RGC-5 cells after the treatment was similar to those seen in primary cultures of rat retinal ganglion cells [25]. Moreover, the morphology of RGC-5 cells differentiated by our method was similar to the ones induced by a broad-spectrum protein kinase inhibitor staurosporine [26]. Nevertheless, Frassetto and coworkers did not conclude this to be the possible differentiation mechanism. This secretome map is a preliminary study to unveil the mechanism since the differentiation is probably the consequence of the action of several proteins and/or enzymes. It was also noted that the differentiation treatment led to decreased culture density compared with the control cells. This finding is consistent with the study from Wood et al. [27]. For subsequent studies, the conditioned medium from confluent flasks containing HNPE cells was used and found to be equally effective in promoting differentiation of RGC-5 cells.

(a)

(b)

Figure 1: Morphological changes in RGC-5 cells after treatment with HNPE conditioned SF-medium (40x) (a) before, and (b) after. The RGC-5 cells treated with HNPE conditioned SF-medium induced morphological changes, including longer axons and more neurite outgrowth (Figure 1(b)), compared to RGC-5 cells without treatment (Figure 1(a)).

Thy-1 expression in undifferentiated RGC-5 cells was used as a marker to identify retinal ganglion cells [28]. After treatment with HNPE cell conditioned SF-medium, RGC-5 cells have an enhanced Thy-1 expression, compared to the undifferentiated cells (Figure 2). In the retina, the class IV POU domain transcription factor, Brn-3b, was expressed almost exclusively in subpopulations of ganglion cells and used to identify RGCs [29]. Brn-3b was regarded as a marker for differentiation of RGCs, since Brn-3 factors were not necessary for the initial specification of sensory neurons, but were essential for their normal differentiation and survival [30]. Specifically, Brn-3b was localized in the nuclear in RGC-5 cells; however, upon treatment with HNPE cell conditioned SF-medium, RGC-5 cells express Brn-3b in a more punctate cytosolic manner (Figure 2).

(a)

(b)

(c)

(d)

Figure 2: Immunocytochemical analysis of Thy-1 and Brn-3b expression in RGC-5 cells differentiated by treatment with HNPE cell conditioned SF-medium. Staining with antibodies to the cell surface glycoprotein, Thy-1, have been commonly used as a marker to identify retinal ganglion cells. After cultivation with HNPE conditioned medium, RGC-5 cells have an enhanced Thy-1 expression, compared to the undifferentiated cells. RGC-5 cells without cultivation with HNPE conditioned medium express Brn-3b in a different pattern compared with treated RGC-5 cells. Specifically, Brn-3b has a nuclear localization in RGC-5 cells without cultivation with HNPE conditioned medium; however, upon treatment, RGC-5 cells express Brn-3b in a more punctate cytosolic manner.

3.2. Proteome Analysis

The SDS-PAGE followed by silver staining resolved the protein bands from HNPE cell conditioned SF-medium. Figure 3 shows the silver-stained 1D SDS-PAGE of secreted proteins from HNPE cells. Five micrograms of secreted protein was loaded on a gel for visualization, and more than 30 protein bands were detected in the HNPE conditioned SF-medium using the image analysis software. To identify the proteins, the position of the 1D SDS-PAGE lane was excised from the gel, washed to remove the stain, and subjected to tryptic digestion. The resulting peptides were characterized by nano-HPLC-MS/MS for protein identification. When a protein was identified by three or more unique peptides possessing MASCOT scores, no visual assessment of spectra was conducted and the protein was considered present in the sample.

Figure 3: 1D SDS-PAGE image of HNPE conditioned SF-medium (5 μg/well, silver stained, left-hand side: molecular weight marker, kDa). The gel bands on the middle lane with serial numbers were analyzed by nano-HPLC-ESI-MS/MS. In the 30 bands, 132 proteins were identified. The gel bands on the right-hand side were the cell lysised proteins.

In this study, all MS/MS spectra were manually confirmed (even if the above criteria were passed) by the visual assessment for their overall quality. In addition, the criteria for manual validation reported by Jaffe et al., which requires a readily observable series of at least four y-ions, was used [31]. Thus, the criteria should be enough for the validation of the identified proteins. By using this strategy, 132 unique proteins with at least three unique peptide sequences matched were identified, and a summary of the protein identifications achieved is listed in Table 1.

Table 1: Proteins identified in HNPE conditioned SF-medium by proteomic analyses.

Serial No.
SwissProt No.
Protein name
MW
Score
Subcellular location
Sequence coverage
Molecular function
Biological process

1-1
P02751
Fibronectin precursor
262442
1728
Secreted
25%
Collagen/heparin binding
cell adhesion/migration

1-2
Q08378
Golgin subfamily A member 3
167252
37
Cytoplasm
4%
Transporter activity
Intra-Golgi vesicle-mediated transport

P02751
Fibronectin precursor
262442
810
Secreted
11%
Collagen/heparin binding
Cell adhesion/migration

P02452
Collagen α-1(I) chain precursor
138799
163
Secreted
3%
Protein binding
Skeletal/epidermis development

P11047
Laminin γ-1 chain precursor
177492
74
Secreted
2%
Extracellular matrix structural constituent
Cell adhesion/migration

P07996
Thrombospondin-1 precursor
129330
50
Secreted
1%
Signal transducer activity
Multicellular organismal development

O95239
Chromosome-associated kinesin KIF4A
139794
46
Nucleus
2%
Protein binding
Anterograde axon cargo transport

Q5VTR2
Ubiquitin-protein ligase BRE1A
113592
39
Nucleus
1%
Transcription coactivator activity, ubiquitin-protein ligase activity and binding, zinc ion binding
Regulation of gene-specific transcription, protein polyubiquitination, negative regulation of cell migration

1-3
Q8TF76
Serine/threonine-protein kinase Haspin
88405
36
Nucleus
6%
ATP binding/protein kinase activity
Protein amino acid phosphorylation

P07996
Thrombospondin-1 precursor
129330
427
Secreted
11%
Signal transducer activity
Multicellular organismal development

P02452
Collagen α-1(I) chain precursor
138799
231
Secreted
8%
Protein binding
Epidermis/ skeletal development

P01024
Complement C3 precursor
187046
145
Secreted
2%
Receptor binding
Complement activation

P02751
Fibronectin precursor
262442
86
Secreted
3%
Collagen/heparin binding
Cell adhesion/migration

Q14980
Nuclear mitotic apparatus protein 1
238130
50
Nucleus
4%
Protein binding/structural molecule activity
Mitotic anaphase

P35442
Thrombospondin-2 precursor
129872
48
Secreted
1%
Heparin binding

P07814
Bifunctional aminoacyl-tRNA synthetase
162923
39
Cytoplasm
1%
Protein binding
Protein complex assembly

P81274
G-protein signaling modulator 2
75798
56
Cytoplasm
5%
Identical protein binding
G-protein coupled receptor protein signaling pathway

O14686
Myeloid/lymphoid or mixed-lineage leukemia protein 2
563831
48
Nucleus
1%
Protein/DNA binding
Regulation of transcription, DNA-dependent

Q9UQ26
Regulating synaptic membrane exocytosis protein 2
160303
43
Cell membrane
2%
Zinc ion binding, Rab GTPase binding
Intracellular protein transport

Q9UM54
Myosin-6
148620
35
Golgi apparatus
4%
ADP/calmodulin binding
DNA damage response, intracellular protein transport

O15020
Spectrin β chain, brain 2
271127
38
Cytoplasm, cytoskeleton
2%
Actin binding
Vesicle-mediated transport

1-4
O00339
Matrilin-2 precursor
106768
109
Secreted
3%
Calcium ion binding

1-5
Q08380
Galectin-3-binding protein precursor
65289
404
Secreted
23%
Protein binding/scavenger receptor activity
Cellular defense response/signal transduction

O94985
Calsyntenin-1 precursor
109724
55
Endoplasmic reticulum membrane, nucleus, Golgi membrane
1%
Calcium ion binding, protein binding
Homophilic cell adhesion

P13569
Cystic fibrosis transmembrane conductance regulator
168066
37
Membrane
1%
ATP-binding and phosphorylation-dependent chloride channel activity
Respiratory gaseous exchange, transport

P12814
α-actinin-1
102993
168
Cytoplasm
7%
Integrin binding
Regulation of apoptosis

1-6
O43707
α-actinin-4
104788
544
Nucleus
11%
Integrin binding
Regulation of apoptosis

P12814
α-actinin-1
102993
521
Cytoplasm
14%
Integrin binding
Regulation of apoptosis

Q08380
Galectin-3-binding protein precursor
65289
467
Secreted
18%
Protein binding/scavenger receptor activity
Cellular defense response/signal transduction

P35609
α-actinin-2
103788
165
Cytoplasm
6%
Integrin binding
Regulation of apoptosis

P34932
Heat shock 70 kDa protein 4
94240
82
Cytoplasm
4%
ATP binding
Response to unfolded protein

P35711
Transcription factor SOX-5
83973
40
Nucleus
8%
Transcription factor activity
Transcription from RNA polymerase II promoter

1-7
Q08380
Galectin-3-binding protein precursor
65289
277
Secreted
15%
Protein binding/scavenger receptor activity
cellular defense response/signal transduction

O43707
α-actinin-4
104788
163
Nucleus
10%
Integrin binding
Regulation of apoptosis

P08238
Heat shock protein HSP 90-β
83081
159
Cytoplasm
5%
Nitric-oxide synthase regulator activity
Response to unfolded protein

P29400
Collagen α-5(IV) chain precursor
160943
47
Secreted
4%
Binding, extracellular matrix structural constituent

Q13740
CD166 antigen precursor
65091
40
Membrane
4%
Receptor binding
Cell adhesion/signal transduction

P07900
Heat shock protein HSP 90- α
84476
225
Cytoplasm
5%
ATP binding/nitric-oxide synthase regulator activity
Response to unfolded protein/signal transduction

P12814
α-actinin-1
102993
194
Cytoplasm
6%
Integrin binding
Regulation of apoptosis

P35609
α-actinin-2
103788
74
Cytoplasm
3%
Integrin binding
Regulation of apoptosis

O00469
Procollagen-lysine,2-oxoglutarate 5-dioxygenase 2 precursor
84632
48
Rough endoplasmic reticulum membrane
4%
Protein binding
Protein modification process/response to hypoxia

P34932
Heat shock 70 kDa protein 4
94240
44
Cytoplasm
3%
ATP binding
Response to unfolded protein

Q13045
Protein flightless-1 homolog
144659
43
Nucleus
3%
Actin binding
Muscle contraction

1-8
P02768
Serum albumin precursor
69321
87
Secreted
3%
Protein binding/pyridoxal phosphate binding
Negative regulation of apoptosis /transport

Q8TEU7
Rap guanine nucleotide exchange factor 6
179294
44
Cytoplasm
3%
GTP-dependent protein binding
Ras protein signal transduction

1-9
O95248
SET-binding factor 1
208125
41
Nucleus
1%
Protein tyrosine/serine/threonine phosphatase activity
Protein amino acid dephosphorylation

Q9UPQ9
Trinucleotide repeat-containing 6B protein
182703
36
Cytoplasmic mRNA processing body
2%
RNA binding, nucleotide binding
Gene silencing by RNA, regulation of translation

P29401
Transketolase
67835
134
Cytosol
7%
Protein binding
Transketolase activity

P02768
Serum albumin precursor
69321
82
Secreted
5%
Protein binding/pyridoxal phosphate binding
Negative regulation of apoptosis/transport

O60333
Kinesin-like protein KIF1B
204305
58
Cytoplasmic vesicle
1%
ATPase activity/microtubule motor activity
Neuromuscular synaptic transmission/nerve-nerve synaptic transmission

O75095
Multiple epidermal growth factor-like domains 6 precursor
128524
46

6%
Calcium ion binding

1-10
P06744
Glucose-6-phosphate isomerase
62976
334
Cytoplasm
8%

Humoral immune response/carbohydrate metabolic process

P27797
Calreticulin precursor
48112
67
Endoplasmic reticulum lumen, Cytoplasm, Secreted
5%
DNA/protein binding
Regulation of apoptosis/transcription, DNA-dependent, protein export from nucleus

P09493
Tropomyosin 1 α chain
32689
44
Cytoplasm
8%
Structural constituent of muscle
Cell motility, regulation of heart/muscle contraction

1-11
Q06495
Sodium-dependent phosphate transport protein 2A
68893
49
Membrane
1%
Sodium-dependent phosphate transmembrane transporter activity
Body fluid secretion, phosphate transport

P37268
Squalene synthetase
48084
40
Endoplasmic reticulum membrane
5%
Farnesyl-diphosphate farnesyltransferase activity
Steroid biosynthetic process

Q12799
T-complex protein 10A homolog
45440
39
Cytosol
7%

O75095
Multiple epidermal growth factor-like domains 6 precursor
128524
46

1%
Calcium ion binding

Q9UM47
Neurogenic locus notch homolog protein 3 precursor
243496
37
Cell membrane
5%
Protein binding

P39191
Alu subfamily SB2 sequence contamination warning entry
65263
59

5%

Q12841
Follistatin-related protein 1 precursor
34963
52
Secreted
2%
Heparin binding
BMP signaling pathway

Q14980
Nuclear mitotic apparatus protein 1
238130
41
Nucleus
2%
Protein binding/structural molecule activity
Mitotic anaphase/nuclear organization and biogenesis

1-12
P14136
Glial fibrillary acidic protein, astrocyte
49850
80
Cytoplasm
7%
Structural constituent of cytoskeleton

P78527
DNA-dependent protein kinase catalytic subunit
468788
39
Nucleus
1%
DNA-dependent protein kinase activity
Peptidyl-serine phosphorylation

Q9UPQ9
Trinucleotide repeat-containing 6B protein
182703
49

4%

P06744
Glucose-6-phosphate isomerase
62976
401
Cytoplasm
16%

Humoral immune response/carbohydrate metabolic process

P14618
Pyruvate kinase isozymes M1/M2
57769
145
Cytosol
7%
Pyruvate kinase activity
Glycolysis

P00390
Glutathione reductase, mitochondrial precursor
56221
69
Mitochondrion
2%
Glutathione-disulfide reductase activity/electron carrier activity

P09622
Dihydrolipoyl dehydrogenase, mitochondrial precursor
54116
47
Mitochondrion matrix
4%
Dihydrolipoyl dehydrogenase activity

P30154
Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A β isoform
66159
42

3%
Protein heterodimerization activity

O95271
Tankyrase-1
141922
37
Cytoplasm
2%
NAD+ ADP-ribosyltransferase activity
Peptidyl-serine/threonine phosphorylation

1-13
P06733
α-enolase
47008
697
Cytoplasm
27%
Transcription factor activity, phosphopyruvate hydratase activity
Negative regulation of cell growth

Q15113
Procollagen C-endopeptidase enhancer 1 precursor
47942
114
Secreted
10%

Multicellular organismal development

P13929
β-enolase
46826
242
Cytoplasm
10%
Phosphopyruvate hydratase activity

P09104
γ-enolase
47108
135
Cytoplasm
4%
Phosphopyruvate hydratase activity

Q7Z3E2
Protein C10orf118
103623
42

2%

P11117
Lysosomal acid phosphatase precursor
48285
42
Lysosome
2%
Acid phosphatase activity

P02686
Myelin basic protein
33097
40
Myelin membrane
7%

Axon ensheathment, immune response, central nervous system development

P50395
Rab GDP dissociation inhibitor β
50631
40
Cytoplasm
2%
Rab GDP-dissociation inhibitor activity
Signal transduction

Q13371
Phosducin-like protein
34260
40
Cytoplasm
8%
Regulator of G-protein signaling activity
Signal transduction

P35573
Glycogen debranching enzyme
174523
39
Cytosol
1%
Amylo-α-1,6-glucosidase activity

Q6ZU80
Protein C14orf145
73526
38

4%

P49746
Thrombospondin-3 precursor
104135
36
Secreted
1%
Calcium ion binding
Cell-matrix adhesion

Q7L1I2
Synaptic vesicle glycoprotein 2B
77393
35
Cytoplasmic vesicle
3%

1-14
P09486
SPARC precursor
34610
36
Secreted
14%
Calcium/calcium ion binding
Ossification, transmembrane receptor protein tyrosine kinase signaling pathway

1-15
P04075
Fructose-bisphosphate aldolase A
39264
341
Cytoskeleton
37%
Fructose-bisphosphate aldolase activity
Fructose metabolic process, glycolysis

P09972
Fructose-bisphosphate aldolase C
39300
134
Cytoskeleton
13%
Fructose-bisphosphate aldolase activity
Fructose metabolic process

P00505
Aspartate aminotransferase, mitochondrial precursor
47445
45
Mitochondrion matrix
3%
Aspartate transaminase activity
Fatty acid transport, response to ethanol

1-16
P40925
Malate dehydrogenase, cytoplasmic
36272
40
Cytoplasm
11%
L-malate dehydrogenase activity, malic enzyme activity

O00623
Peroxisome assembly protein 12
40771
37
Peroxisome membrane
5%
Zinc ion/protein C-terminus binding
Protein import into peroxisome matrix

Q9NVP4
Protein C20orf12
62868
33
Intracellular
2%
zinc ion binding

P07195
L-lactate dehydrogenase B chain
36484
52
Cytoplasm
5%
L-lactate dehydrogenase activity

P10909
Clusterin precursor
52461
42
Secreted
5%
Protein binding
Complement activation, lipid metabolic process

1-17
P11117
Lysosomal acid phosphatase precursor
48285
44
Lysosome
2%
Acid phosphatase activity

P09493
Tropomyosin 1 α chain
32689
59
Cytoplasm
3%
Structural constituent of muscle
Cell motility, regulation of heart/muscle contraction

1-18
P18669
Phosphoglycerate mutase 1
28655
151
Cytosol
16%
Bisphosphoglycerate 2-phosphatase activity
Glycolysis

P63104
14-3-3 protein zeta/delta
27728
110
Cytoplasm
12%
Transcription factor binding
Antiapoptosis, signal transduction

O60242
Brain-specific angiogenesis inhibitor 3 precursor
171379
38
Cell membrane
1%

O00459
Phosphatidylinositol 3-kinase regulatory subunit β
81574
34
Cytosol
2%
Protein binding
Negative regulation of antiapoptosis

P46940
Ras GTPase-activating-like protein IQGAP1
189134
34
Cell membrane
2%
GTPase activator/inhibitor activity, calmodulin binding
Signal transduction

1-19
P60174
Triosephosphate isomerase
26522
156
Cytosol
18%
Triose-phosphate isomerase activity

P26232
α-2 catenin
105085
34
Cytoplasm
1%
Structural constituent of cytoskeleton
Cell adhesion

1-20
P60174
Triosephosphate isomerase
26522
373
Cytosol
37%
Triose-phosphate isomerase activity

Q06495
Sodium-dependent phosphate transport protein 2A
68893
49
Membrane
1%
Sodium-dependent phosphate transmembrane transporter activity, protein binding
Body fluid secretion, phosphate metabolic process and transport

Q14980
Nuclear mitotic apparatus protein 1
238115
44
Nucleus
4%
Protein binding, structural molecule activity
Mitotic anaphase, nuclear organization and biogenesis

P78527
DNA-dependent protein kinase catalytic subunit
468788
36
Nucleus
1%
DNA-dependent protein kinase activity/protein binding
Peptidyl-serine phosphorylation

1-21
Q06830
Peroxiredoxin-1
22096
231
Cytoplasm, melanosome
19%
Peroxidase activity/protein binding
Cell proliferation, hydrogen peroxide catabolic process, skeletal development

P30086
Phosphatidylethanolamine-binding protein 1
20913
122
Cytoplasm
10%
Phosphatidylethanolamine binding/protein binding

P16035
Metalloproteinase inhibitor 2 precursor
24383
104
Secreted
13%
Metalloendopeptidase inhibitor activity/protein binding

P04179
Superoxide dismutase
24707
66
Mitochondrion matrix
6%
Superoxide dismutase activity
Age-dependent response to reactive oxygen species, regulation of transcription from RNA polymerase II promoter, response to superoxide, superoxide metabolic process

P53618
Coatomer subunit β
107071
51
Cytoplasm
2%
Protein binding
COPI coating of Golgi vesicle, intra-Golgi vesicle-mediated transport, retrograde vesicle-mediated transport, Golgi to ER

O43395
U4/U6 small nuclear ribonucleoprotein Prp3
77481
45
Nucleus speckle
3%
Protein binding/RNA splicing factor activity, transesterification mechanism
Nuclear mRNA splicing, via spliceosome

O15240
Neurosecretory protein VGF precursor
67247
44
Secreted
7%

Response to cAMP

Q06495
Sodium-dependent phosphate transport protein 2A
68893
42
Membrane
1%
Protein binding/sodium-dependent phosphate transmembrane transporter activity
Body fluid secretion, phosphate metabolic process, phosphate transport

Q9Y587
AP-4 complex subunit sigma-1
16994
41
Golgi apparatus
9%
Transporter activity

Q04760
Lactoylglutathione lyase
20575
40
Cytoplasm
4%
Lactoylglutathione lyase activity
Antiapoptosis, carbohydrate metabolic process

P11117
Lysosomal acid phosphatase precursor
48285
40
Lysosome
2%
Acid phosphatase activity

Q9P0K1
ADAM 22 precursor
100368
39
Membrane
5%
Integrin binding
Central nervous system development, negative regulation of cell adhesion

Q96Q42
Alsin
183550
38
Cytosol
1%
Protein homodimerization activity/protein serine/threonine kinase activator activity/Rab GTPase binding/Rac guanyl-nucleotide exchange factor activity
Cell death, endosome organization, neuron projection morphogenesis, positive regulation of Rac GTPase activity, positive regulation of Rac protein signal transduction, positive regulation of protein kinase activity, regulation of endosome size

1-22
Q06830
Peroxiredoxin-1
22096
110
Cytoplasm, Melanosome
21%
Peroxidase activity/protein binding
Cell proliferation, hydrogen peroxide catabolic process, skeletal development

Q8N1I0
Dedicator of cytokinesis protein 4
225005
41
Intracytoplasmic membrane
1%
PDZ domain binding/Rac GTPase activator activity/Rac GTPase binding

P11117
Lysosomal acid phosphatase precursor
48285
39
Lysosome
2%
Acid phosphatase activity

1-23
O95274
Ly6/PLAUR domain-containing protein 3 precursor
35948
38
Cell membrane
8%

P00441
Superoxide dismutase [Cu-Zn]
15795
73
Cytoplasm, cytosol, nucleus
4%
Chaperone/phosphatase 2B/copper ion/zinc ionbinding, protein homodimerization activity, superoxide dismutase activity/antioxidant activity
Cell aging, oxidation reduction, regulation of organ growth, positive regulation of apoptosis

O15516
Circadian locomoter output cycles protein kaput
95244
55
Cytoplasm, nucleus
1%
Transcription factor activity
Circadian rhythm, photoperiodism, positive regulation of transcription from RNA polymerase II promoter, signal transduction

O00160
Myosin If
124725
39

2%
Actin binding/ATP binding/calmodulin binding

Q13075
Baculoviral IAP repeat-containing protein 1
159479
39
Intracellular
1%
Nucleoside-triphosphatase activity, nucleotide binding, zinc ion binding
Antiapoptosis, nervous system development

1-24
Q6ZUB1
Protein C9orf79
157037
35
Membrane
1%

Q9BXM0
Periaxin
154906
35
cytoplasm, nucleus
1%
Protein binding
Axon ensheathment

Q15084
Protein disulfide-isomerase A6 precursor
48091
38
Endoplasmic reticulum lumen, melanosome
2%
Protein binding/protein disulfide isomerase activity
Protein folding

Q13136
Liprin-α-1
135695
42
Cytoplasm
2%
Protein binding/signal transducer activity
Cell-matrix adhesion, signal transduction

Q32MQ0
Protein ZNF750
77312
44
Intracellular
5%
Zinc ion binding

Q15113
Procollagen C-endopeptidase enhancer 1 precursor
47942
48
Secreted
3%

Multicellular organismal development

P15531
Nucleoside diphosphate kinase A
17138
67
Cytoplasm, nucleus
17%
Deoxyribonuclease activity, DNA/GTP/magnesium ion binding, nucleoside diphosphate kinase activity
Negative regulation of cell proliferation, positive regulation of DNA binding, positive regulation of epithelial cell proliferation, regulation of apoptosis

P00441
Superoxide dismutase [Cu-Zn]
15795
70
Cytoplasm, cytosol, nucleus
19%
Chaperone/phosphatase 2B/copper ion/zinc ionbinding, protein homodimerization activity, superoxide dismutase activity/antioxidant activity
Cell aging, oxidation reduction, regulation of organ growth, positive regulation of apoptosis

P23528
Cofilin-1
18360
139
Nucleus matrix, cytoplasm
17%
Protein binding
Actin cytoskeleton organization and biogenesis, antiapoptosis, Rho protein signal transduction

1-25
Q96EZ8
Microspherule protein 1
51771
35
Nucleus
2%
Protein binding
Protein modification process

O95271
Tankyrase 1
141922
35
Cytoplasm, Golgi apparatus membrane, nucleus
1%
NAD+ ADP-ribosyltransferase activity
Peptidyl-serine phosphorylation, peptidyl-threonine phosphorylation, telomere maintenance via telomerase

Q14980
Nuclear mitotic apparatus protein 1
238115
35
Nucleus
1%
Protein binding, structural molecule activity
Mitotic anaphase, nuclear organization and biogenesis

P17612
cAMP-dependent protein kinase, α-catalytic subunit
40433
37
Cytoplasm, nucleus
1%
ATP binding, cAMP-dependent protein kinase activity
Hormone-mediated signaling, protein amino acid phosphorylation, protein kinase cascade

P11117
Lysosomal acid phosphatase precursor
48285
39
Lysosome
2%
acid phosphatase activity

P10071
Zinc finger protein GLI3
169743
39
Nucleus
1%
Protein binding, transcription factor activity
Anatomical structure morphogenesis, multicellular organismal development, protein import into nucleus, translocation, signal transduction

P78527
DNA-dependent protein kinase catalytic subunit
468788
40
Nucleus
1%
DNA-dependent protein kinase activity
Peptidyl-serine phosphorylation

Q06495
Sodium-dependent phosphate transport protein 2A
68893
40
Membrane
1%
Protein binding, sodium-dependent phosphate transmembrane transporter activity
Body fluid secretion, phosphate metabolic process, phosphate transport

P15531
Nucleoside diphosphate kinase A
17138
86
Cytoplasm, nucleus
17%
Deoxyribonuclease activity, DNA/GTP/magnesium ion binding, nucleoside diphosphate kinase activity
Negative regulation of cell proliferation, positive regulation of DNA binding, positive regulation of epithelial cell proliferation, regulation of apoptosis

P62937
Peptidyl-prolyl cis-trans isomerase A
17870
422
Cytoplasm
37%
Unfolded protein binding, virion binding
Initiation of viral infection, protein folding, provirus integration, regulation of viral genome replication

1-26
P62937
Peptidyl-prolyl cis-trans isomerase A
17870
171
Cytoplasm
32%
Unfolded protein binding, virion binding
Initiation of viral infection, protein folding, provirus integration, regulation of viral genome replication

O95352
Autophagy-related protein 7
77909
34
Cytoplasm
2%
Protein homodimerization activity, ubiquitin activating enzyme activity
Membrane fusion, positive regulation of protein modification process, protein amino acid lipidation

1-27
Q9UPT6
C-jun-amino-terminal kinase-interacting protein 3
146962
54
Cytoplasm
2%
kinesin/protein kinase binding, MAP-kinase scaffold activity
Regulation of JNK cascade, vesicle-mediated transport

Q8IWJ2
GRIP and coiled-coil domain-containing protein 2
184545
38
Cytoplasm, Golgi apparatus membrane
1%
Identical protein binding

Q9NX63
Coiled-coil-helix-coiled-coil-helix domain-containing protein 3
26136
39
Mitochondrion
13%
Protein binding

P02452
Collagen α-1(I) chain precursor
138799
44
Secreted
3%
Protein binding
Epidermis development, skeletal development

P59998
Actin-related protein 2/3 complex subunit 4
19523
45
Cytoplasm
2%
Protein binding, structural constituent of cytoskeleton
Actin nucleation

P01034
Cystatin C precursor
15789
56
Secreted
7%
Cysteine protease inhibitor activity, protein homodimerization activity

P23528
Cofilin-1
18360
178
Nucleus matrix, cytoplasm
19%
Protein binding
Actin cytoskeleton organization and biogenesis, antiapoptosis, Rho protein signal transduction

P07737
Profilin-1
14914
478
Cytoplasm
45%
Actin binding, proline-rich region binding
Actin cytoskeleton organization

1-28
P09382
Galectin-1
14575
41
Cytoplasm
7%
Protein binding, signal transducer activity
Positive regulation of I-κB kinase, NF-κB cascade, regulation of apoptosis

P10599
Thioredoxin
11599
59
Cytoplasm
13%
Protein binding
Cell motility, cell proliferation, cell-cell signaling, signal transduction

P04080
Cystatin B
11133
61
Cytoplasm, nucleus
12%
Endopeptidase inhibitor activity, protein binding

P07737
Profilin-1
14914
320
Cytoplasm
42%
Actin binding, proline-rich region binding
Actin cytoskeleton organization

1-29
Q9UPQ9
Trinucleotide repeat-containing 6B protein
182703
34
Cytoplasmic mRNA processing body
4%
RNA binding, nucleotide binding
Gene silencing by RNA, regulation of translation

P78527
DNA-dependent protein kinase catalytic subunit
468788
35
Nucleus
1%
DNA-dependent protein kinase activity, protein binding
Peptidyl-serine phosphorylation

P81605
Dermcidin precursor
11277
45
Secreted
12%
Protein binding

P10599
Thioredoxin
11599
76
Cytoplasm
19%
Protein binding
Cell motility, cell proliferation, cell-cell signaling, signal transduction

1-30
Q6NUM9
All-trans-retinol 13,14-reductase precursor
66777
39
Endoplasmic reticulum membrane
3%
All-trans-retinol 13,14-reductase activity
Oxidation reduction, retinol metabolic process

P14174
Macrophage migration inhibitory factor
12337
39
Cytoplasm, secreted
11%
Cytokine activity
Cell proliferation, cell surface receptor linked signal transduction, negative regulation of apoptosis, prostaglandin biosynthetic process, regulation of macrophage activation

Q6KC79
Nipped-B-like protein
315854
36
Nucleus
1%
Protein C-terminus binding
Maintenance of mitotic sister chromatid cohesion

P82279
Crumbs homolog 1 precursor
154081
36
Secreted
1%
Calcium ion binding
Cell-cell signaling, establishment and/or maintenance of cell polarity, response to stimulus

Q9NX63
Coiled-coil-helix-coiled-coil-helix domain-containing protein 3
26136
39
Mitochondrion
13%
Protein binding

Q8N0X7
Spartin
72788
41

2%

Cell death

P14174
Macrophage migration inhibitory factor
12337
60
Cytoplasm, secreted
5%
Cytokine activity
Cell proliferation, cell surface receptor linked signal transduction, negative regulation of apoptosis, prostaglandin biosynthetic process, regulation of macrophage activation

P58546
Myotrophin
12756
71
Cytoplasm
7%
Protein binding
Cell growth, neuron differentiation

1-31
Q8WUT4
Uncharacterized protein C20orf75 precursor
78794
37
Membrane
5%
Protein binding

O94851
Protein MICAL-2
126609
40
Cytoplasm
1%
Monooxygenase activity, zinc ion binding
Metabolic process

Q9Y333
U6 snRNA-associated Sm-like protein LSm2
10828
69
Nucleus
20%
Protein/U6 snRNA binding
Nuclear mRNA splicing

These serial numbers are designated as in Figure 3. Swiss-Prot/TrEMBL accession number was given from http://us.expasy.org/.

In this study, 47 proteins (35.6%) were known to be present in cytoplasm. Twenty-two proteins (16.7%) were known to be secreted into the extracellular space. Twenty-five proteins (18.9%) were known to be nuclear proteins. Eleven proteins (8.3%) were known to be membrane proteins. Ten proteins (7.6%) were known to be cytosol proteins. A few mitochondrial, endoplasmic reticulum, intracellular, cytoskeleton, and golgi apparatus proteins were also identified. A considerable portion of the identified proteins (6%, 8 proteins) has not been reported for their synthesized locations. Some proteins were described as found in different subcellular locations, which explains the total sum being substantially larger than 100%.

Some identified proteins in the distribution of cellular location were not secreted proteins, but they were still present in the secreted medium. To clarify the puzzle, a cell viability test was applied. The survival rate of HNPE cells was determined by the dimethylthiazol-diphenyltetrazolium bromide (MTT) assay, which was about 97%. Thus, those identified proteins were not corresponding to released proteins from dead cells. Also, according the protein profiles in Figure 3, the protein patterns obtained from secreted medium and cell lysate were very different. As a result, these proteins identified in this study can be considered as secreted proteins, which may have been synthesized inside the cells and transferred out.

Based on the functional categories in the Swiss-Prot and TrEMBL protein database, the identified proteins were classified into several groups. The Swiss-Prot identifiers could be employed for linkages of proteins to defined vocabulary of terms describing the cellular components, biological processes, and molecular functions of known gene ontology (GO). Gene Ontology Consortium provides annotations of each protein and its structure, which allowed us to organize selected proteins into biologically relevant groups. These groupings can be utilized as the basis for identifying biological information showing correlated protein changes [20, 32]. Such protein functions were listed in Table 2.

Table 2: The functions of 132 proteins identified in this study were presented into functional categories based on their annotations in the GO database.

SwissProt No.
Protein name
Protein function

O00160
Myosin If
Myosins are actin-based motor molecules with ATPase activity. Unconventional myosins serve in intracellular movements

O00339
Matrilin-2 precursor
Involved in matrix assembly

O00459
Phosphatidylinositol 3-kinase regulatory subunit β
Binds to activated (phosphorylated) protein-tyrosine kinases, through its SH2 domain, and acts as an adapter, mediating the association of the p110 catalytic unit to the plasma membrane.

O00469
Procollagen-lysine,2-oxoglutarate 5-dioxygenase 2 precursor
Forms hydroxylysine residues in -Xaa-Lys-Gly- sequences in collagens. These hydroxylysines serve as sites of attachment for carbohydrate units and are essential for the stability of the intermolecular collagen cross-links

O00623
Peroxisome assembly protein 12
Required for protein import into peroxisomes

O14686
Myeloid/lymphoid or mixed-lineage leukemia protein 2
Histone methyltransferase. Methylates “Lys-4” of histone H3. H3 “Lys-4” methylation represents a specific tag for epigenetic transcriptional activation. Plays a central role in β-globin locus transcription regulation by being recruited by NFE2. Acts as a coactivator for estrogen receptor by being recruited by ESR1, thereby activating transcription

O15020
Spectrin β chain, brain 2
Probably plays an important role in neuronal membrane skeleton

O15240
Neurosecretory protein VGF precursor
May be involved in the regulation of cell-cell interactions or in synaptogenesis during the maturation of the nervous system

O15516
Circadian locomotors output cycles protein kaput
ARNTL/2-CLOCK heterodimers activate E-box element (3′-CACGTG-5′) transcription of a number of proteins of the circadian clock. Activates transcription of PER1 and PER2. This transcription is inhibited in a feedback loop by PER and CRY proteins. Has intrinsic histone acetyltransferase activity and this enzymatic function contributes to chromatin-remodeling events implicated in circadian control of gene expression

O43395
U4/U6 small nuclear ribonucleoprotein Prp3
Participates in pre-mRNA splicing. May play a role in the assembly of the U4/U5/U6 tri-snRNP complex

O43707
α-actinin-4
F-actin cross-linking protein which is thought to anchor actin to a variety of intracellular structures. This is a bundling protein

O60242
Brain-specific angiogenesis inhibitor 3 precursor
Might be involved in angiogenesis inhibition and suppression of glioblastoma

O60333
Kinesin-like protein KIF1B
Motor for anterograde transport of mitochondria. Has a microtubule plus end-directed motility

O75095
Multiple epidermal growth factor-like domains 6 precursor

O94851
Protein MICAL-2

O94985
Calsyntenin-1 precursor
Induces KLC1 association with vesicles and functions as a cargo in axonal anterograde transport. Complex formation with APBA2 and APP, stabilizes APP metabolism and enhances APBA2-mediated suppression of β-APP40 secretion, due to the retardation of intracellular APP maturation. In complex with APBA2 and C99, a C-terminal APP fragment, abolishes C99 interaction with PSEN1 and thus APP C99 cleavage by γ-secretase, most probably through stabilization of the direct interaction between APBA2 and APP. The intracellular fragment AlcICD suppresses APBB1-dependent transactivation stimulated by APP C-terminal intracellular fragment (AICD), most probably by competing with AICD for APBB1-binding. May modulate calcium-mediated postsynaptic signals

O95239
Chromosome-associated kinesin KIF4A
Motor protein that translocates PRC1 to the plus ends of interdigitating spindle microtubules during the metaphase to anaphase transition, an essential step for the formation of an organized central spindle midzone and midbody and for successful cytokinesis. May play a role in mitotic chromosomal positioning and bipolar spindle stabilization

O95248
SET-binding factor 1
Probable pseudophosphatase. Lacks several amino acids in the catalytic pocket which renders it catalytically inactive as a phosphatase. The pocket is, however, sufficiently preserved to bind phosphorylated substrates, and may be protect them from phosphatases. Inhibits myoblast differentiation in vitro and induces oncogenic transformation in fibroblasts

O95271
Tankyrase 1
Regulate vesicle trafficking and modulate the subcellular distribution of SLC2A4/GLUT4-vesicles. Has PARP activity and can modify TERF1, and thereby contribute to the regulation of telomere length

O95274
Ly6/PLAUR domain-containing protein 3 precursor
Supports cell migration. May be involved in urothelial cell-matrix interactions. May be involved in tumor progression

O95352
Autophagy-related protein 7
E1 enzyme essential for multisubstrates such as GABARAPL1 and ATG12

P00390
Glutathione reductase, mitochondrial precursor
Maintains high levels of reduced glutathione in the cytosol

P00441
Superoxide dismutase (Cu-Zn)
Destroys radicals which are normally produced within the cells and which are toxic to biological systems

P00505
Aspartate aminotransferase, mitochondrial precursor
Facilitates cellular uptake of long-chain free fatty acids

P01024
Complement C3 precursor
C3 plays a central role in the activation of the complement system. Its processing by C3 convertase is the central reaction in both classical and alternative complement pathways. After activation C3b can bind covalently, via its reactive thioester, to cell surface carbohydrates or immune aggregates. Derived from proteolytic degradation of complement C3, C3a anaphylatoxin is a mediator of local inflammatory process. It induces the contraction of smooth muscle, increases vascular permeability, and causes histamine release from mast cells and basophilic leukocytes

P01034
Cystatin C precursor
As an inhibitor of cysteine proteinases, this protein is thought to serve an important physiological role as a local regulator of this enzyme activity

P02452
Collagen α-1(I) chain precursor
Type I collagen is a member of group I collagen (fibrillar forming collagen)

P02686
Myelin basic protein
The classic group of MBP isoforms (isoform 4-isoform 14) are with PLP the most abundant protein components of the myelin membrane in the CNS. They have a role in both its formation and stabilization. The smaller isoforms might have an important role in remyelination of denuded axons in multiple sclerosis. The nonclassic group of MBP isoforms (isoform 1-isoform 3/Golli-MBPs) may preferentially have a role in the early developing brain long before myelination, maybe as components of transcriptional complexes, and may also be involved in signaling pathways in T-cells and neural cells. Differential splicing events combined with optional posttranslational modifications give a wide spectrum of isomers, with each of them potentially having a specialized function. Induces T-cell proliferation

P02751
Fibronectin precursor
Fibronectins bind cell surfaces and various compounds including collagen, fibrin, heparin, DNA, and actin. Fibronectins are involved in cell adhesion, cell motility, opsonization, wound healing, and maintenance of cell shape. Interaction with TNR mediates inhibition of cell adhesion and neurite outgrowth

P02768
Serum albumin precursor
Serum albumin, the main protein of plasma, has a good binding capacity for water, Ca(2+), Na(+), K(+), fatty acids, hormones, bilirubin, and drugs. Its main function is the regulation of the colloidal osmotic pressure of blood

P04075
Fructose-bisphosphate aldolase A

P04080
Cystatin B
Intracellular thiol proteinase inhibitor

P04179
Superoxide dismutase
Destroys radicals which are normally produced within the cells and which are toxic to biological systems

P06733
α-enolase
Multifunctional enzyme that, as well as its role in glycolysis, plays a part in various processes such as growth control, hypoxia tolerance, and allergic responses. May also function in the intravascular and pericellular fibrinolytic system due to its ability to serve as a receptor and activator of plasminogen on the cell surface of several cell-types such as leukocytes and neurons. May be a tumor suppressor

P06744
Glucose-6-phosphate isomerase
Neuroleukin is a neurotrophic factor for spinal and sensory neurons

P07195
L-lactate dehydrogenase B chain

P07737
Profilin-1
Binds to actin and affects the structure of the cytoskeleton. At high concentrations, profilin prevents the polymerization of actin, whereas it enhances it at low concentrations. By binding to PIP2, it inhibits the formation of IP3 and DG

P07814
Bifunctional aminoacyl-tRNA synthetase
Catalyzes the attachment of the cognate amino acid to the corresponding tRNA in a two-step reaction: the amino acid is first activated by ATP to form a covalent intermediate with AMP and is then transferred to the acceptor end of the cognate tRNA

P07900
Heat shock protein HSP 90-α
Molecular chaperone. Has ATPase activity

P07996
Thrombospondin-1 precursor
Adhesive glycoprotein that mediates cell-to-cell and cell-to-matrix interactions. Can bind to fibrinogen, fibronectin, laminin, type V collagen, and integrins α-V/ β-1, α-V/ β-3 and α-IIb/ β-3

P08238
Heat shock protein HSP 90- β
Molecular chaperone. Has ATPase activity.

P09104
γ-enolase
Has neurotrophic and neuroprotective properties on a broad spectrum of central nervous system (CNS) neurons. Binds, in a calcium-dependent manner, to cultured neocortical neurons and promotes cell survival

P09382
Galectin-1
Cell apoptosis and cell differentiation

P09486
SPARC precursor
Appears to regulate cell growth through interactions with the extracellular matrix and cytokines. Binds calcium and copper, several types of collagen, albumin, thrombospondin, PDGF and cell membranes. There are two calcium binding sites; an acidic domain that binds 5 to 8 Ca(2+) with a low affinity and an EF-hand loop that binds a Ca(2+) ion with a high affinity

P09493
Tropomyosin 1 α chain
Binds to actin filaments in muscle and nonmuscle cells. Plays a central role, in association with the troponin complex, in the calcium dependent regulation of vertebrate striated muscle contraction. Smooth muscle contraction is regulated by interaction with caldesmon. In non-muscle cells is implicated in stabilizing cytoskeleton actin filaments

P09622
Dihydrolipoyl dehydrogenase, mitochondrial precursor
Lipoamide dehydrogenase is a component of the glycine cleavage system as well as of the α-ketoacid dehydrogenase complexes

P09972
Fructose-bisphosphate aldolase C

P10071
Zinc finger protein GLI3
A role in limb and brain development

P10599
Thioredoxin
Participates in various redox reactions through the reversible oxidation of its active center dithiol to a disulfide and catalyzes dithiol-disulfide exchange reactions

P10909
Clusterin precursor
Not yet clear. It is known to be expressed in a variety of tissues, and it seems to be able to bind to cells, membranes, and hydrophobic proteins. It has been associated with programmed cell death (apoptosis)

P11047
Laminin γ-1 chain precursor
Binding to cells via a high-affinity receptor, laminin is thought to mediate the attachment, migration, and organization of cells into tissues during embryonic development by interacting with other extracellular matrix components

P11117
Lysosomal acid phosphatase precursor

P12814
α-actinin-1
F-actin cross-linking protein which is thought to anchor actin to a variety of intracellular structures. This is a bundling protein

P13569
Cystic fibrosis transmembrane conductance regulator
Involved in the transport of chloride ions. May regulate bicarbonate secretion and salvage in epithelial cells by regulating the SLC4A7 transporter

P13929
β-enolase
Appears to have a function in striated muscle development and regeneration

P14136
Glial fibrillary acidic protein, astrocyte
GFAP, a class-III intermediate filament, is a cell-specific marker that, during the development of the central nervous system, distinguishes astrocytes from other glial cells

P14174
Macrophage migration inhibitory factor
Mediator in regulating the function of macrophage in host defense

P14618
Pyruvate kinase isozymes M1/M2
Glycolytic enzyme that catalyzes the transfer of a phosphoryl group from phosphoenolpyruvate (PEP) to ADP, generating ATP

P15531
Nucleoside diphosphate kinase A
Synthesis of nucleoside triphosphates other than ATP

P16035
Metalloproteinase inhibitor 2 precursor
Complexes with metalloproteinases (such as collagenases) and irreversibly inactivates them. Known to act on MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-13, MMP-14, MMP-15, MMP-16, and MMP-19

P17612
cAMP-dependent protein kinase, α-catalytic subunit
Phosphorylates a large number of substrates in the cytoplasm and the nucleus

P18669
Phosphoglycerate mutase 1
Interconversion of 3- and 2-phosphoglycerate with 2,3-bisphosphoglycerate as the primer of the reaction. Can also catalyze the reaction of synthase and phosphatase, but with a reduced activity

P23528
Cofilin-1
Controls reversibly actin polymerization and depolymerization

P26232
α-2 catenin

P27797
Calreticulin precursor
Molecular calcium binding chaperone promoting folding, oligomeric assembly and quality control in the ER via the calreticulin/calnexin cycle. This lectin interacts transiently with almost all of the monoglucosylated glycoproteins that are synthesized in the ER. Interacts with the DNA-binding domain of NR3C1 and mediates its nuclear export

P29400
Collagen α-5(IV) chain precursor
Type IV collagen is the major structural component of glomerular basement membranes (GBM), forming a “chicken-wire” meshwork together with laminins, proteoglycans and entactin/nidogen

P29401
Transketolase

P30086
Phosphatidylethanolamine-binding protein 1
Binds ATP, opioids, and phosphatidylethanolamine. Has lower affinity for phosphatidylinositol and phosphatidylcholine. Serine protease inhibitor which inhibits thrombin, neuropsin, and chymotrypsin but not trypsin, tissue type plasminogen activator, and elastase/HCNP may be involved in the function of the presynaptic cholinergic neurons of the central nervous system. HCNP increases the production of choline acetyltransferase but not acetylcholinesterase. Seems to be mediated by a specific receptor

P30154
Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A β isoform
The PR65 subunit of protein phosphatase 2A serves as a scaffolding molecule to coordinate the assembly of the catalytic subunit and a variable regulatory B subunit

P34932
Heat shock 70 kDa protein 4

P35442
Thrombospondin-2 precursor
Adhesive glycoprotein that mediates cell-to-cell and cell-to-matrix interactions. Can bind to fibrinogen, fibronectin, laminin, and type V collagen

P35573
Glycogen debranching enzyme
Multifunctional enzyme acting as 1,4-α-D-glucan:1,4-α-D-glucan 4-α-D-glycosyltransferase and amylo-1,6-glucosidase in glycogen degradation

P35609
α-actinin-2
F-actin cross-linking protein which is thought to anchor actin to a variety of intracellular structures. This is a bundling protein

P35711
Transcription factor SOX-5
Binds specifically to the DNA sequence 5′-AACAAT-3′. Activates transcription of COL2A1 and AGC1 in vitro

P37268
Squalene synthetase

P39191
Alu subfamily SB2 sequence contamination warning entry

P40925
Malate dehydrogenase, cytoplasmic

P46940
Ras GTPase-activating-like protein IQGAP1
Binds to activated CDC42 but does not stimulate its GTPase activity. It associates with calmodulin. Could serve as an assembly scaffold for the organization of a multimolecular complex that would interface incoming signals to the reorganization of the actin cytoskeleton at the plasma membrane. May promote neurite outgrowth

P49746
Thrombospondin-3 precursor
Adhesive glycoprotein that mediates cell-to-cell and cell-to-matrix interactions. Can bind to fibrinogen, fibronectin, laminin, and type V collagen

P50395
Rab GDP dissociation inhibitor β
Regulates the GDP/GTP exchange reaction of most Rab proteins by inhibiting the dissociation of GDP from them, and the subsequent binding of GTP to them

P53618
Coatomer subunit β
The coatomer is a cytosolic protein complex that binds to dilysine motifs and reversibly associates with Golgi nonclathrin-coated vesicles, which further mediate biosynthetic protein transport from the ER, via the Golgi up to the trans Golgi network. Coatomer complex is required for budding from Golgi membranes, and is essential for the retrograde Golgi-to-ER transport of dilysine-tagged proteins. In mammals, the coatomer can only be recruited by membranes associated to ADP-ribosylation factors (ARFs), which are small GTP-binding proteins; the complex also influences the Golgi structural integrity, as well as the processing, activity, and endocytic recycling of LDL receptors

P58546
Myotrophin
Cerebellar morphogenesis

P59998
Actin-related protein 2/3 complex subunit 4
Actin-binding component of the Arp2/3 complex which is involved in regulation of actin polymerization and together with an activating nucleation-promoting factor (NPF) mediates the formation of branched actin networks

P60174
Triosephosphate isomerase

P62937
Peptidyl-prolyl cis-trans isomerase A
PPIases accelerate the folding of proteins

P63104
14-3-3 protein zeta/delta
Adapter protein implicated in the regulation of a large spectrum of both general and specialized signaling pathway. Binds to a large number of partners, usually by recognition of a phosphoserine or phosphothreonine motif. Binding generally results in the modulation of the activity of the binding partner

P78527
DNA-dependent protein kinase catalytic subunit
Serine/threonine-protein kinase that acts as a molecular sensor for DNA damage. Involved in DNA nonhomologous end joining (NHEJ) required for double-strand break (DSB) repair and V(D)J recombination. Must be bound to DNA to express its catalytic properties. Promotes processing of hairpin DNA structures in V(D)J recombination by activation of the hairpin endonuclease artemis (DCLRE1C). The assembly of the DNA-PK complex at DNA ends is also required for the NHEJ ligation step. Required to protect and align broken ends of DNA. May also act as a scaffold protein to aid the localization of DNA repair proteins to the site of damage. Found at the ends of chromosomes, suggesting a further role in the maintenance of telomeric stability and the prevention of chromosomal end fusion. Also involved in modulation of transcription. Recognizes the substrate consensus sequence (ST)-Q. Phosphorylates “Ser-139” of histone variant H2AX/H2AFX, thereby regulating DNA damage response mechanism. Phosphorylates DCLRE1C, c-Abl/ABL1, histone H1, HSPCA, c-jun/JUN, p53/TP53, PARP1, POU2F1, DHX9, SRF, XRCC1, XRCC1, XRCC4, XRCC5, XRCC6, WRN, c-myc/MYC, and RFA2. Can phosphorylate C1D not only in the presence of linear DNA but also in the presence of supercoiled DNA. Ability to phosphorylate TP53/p53 in the presence of supercoiled DNA is dependent on C1D

P81274
G-protein signaling modulator 2
Plays an important role in spindle pole orientation. Interacts and contributes to the functional activity of G(i) α proteins. Acts to stabilize the apical complex during neuroblast divisions

P81605
Dermcidin precursor
DCD-1 displays antimicrobial activity thereby limiting skin infection by potential pathogens in the first few hours after bacterial colonization. Highly effective against E. coli, E. faecalis, S. aureus and C. albicans. Optimal pH and salt concentration resemble the conditions in sweat. Survival-promoting peptide promotes survival of neurons and displays phosphatase activity. It may bind IgG

P82279
Crumbs homolog 1 precursor
Photoreceptor morphogenesis in the retina/May maintain cell polarization and adhesion

Q04760
Lactoylglutathione lyase
Catalyzes the conversion of hemimercaptal, formed from methylglyoxal and glutathione, to S-lactoylglutathione

Q06495
Sodium-dependent phosphate transport protein 2A
May be involved in actively transporting phosphate into cells via Na(+) cotransport in the renal brush border membrane. Probably mediates 70–80% of the apical influx

Q06830
Peroxiredoxin-1
Involved in redox regulation of the cell. Reduces peroxides with reducing equivalents provided through the thioredoxin system but not from glutaredoxin. May play an important role in eliminating peroxides generated during metabolism. Might participate in the signaling cascades of growth factors and tumor necrosis factor-α by regulating the intracellular concentrations of H₂O₂

Q08378
Golgin subfamily A member 3
Golgi autoantigen; probably involved in maintaining Golgi structure

Q08380
Galectin-3-binding protein precursor
Promotes integrin-mediated cell adhesion. May stimulate host defense against viruses and tumor cells

Q12799
T-complex protein 10A homolog

Q12841
Follistatin-related protein 1 precursor
May modulate the action of some growth factors on cell proliferation and differentiation. Binds heparin

Q13045
Protein flightless-1 homolog
May play a role as coactivator in transcriptional activation by hormone-activated nuclear receptors (NR) and acts in cooperation with NCOA2 and CARM1. Involved in estrogen hormone signaling. Involved in early embryonic development (By similarity). May play a role in regulation of cytoskeletal rearrangements involved in cytokinesis and cell migration

Q13075
Baculoviral IAP repeat-containing protein 1
Prevents motor-neuron apoptosis induced by a variety of signals

Q13136
Liprin-α-1
Regulate the disassembly of focal adhesions/may localize receptor-like tyrosine phosphatases type 2A at specific sites on the plasma membrane, possibly regulating their interaction with the extracellular environment and their association with substrates

Q13371
Phosducin-like protein

Q13740
CD166 antigen precursor
Cell adhesion molecule that binds to CD6. Involved in neurite extension by neurons via heterophilic and homophilic interactions. May play a role in the binding of T- and B-cells to activated leukocytes, as well as in interactions between cells of the nervous system

Q14980
Nuclear mitotic apparatus protein 1
May be a structural component of the nucleus

Q15084
Protein disulfide-isomerase A6 precursor
Catalyzes the rearrangement of -S-S- bonds in proteins

Q15113
Procollagen C-endopeptidase enhancer 1 precursor
Binds to the C-terminal propeptide of type I procollagen and enhances procollagen C-proteinase activity

Q32MQ0
Protein ZNF750

Q5VTR2
Ubiquitin-protein ligase BRE1A
E3 ubiquitin-protein ligase that mediates monoubiquitination of “Lys-120” of histone H2B. H2B “Lys-120” ubiquitination gives a specific tag for epigenetic transcriptional activation and is also prerequisite for histone H3 “Lys-4” and “Lys-79” methylation. Forms a ubiquitin ligase complex in cooperation with the E2 enzyme UBE2E1/UBCH6. It thereby plays a central role in histone code and gene regulation. Required for transcriptional activation of Hox genes. Recruited to the MDM2 promoter, probably by being recruited by p53/TP53, and thereby acts as a transcriptional coactivator

Q6KC79
Nipped-B-like protein
Probably plays a structural role in chromatin. Involved in sister chromatid cohesion, possibly by interacting with the cohesin complex

Q6NUM9
All-trans-retinol 13,14-reductase precursor
Play a role in the metabolism of vitamin A

Q6ZU80
Protein C14orf145

Q6ZUB1
Protein C9orf79

Q7L1I2
Synaptic vesicle glycoprotein 2B
Probably plays a role in the control of regulated secretion in neural and endocrine cells

Q7Z3E2
Protein C10orf118

Q8IWJ2
GRIP and coiled-coil domain-containing protein 2
Function probably involved in maintaining Golgi structure

Q8N0X7
Spartin
May be implicated in endosomal trafficking, or microtubule dynamics, or both.

Q8N1I0
Dedicator of cytokinesis protein 4
Involved in regulation of adherens junction between cells. Functions as a guanine nucleotide exchange factor (GEF), which activates Rap1 small GTPase by exchanging bound GDP for free GTP

Q8TEU7
Rap guanine nucleotide exchange factor 6
Guanine nucleotide exchange factor (GEF) for Rap1A, Rap2A, and M-Ras GTPases. Does not interact with cAMP

Q8TF76
Serine/threonine-protein kinase Haspin
Required for normal alignment of chromosomes at metaphase. Phosphorylates histone H3 “Thr-3” during mitosis

Q8WUT4
Uncharacterized protein C20orf75 precursor
May play an important role in hippocampus-dependent long-lasting memory

Q96EZ8
Microspherule protein 1
Modulates the transcription repressor activity of DAXX by recruiting it to the nucleolus. May be an inhibitor of TERT telomerase activity

Q96Q42
Alsin
May act as a GTPase regulator. Controls survival and growth of spinal motoneurons

Q9BXM0
Periaxin
Is required for maintenance of peripheral nerve myelin sheath/may have a role in axon-glial interactions

Q9NVP4
Protein C20orf12

Q9NX63
Coiled-coil-helix-coiled-coil-helix domain-containing protein 3

Q9P0K1
ADAM 22 precursor
Probable ligand for integrin in the brain. This is a noncatalytic metalloprotease-like protein. Involved in regulation of cell adhesion and spreading and in inhibition of cell proliferation

Q9UM47
Neurogenic locus notch homolog protein 3 precursor
Functions as a receptor for membrane-bound ligands Jagged1, Jagged2, and Delta1 to regulate cell-fate determination. Upon ligand activation through the released notch intracellular domain (NICD) it forms a transcriptional activator complex with RBP-Jκ and activates genes of the enhancer of split locus. Affects the implementation of differentiation, proliferation, and apoptotic programs

Q9UM54
Myosin-6
Myosins are actin-based motor molecules with ATPase activity. Unconventional myosins serve in intracellular movements. Myosin 6 is a reverse-direction motor protein that moves towards the minus-end of actin filaments. Has slow rate of actin-activated ADP release due to weak ATP binding. Functions in a variety of intracellular processes such as vesicular membrane trafficking and cell migration. Required for the structural integrity of the Golgi apparatus via the p53-dependent prosurvival pathway. Appears to be involved in a very early step of clathrin-mediated endocytosis in polarized epithelial cells. May act as a regulator of F-actin dynamics. May play a role in transporting DAB2 from the plasma membrane to specific cellular targets. Required for structural integrity of inner ear hair cells

Q9UPQ9
Trinucleotide repeat-containing 6B protein
Plays a role in RNA-mediated gene silencing by both micro-RNAs (miRNAs) and short interfering RNAs (siRNAs). Required for miRNA-dependent translational repression and siRNA-dependent endonucleolytic cleavage of complementary mRNAs by argonaute family proteins

Q9UPT6
C-jun-amino-terminal kinase-interacting protein 3
Mediates JNK signaling by aggregating specific components of the MAPK cascade to form a functional JNK signaling module. May function as a regulator of vesicle transport, through interactions with the JNK-signaling components and motor proteins

Q9UQ26
Regulating synaptic membrane exocytosis protein 2
Rab effector involved in exocytosis. May act as scaffold protein.

Q9Y333
U6 snRNA-associated Sm-like protein LSm2
Binds specifically to the 3′-terminal U-tract of U6 snRNA. May be involved in pre-mRNA splicing

Q9Y587
AP-4 complex subunit sigma-1
Subunit of novel type of clathrin- or nonclathrin-associated protein coat involved in targeting proteins from the trans-Golgi network (TGN) to the endosomal-lysosomal system

*sort by SwissProt No.

In this study, some of the proteins secreted by HNPE cells, which were confirmed by the Western blotting method, may be candidate factors responsible for promoting differentiation of RGC-5 cells including thrombospondin-1, 2, 3 precursor (1-2, 1-3, 1-13), galectin-3-binding protein (1-5~1-7), neurogenic locus notch homolog protein 3 (Notch-3, 1-11), follistatin-related protein 1 precursor (1-11), sPARC precursor (1-14), peroxiredoxin-1 (1-21, 1-22), cofilin 1 (1-24, 1-27), profilin 1 (1-27, 1-28), galectin-1 (1-28), and myotrophin (1-30). Cell differentiation is directed by a variety of intra- and extracellular events including signals generated by extracellular matrix (ECM) components, which mediate adhesive cell-to-cell interactions and trigger a cascade of post-receptor intracellular signaling pathways. The roles for ECM proteins in cell growth and differentiation can be indicated by their abilities to modulate a variety of growth factors [33].

Thrombospondin (TSP, MW~420 kDa), which belongs to a multigene family of modular modular glycoproteins, is composed of three identical subunits within a disulfide linkage. TSP is synthesized by several matrix-forming cells and is incorporated into their extracellular matrix. In several cell types, this protein supports cell growth and proliferation. As a component of ECM, TSP is involved in the regulation of mediate platelet aggregation, inflammation, and angiogenesis as well as adhesion, migration, growth, and differentiation of a number of normal and transformed cells [34, 35]. The expression of the TSP has been also investigated during the process of differentiation of embryonal carcinoma cells, granulose cells and HL-60 cells in vitro [36–39]. Although the TSP is prevalent in differentiated cells, the induced TSP syntheses during the differentiation may function differently during neurogenesis.

In the eye, TSP-1 is localized in the epiretinal membrane and between the retinal pigment epithelial layer and Bruch’s membrane, which is a cell-attachment factor with cell-specific affinity. TSP-1 production by retinal pigment epithelial cells is affected by the state of proliferation and cell density. With its anti-angiogenic activity, TSP-1 may play several biologic roles on Bruch’s membrane [35]. In another report, the authors evaluated the bone marrow stromal cells (BMSCs) secretion of TSP-1, which is a putative mechanistic agent acting on RGCs for survival and growth [40]. The BMSC-derived TSP-1 is identified as a specific mediator of reparative processes in neurons, which functions included enhanced RGC neurite formation, cell survival, and expression of synaptophysin. It suggested that the TSP-1 signaling pathway might be an important role in neural-like differentiation in BMSCs and outgrowth in RGCs [40]. These observations suggest that the synthesis of TSP contributes to the differentiation options/alternatives of RGC-5 cells toward a neural fate, reminiscent of their neural crest origin.

TSP-2 and SPARC (secreted protein, acidic and rich in cysteine) are classified as matricellular proteins. TSP-2 appears to play a role in reducing proliferation, while SPARC may have a positive role in progenitor cell expansion. TSP-2 and SPARC have been shown to positively influence osteoblast differentiation, with the ability to limit adipogenesis [41, 42].

TSP-3 is structurally similar to cartilage oligomeric matrix protein (COMP/TSP-5), and was a recently described member with the calcium binding Type 3 repeats. Like Type 1 and 2 repeats, TSP-3 is absence of the complement and contains four epidermal growth factor receptors with a distinct N terminus that has no significant homology to other TSPs. TSP-3 is also an oligomeric heparin binding protein present in both the cell layer and medium [43].

Galectin-3-binding protein (G3BP), also known as Mac-2 binding protein, is a secreted glycoprotein with a molecular mass of ~90 kDa present in the extracellular matrix of cells. Gelectins and their binding proteins have primarily been described in cell-cell and cell-matrix interactions and play roles in autoimmunity, inflammation and tumor progression or metastasis [44]. G3BP promotes integrin-mediated cell adhesion and functions in cancer progression of human tumor cells. It also binds to multiple proteins in the extracellular matrix including collagen, fibronectin, and nidogen, and to molecules mediating cell-cell and cell-matrix adhesions that are critical during tumor cell invasion and migration [45–48].

Notch-3 was the third discovered human homologue of the Drosophila melanogaster type I membrane protein notch. In Drosophila, the interaction of notch with its cell-bound ligands (delta and serrate) establishes an intercellular signaling pathway that plays a key role in neural development. Members of the Notch gene family were thought to be involved as receptors for membrane-bound ligands Jagged1, Jagged2, and Delta1 in the regulation of cell fate in a variety of neurogenesis of embryos, particularly in the developing central nervous system (CNS) from the homogenous cell population of the neural tube [49, 50]. The Notch-3 activation induces the increase of the progenitor cell number in the CNS and affected CNS development. The Notch-3 mutation may lead to cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). CADASIL leads to stroke and dementia and is the main feature of recurrent subcortical ischemic events and vascular dementia. Such mutations affect highly-conserved cysteine residues in epidermal growth factor- (EGF-) like repeat domain in the extracellular part of the receptor [51, 52].

Follistatin-related protein (FSRP) is a recently discovered glycoprotein that is highly homologous in both primary sequence and exon/intron domain structure to the activin-binding protein, follistatin (FS). FS is a secreted monomeric glycoprotein and a member of a large group of proteins containing a highly conserved module of cysteine-rich sequence termed the follistatin domain. It was first isolated from ovarian follicular fluid on the basis of its ability to suppress FSH secretion by pituitary cells in vitro [53]. This follistatin gene family includes follistatin, follistatin-related gene (FLRG) protein, follistatin-related protein (FSRP), agrin, secreted protein acidic, and it is rich in cysteine (SPARC), and Mac25 [54]. A follistatin-like sequence containing 10 conserved cysteine residues may modulate the action of some growth factors on cell proliferation and differentiation. It was also thought to be an autoantigen associated with rheumatoid arthritis [55].

SPARC, also known as osteonectin, 43 K protein, or BM-40, is a 32.7 kDa calcium- and copper-binding glycoprotein, which is a product of natural synthesis from osteoblasts, endothelial cells, and megakaryocytes. It functions as a counteradhesive protein, as a modulator of growth factor activity, and as a cell-cycle inhibitor [56]. SPARC belongs to matrix-associated factors that mediate cell-matrix interactions. Other members of this group include TSP-1 and -2, osteopontin (OPN), tenascins, and the SPARC-related proteins. Expressed during many stages of development in a variety of organisms, the expression of this matricellular protein, SPARC, is restricted in adult vertebrates primarily to tissues that undergo consistent turnover or to sites of injuries and diseases [56]. Vertebrate SPARC binds to a number of different ECM components including albumin, thrombospondin 1, PDGF, vitronectin, entactin/nidogen, fibrillar collagens (types I, II, III, and V), and collagen type IV, the prevalent collagen in basement membranes [57]. The ability of SPARC to bind to several resident ECM proteins affects the expression of matrix metalloproteinases and adjusts effects of growth factors; as a counteradhesive factor of cell shape change, this supports SPARC to regulate cell interactions during their development [57]. SPARC appears to regulate cell growth through interactions with the extracellular matrix and cytokines. It is also a matricellular protein that modulates cell adhesion and proliferation and is thought to function in tissue remodeling and angiogenesis [58, 59].

Peroxiredoxin (PRDX) is a recently identified family of antioxidative proteins that includes six isoforms in mammals. They share a common reactive Cys residue in the N-terminal region and are capable of serving as a peroxidase, involving thioredoxin and/or glutathione as the electron donor. PRDX 1–4 have an additional reactive Cys residue in the conserved C-terminal region and show >70% amino acid sequence homology. In this capacity, they may be involved in the protection of cells from oxidative stress. Peroxiredoxin1 (PRDX1) is ubiquitously expressed and functions as an antioxidant enzyme, which reduces hydrogen peroxide and alkyl hydroperoxide and is involved in cellular proliferation, differentiation, apoptosis, and innate immunity [60]. PRDX1 may participate in the signal cascades of growth factors and tumor necrosis factor-α by regulating the intracellular concentrations of hydrogen peroxide [61–63]. A previous study also applied a proteomic approach to study PRDX1, -2, and -3 expressions in Alzheimer’s diseases and Down’s syndrome, and found a significant increase in PRDX1 expression associated with the neurodegenerative diseases [64].

The human cofilin protein has a molecular weight of approximately 21 kDa. It is a member of the actin depolymerization factor (ADF)/cofilin family. Cofilin is an essential cellular protein that can bind the barbed end of actin and is required for cell viability [65]. In cells, cofilin acts in harmony with other regulatory proteins to mediate the response of the actin cytoskeleton to extracellular signals. In vertebrates, cofilin is regulated by pH, phosphorylation and phosphoinositides. It is involved in the translocation of the actin-cofilin complex from cytoplasm to nucleus. Cofilin plays an essential role in actin filament dynamics by enhancing depolymerization and severance of actin filaments [66]. These activities of cofilin can be abolished by phosphorylation at Ser-3; therefore, phosphorylation/dephosphorylation of cofilin at Ser-3 is regarded as one of the important mechanisms for regulating cofilin activities and actin filament dynamics [67]. Sinha et al. reported that the suppression of cofilin might lead to cancer regression [68].

Profilin-1 (PFN1) is a widely and highly expressed 14- to 17-kDa cytoplasmic and nuclear ligand protein of the microfilament system. It is a ubiquitous actin monomer-binding protein involved in actin polymerization in response to extracellular signaling pathways. PFN1 plays a central role in the regulation of de novo actin assembly by preventing spontaneous actin polymerisation through the binding of actin monomers and addition of monomeric actin to the barbed actin-filament ends [69]. The importance of profilins for normal cell proliferation, differentiation, cellular survival, motility, adhesion, migration, and cytoskeleton remodelling has been verified [69–72]. PFN1 may be a tumor suppressor because its expression was reduced in several types of invasive cancers and it was able to suppress tumorigenicity when overexpressed [73]. In addition, the immunohistochemistry analysis also showed low levels of PFN1 in several human breast cancers. Other than being a tumor suppressor, PFN1 was reported as a necessary element for differentiation of human epithelial cells [74].

Galectins are a family of structurally related carbohydrate-binding proteins and widely distributed in nematodes, insects, and porifer, as well as vertebrates and fungi [75]. They are defined by their affinity for poly-N-acetyllactosamine-enriched glycoconjugates and sequence similarities in the carbohydrate recognition domain. The galectins are a family of β-galactoside-binding proteins implicated in modulating cell-cell and cell-matrix interactions, which would be required for protein secretion through the classical secretory pathways found in the extracellular space [76].

Galectin-1 is expressed during human embryogenesis, and many adult cell types express and secrete galectin-1 into the extracellular matrix [76]. Galectin-1 contributes to different events associated with cancer biology, including tumour transformation, proliferation, differentiation, cell cycle regulation, growth arrest, apoptosis, cell adhesion, migration, inflammation, and inhibition of full cell activation [77]. A previous study has shown that galectin-1 induced sustained exposure of phosphatidylserine on the cell surface in a carbohydrate-dependent fashion, but phosphatidylserine exposure is not associated with cell death by apoptosis and does not affect cell viability. There is evidence that galectin-1 contributes to tumour evasion of immune responses [78].

A positive correlation has recently been shown between galectin-3 expression and the degree of malignant transformation in certain types of cell lines, and the amount of galectin-3 expression is expected to possibly serve as an index of degree for neoplastic transformation, tumor cell survival, angiogenesis, tumor metastasis, and tumor malignancy [79, 80]. Recent studies have revealed that intracellular galectin-3 exhibits the activity to suppress drug-induced apoptosis and anoikis that contribute to cell survival. Resistance to apoptosis is essential for cancer cell survival and plays a role in tumor progression [81].

Moreover, both galectin-1 and galectin-3 expressions are necessary for the initiation of the transformed phenotype of tumors. Inhibition of galectin-1 expression can suppress the transformed phenotype of human glioma cells [82]. In addition, following the inhibition of galectin-3 expression, breast carcinoma cells and thyroid papillary carcinoma cells lose their transformed characteristic phenotypes in cell culture [83, 84].

Myotrophin, a 12 kDa protein consists of 117 amino acids, has a potential role in cerebellar morphogenesis and may be involved in differentiation of cerebellar neurons, particularly of granule cells, and associated with cardiac hypertrophy. It appears to be a primary modulator for myocardial cell growth and differentiation [85]. Myotrophin accelerates myocyte growth by stimulating protein synthesis and may be correlated with cardiac hypertrophy in the pathogenesis, where it is involved in the conversion of NF-κ B p50-p65 heterodimers to p50-p50 and p65-p65 homodimers as well as in the normal development of cardiac myocytes [86]. A previous study also indicated that myotrophin may be involved in the upregulation of myofibrillar protein and the activation of cardiac gene transcription during the growth and hypertrophy of myocardium; thus, the induction of early response of gene expression may be linked to this response [87].

The 132 proteins identified in this study may be involved in some biologic processes that are associated with cell differentiation, proliferation, and adhesion. We have tested some proteins incorporated into the medium; however, none of those proteins can solely induce cell differentiation. The results form a database with a diversity and relative abundance of various proteins found in the HNPE cell-secreted proteins. The database provides not only information on the nature of protein contents in HNPE cells but also potential proteins to be examined in further investigations.

4. Conclusions

In this study, we established the first secretome database for HNPE cells. The experimental results obtained by SDS-PAGE and nano-high performance liquid chromatography electrospray ionization tandem mass spectrometry (nano-HPLC-ESI-MS/MS) system revealed the identification of 132 unique proteins from HNPE cell secretome. Among these 132 proteins identified with higher confidence levels, some proteins have been reported involving in cell differentiation, such as thrombospondin-1, 2, 3 precursor, galectin-3-binding protein, neurogenic locus notch homolog protein 3, follistatin-related protein 1 precursor, sPARC precursor, peroxiredoxin-1, cofilin 1, profilin 1, galectin-1, and myotrophin. However, none of those proteins can induce cell differentiation solely. This list serves as a starting point for buildingup a comprehensive database of the proteome of this cell-line. The database can include diverse repertoires of proteins expressed by HNPE cells. All of this data will enhance our understanding of the molecular mechanisms involved in maintaining the differentiated states of HNPE cells and directing their differentiation and, in turn, will bring us closer to fulfill the vast clinical potentials of the cells.

In conclusion, we have demonstrated that RGC-5 cells upon coculturing with HNPE cell conditioned SF-medium developed a differentiated morphology and continued to express the necessary RGC markers. The differentiated RGC-5 cells would therefore be useful to study apoptotic pathways of retinal ganglion cell death. The findings from this study may have significant impacts on HNPE cell biology and cell engineering.

Acknowledgments

This paper was supported by research Grants Q097004 from the Kaohsiung Medical University Research Foundation, NSC96-2321-B-037-006, NSC-099-2811-E-224-002, and NSC97-2320-B-037-012-MY3 from the National Science Council, Taiwan.

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