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ISSN 0582-9879 ACTA BIOCHIMICA et BIOPHYSICA SINICA 2003, 35(6): 522-528 CN 31-1300/Q

Differential Protein Expression Induced by Transient Transfection of Metallothionein-3 Gene in SH-SY5Y Neuroblastoma Cell Line

ZHOU Bo, YANG Wei, JI Jian-Guo*, RU Bing-Gen*

( Proteome Group, National Laboratory of Protein Engineering, College of Life Sciences, Peking University, Beijing 100871, China )

Abstract Metallothionein-3(MT-3), also known as growth inhibitory factor (GIF), is predominantly expressed in central nervous system (CNS). It belongs to the family of metallothionein(MT) but has several unique properties that are not shared by other family members such as MT-1/MT-2. In the past few years, MT-3 had been postulated to be a multipurpose protein which could play important neuromodulatory and neuroprotective roles in CNS besides the common roles of MTs. However, the primary function of MT-3 and the mechanism underlying its multiple functions were not elucidated so far. In present study, human neuroblastoma cell line SH-SY5Y was employed to study the overall cellular protein changes induced by transient transfection of MT-3 gene, based on comparative proteome analysis. Averagely about 750 spots were visualized by Coomassie staining in one 2D gel, in which 17 proteins were shown to display significant and reproducible changes by semiquantitative analysis with ImageMaster 2D Elite software. Among them, 12 proteins were up-regulated while other 5 proteins were down-regulated. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, 10 proteins were further identified to be zinc finger protein, glutamate transporter, and enhancer protein, etc., which were involved in several important pathways regulating the functions of central nervous system. The results showed that MT-3 might exert its unique functions by regulating the expression of these proteins.

Key words metallothionein-3; cell transfection; 2-DE; MALDI-TOF-MS

The metallothionein(MT) family is a class of low molecular, intracellular, and cysteine-rich proteins with a high affinity for metals[1]. Four major isomers, MT-1 and MT-2 known previously, and MT-3[2] and MT-4[3] found recently, had been identified in mammals. Since it was discovered in 1991, metallothionein-3, also called nerve growth inhibitory factor (GIF)[4], had aroused great interest due to its close correlation with Alzheimer's disease (AD). During the past ten years or so, it had been proven that MT-3 could not only inhibit neuronal cell growth in the presence of AD brain extracts[4－6] but also protect cells from glutamate neurotoxicity[7]. Besides, MT-3 might also participate in the processes of heavy metal detoxification[8], metabolism regulation[9], and protection from oxidative free radicals damage[10] in central nervous system (CNS) like other MTs. However, the primary function of MT-3 and the related mechanisms remain obscure so far[11]. Proteomics[12,13], an emerging technology platform integrating two-dimensional gel electrophoresis (2-DE), mass spectrometry (MS) and bioinformatics, can provide useful information for discovery-based science and will contribute greatly to understanding of gene function in the post-genomic era[14]: 2-DE allows separation of thousands of cellular proteins in one sample with unparalleled resolution; MS provides a fast and reliable way of characterizing proteins of interest, especially when the gene sequence of the source organism is known. Comparative proteome analysis, one important part of proteomic research, can give us new insights into the molecular mechanisms by dynamically inspecting the changes of cellular proteins[15,16].

In this experiment, SH-SY5Y, which was a well-characterized model of human neuronal growth and differentiation[17], was transiently transfected with pEGFP-N3-MT-3, with blank vector pEGFP-N3 transfected in a control group. Their proteome profiles were analyzed and compared, and the proteins exhibiting significant changes induced by MT-3 transfection were identified to provide some new insights to decipher the mechanism of MT-3's diverse functions.

1 Materials and Methods

1.1 Chemicals and materials

DMEM and LipofectAMINETM 2000 transfection reagent were purchased from Gibco (Grand Land, NY, USA). Immobiline DryStrips (pH 3－10 L), IPG buffer (pH 3－10) were purchased from Amersham Pharmacia Biotech (Uppsala, Sweden). DTT, iodoacetamide, urea, agarose, glycerol, bromophenol blue, CHAPS, acrylamide, Bis, Tris, glycine, SDS, ammonium persulfate and TEMED were obtained from Sigma (St. Louis, MO, USA). Acetonitrile was from Fisher (Fair Lawn, NJ, USA). TFA was from Merk (Darmstadt, Germany).

1.2 Cell line and cell culture

SH-SY5Y cell line was obtained from Xuanwu Hospital (Beijing, China). SH-SY5Y cells were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 ku/L penicillin, and 100 mg/L streptomycin, in a water-saturated 5% CO2 atmosphere at 37 °C. The medium was changed everyday and cells were passaged every 2－3 d.

1.3 Gene transfection

MT-3 containing plasmid pEGFP-N3-MT-3 was constructed by Dr. Ying Liu of our laboratory. The target gene MT-3 was inserted between restriction sites BamHI/EcoRI of the vector pEGFP-N3 (Clontech, CA). The preparation of plasmid was performed according to manufacturer's protocol of Qiagen plasmid miniprep kits (Qiagen, USA). The purity and concentration of DNA were determined by UV absorbance at 260 nm and 280 nm. SH-SY5Y cells were transiently transfected with pEGFP-N3 or pEGFP-N3-MT-3 using LipofectAMINETM 2000 rea-gent (Gibco BRL) under the instruction of supplier's protocol for 36－48 h and then harvested. The harvested cells were observed to emit green fluorescence with Olympus BH-2 fluorescent microscope (Olympus, Japan). The power of objective was selected as 20×, and five visual fields were observed to calculate the average value of transfection efficiency by ratioing cells with green fluorescence to total cells observed with fluorescent microscope. Four independent experiments were carried out to test the reproducibility of transient transfection. Proteins of transfected SH-SY5Y cells were separated by 12.5% SDS-PAGE, and the target protein was detected by Western blotting with rabbit anti-MT-3 antisera.

1.4 Sample preparation

The cultured cells were harvested using cell scraper, rinsed two times with ice-cold PBS and pelleted by centrifugation at 1000 r/min for 5 min. The cell pellets were then lysed in a buffer containing 7 mol/L urea, 2 mol/L thiourea, 4% CHAPS, 40 mmol/L Tris base and 65 mmol/L DTT, then frozen and thawed instantly for three times, and further centrifuged at 13 000 r/min at 4 ℃ for 20 min to remove the insoluble materials. At least three volumes of cold acetone were added to the supernatant, then precipitation procedure was processed at －20 ℃ overnight.

1.5 2-DE

2-DE was performed as the method described[18]. The first dimension was carried out on an IPGphor isoelectric focusing system (Amersham Pharmacia Biotech). The samples were dissolved in rehydration solution containing 8 mol/L urea, 2% CHAPS, 0.5% IPG buffer and 18 mmol/L DTT. The protein concentration was determined by the Bradford assay[19]. Typically 700 μg protein in 250 μL rehydration solution was loaded onto each 13 cm IPG dry strip, pH 3－10 L, at both the basic and acidic ends of the strips. The rehydration was conducted for 12 h under low voltage (30 V) at 20 °C, then the separation program was automatically processed as the following parameters: 200 V, 1 h; 500 V, 1 h; 1000 V, 1 h; 5000 V, 1 h; 8000 V, 2 h. When the IEF run was complete, the IPG strips were immediately equilibrated for 2×15 min in equilibration buffer containing 50 mmol/L Tris-HCl, pH 6.8, 30% glycerol, 1% SDS, traces of bromophenol blue. The first equilibration was performed in above-mentioned equilibration buffer with 1% DTT followed by a second equilibration with 2.5% iodoacetamide. The strips were subsequently subjected to a second dimensional electrophoresis on 12.5% SDS polyacrylamide gels using a Hoefer SE600 (Amersham Pharmacia Biotech). SDS-PAGE was performed at constant current (30 mA per gel) and temperature (20 ℃) for about 4 h until the dye front reached the bottom of gels. Then the gels were stained with Coomassie brilliant blue R-250.

1.6 Image acquisition and analysis

The Coomassie blue-stained gels were scanned with Sharp color image scanner JX-330 (Sharp, Japan). Spot detection, quantification and matching were performed using an ImageMaster 2D Elite software (Amersham Pharmacia Biotech). The protein level of each spot was expressed as a percentage of total spot volume in the whole gel (%vol). The expression level of proteins with an increase or decrease of >100% over control was considered as significant difference. Student's t-test was also used to compare data from the different treatment groups.

1.7 In-gel protein digestion[20]

Protein spots of interest were excised from gels, and cut into small pieces (about 1 mm2). These gel pieces were destained with 50 % acetonitrile in 25 mmol/L ammonium bicarbonate in siliconized Eppendorf tubes for three times, and then dehydrated with SpeedVac concentrator (Thermo Savant, USA). The dried gel pieces were rehydrated with 20 μL 25 mmol/L ammonium bicarbonate containing 0.01 g/L trypsin at 4 ℃ for 30 min. Then if necessary, certain amount of 25 mmol/L ammonium bicarbonate buffer could be added to the gel slices for recovering to be original size. The gel slices were subsequently incubated at 37 ℃ for 16－18 h. Then the peptide mixture was extracted as follows: 50 μL 5%TFA was added to the tubes, and then incubated at 40 ℃ for 1 h. The supernatant was transferred to another tube and 50 μL 2.5% TFA containing 50% acetonitrile was added to extract again. The combined solution was freeze-dried and stored at －20 ℃ until use.

1.8 MALDI-TOF-MS analysis

The trypsin-digested samples were mixed with the matrix (α-cyano-4-hydroxycinnamic acid dissolved in 50% acetonitrile, 0.1%TFA) and then analyzed in Voyager-DETM pro MALDI-TOF mass spectrometer system (ABI, USA). Mass spectra were recorded in the positive mode with delayed extraction. Monoisotopic masses of peptides were analyzed by using PeptIdent search engine provided by Expasy proteomics server. By combining the observed M_r and pI on the 2-D gel, 10 proteins were finally identified.

Fig.1 Transient expression of pEGFP-N3-MT-3 in SH-SY5Y

(1－3)×10⁵ cells were seeded on a piece of glass coverslip placed in a 35-mm polystyrene dish and allowed to attach overnight. The cells were transfected with 1.2 μg pEGFP-N3-MT-3 using a liposome-based transfection method. After being transfected for 36 h, the cells were observed with fluorescent microscope (Ex=475 nm; 20× objective).

2 Results

2.1 Transient expression of human MT-3 gene in the cell line SH-SY5Y

The SH-SY5Y cells transfected with either pEGFP-N3 or pEGFP-N3-MT-3 were observed with fluorescent microscope. The enhanced green fluorescent protein (EGFP) would be expressed at the C-terminus of MT-3, thus MT-3 expression could be monitored by fluorescence detection. When illuminated by blue light of 475 nm, cells expressing recombinant protein yielded bright green fluorescence that could be seen in Fig.1. The efficiency of transfection was averagely (32.0±4.5)%. The expression of MT-3 was also confirmed by Western blotting detection. The result of Western blotting showed that there was positive interaction with rabbit anti-MT-3 antisera at the position of 32 kD as expected in the pEGFP-N3-MT-3 transfected cell extract, while cells transfected with pEGFP-N3 produced negative reaction as shown in Fig.2.

Fig.2 Western blotting analysis of the expression of MT-3 recombinant protein

1, protein molecular weight standard; 2, untransfected SH-SY5Y cells; 3, SH-SY5Y cells carrying pEGFP-N3 vector; 4, SH-SY5Y cells carrying pEGFP-N3-MT-3 plasmid.

2.2 Proteome profiles comparison of MT-3 transfected SH-SY5Y with normal one

For statistical quantification of expression difference, three pairs of transfected samples from different batches were prepared and parallel experiments were performed. Samples transfected with pEGFP-N3 were used as a control to eliminate the possibility of expression difference resulted from the EGFP. The typical images of 2-DE were shown in Fig.3. The image analysis software averagely detected 752 ± 46 spots in each gel following Commassie blue staining. Most spots distributed in the region of pI 4.0－7.0 and molecular weight 30－66 kD. It could be observed in the gels that a few proteins underwent significant increase or decrease in intensity and/or area. The changed protein spots distributed all over the gels, but mainly in basic regions.

2.3 Image analsis of 2-DE gels

Coomassie blue R-250 stained 2-DE gel images were acquired with Sharp color image scanner JX-330 and subjected to visual assessment in order to detect changes in protein expression between pairs of transfection samples. By gel matching and statistical analysis, 17 protein spots were found to be changed significantly (P<0.05) in proteome of SH-SY5Y cells transfected by pEGFP-N3-MT-3 compared with those of control: 12 proteins underwent significant increasing in volumes, whereas other 5 were significantly down-regulated. These changed protein spots were marked with arrows as shown in Fig.3, and the statistic analysis were summarized in Table 1.

Fig.3 Representative proteome profile of SY5Y cells transiently transfected with MT-3

(A) Transfected with pEGFP-N3-MT-3. (B) Transfected with pEGFP-N3 as a control. Protein spots with significant difference were indicated with arrows and numbered. U1－U12, up-regulated spots; D1－D5, down-regulated spots.

Table 1 Summary of significantly changed proteins

Spot	Observed pI	Observed Mr (kD)	N3 (% vol)		MT-3 (% vol)
				s		s
U1	7.6	10.2	n.d.	-	0.328	0.043
U2	9.2	20.6	0.031	0.005	0.121	0.023
U3	9.9	32.1	n.d.	-	0.180	0.035
U4	8.6	28.4	0.091	0.012	0.198	0.031
U5	8.4	35.5	n.d.	-	0.068	0.011
U6	7.7	31.0	0.076	0.012	0.186	0.013
U7	8.3	38.5	0.110	0.009	0.242	0.026
U8	9.9	38.1	0.185	0.031	0.347	0.051
U9	8.6	42.2	0.073	0.010	0.149	0.012
U10	5.6	29.3	0.084	0.013	0.204	0.022
U11	4.8	16.8	0.061	0.011	0.156	0.018
U12	5.7	66.0	0.140	0.020	0.300	0.044
D1	9.9	20.5	0.230	0.028	n.d.	-
D2	3.1	18.6	0.440	0.039	0.219	0.030
D3	3.1	16.4	0.350	0.033	0.167	0.024
D4	6.5	32.4	0.336	0.028	0.162	0.017
D5	6.4	37.1	0.218	0.042	n.d.	-

SD, standard deviation; n.d., not detected

2.4 MALDI-TOF-MS analysis and protein identification

The proteins with significant difference were excised from the 2-D gels, subjected to tryptic digestion. Then peptide mixtures were extracted and analyzed by MALDI-TOF-MS. A representative PMF spectrum was shown as Fig.4. The maps of peptide mass fingerprinting (PMF) were used to search protein database. Ten proteins, mostly in basic region of 2D-gels, were successfully identified by MALDI-TOF mass spectrometry combined with database mining. Among these identified proteins, eight proteins were up-regulated and two were down-regulated upon MT-3 transfection. The results were summarized in Table 2. Most of these proteins had not yet been positioned on 2-DE maps in SWISS 2D-PAGE.

Fig.4 MALDI-TOF mass spectrum of the spot U7

The protein spot U7 in Fig. 3(A) was in-gel digested with trypsin. After desalting, the peptide mixture was analyzed by MALDI-TOF-MS. Mass spectra was recorded in the positive mode with delayed extraction.

Table 2 Proteins regulated by transfection with MT-3 identified by MALDI-TOF-MS

Spot No.	Accession No.	Match	Coverage (%)	Mr/ pI	Description
U2	Q9BW45	4	41.8	17383/9.93	Similar to solute carrier family 1 (Glutamate transporter), member 7
U3	Q8N188	7	40.2	32088/9.42	Similar to zinc finger protein 1
U4	P49675	9	26.0	28104/8.34	Steroidogenic acute regulatory protein, mitochondria [precursor]
U5	Q99982	7	18.9	36915/8.70	XIAP associated factor-1
U6	Q8NBYO	11	44.4	undefined	Hypothetical protein FLJ90676
U7	Q96A47	8	34.8	39768/8.64	Insulin gene enhencer protein ISL-2
U8	Q14567	7	26.1	41289/9.42	Enhancer protein (fragment)
U9	Q9UPC5	5	25.5	43860/9.89	Probable G protein-coupled receptor GPR34
D1	O00441	6	45.3	20363/9.09	ISG20 protein
D3	P00742	14	45.3	15725/4.59	Factor Xa

3 Discussion

The comparative proteome analysis showed that there was some expression difference caused by the transfection of MT-3 gene. The differential proteins might have direct or indirect functional correlation with MT-3. It is interesting to further examine the properties of these identified proteins.

3.1 Protein similar to solute carrier family 1 (glutamate transporter), member 7

Glutamate transporter was a family of high affinity sodium-dependent transporters for neurotransmitter clearance mediating the amount of glutamate and other excitatory amino acids in the nervous system[21,22]. The up-regulation of glutamate transporter accompanying with overexpression of MT-3 in SH-SY5Y implied that MT-3's neuroprotective effect from glutamate neurotoxicity might work through regulating the glutamate transporter's expression for neurotransmitter clearance.

3.2 Protein similar to zinc finger protein 1

Zinc finger protein was a family of proteins containing a functional domain that required coordination of one or more zinc ions to stabilize its structure that may participate in nucleic acid binding or protein-protein interaction[23,24]. As a protein noted for its high zinc contents, MT-3 might be involved in mechanisms of regulating the concentration and distribution of zinc and as a source of zinc for incorporation into proteins[25]. So, the overexpression of MT-3 might up-regulate the expression of zinc finger protein and in turn regulate the expression of many other proteins.

3.3 XIAP associated factor-1

XIAP was a protein that functions as inhibitor of apoptosis counteracting the cellular apoptosis process[26]. The up-regulation of XIAP associated factor-1 with MT-3 transfection perhaps meant that MT-3 might exert its neuroprotective effect by up-regulating XIAP associated factor-1, which might in turn protect cells from apoptosis in central nervous system (CNS).

3.4 Insulin gene enhancer protein ISL-2

ISL-2 was a kind of LIM-homeodomain protein. LIM box was a specialized conserved cysteine-rich domain, which binded two zinc ions and was involved in protein-protein interaction[27,28]. LIM-homeodomain protein, which works as a transcription factor, played an important role in mediating tissue specific gene expression in nervous system[29]. Therefore, the up-regulation of ISL-2 upon MT-3 transfection implies that MT-3 might participate in affecting the neuronal cells' differentiation and maintenance of differentiated cells through LIM-homeodomain proteins.

3.5 Enhancer protein in hsp70

HSP70 was a member of heat shock proteins (HSP70s) that worked mainly as a molecular chaperone in cells as well as played an important role in protecting the cells from cytotoxic stresses and improving cell survival[30]. In present study, enhancer protein in hsp70 was identified up-regulated with the transfection of MT-3 in cells. It might indicate that MT-3 might exert its neuroprotective role partially by regulating the expression of enhancer protein in hsp70, and then affecting the expression and biological activity of HSP70.

Although the relationships of other proteins to MT-3 were not clear at present, they might still play an important role on cell, involved in mediating several important biological processes such as regulation of gene expression, signal transduction and inhibition of apoptosis. Therefore, the multiple functions of MT-3 could be reflected by the diversity of these changed proteins. Among them, several proteins were related to the neuroprotective role of MT-3 in glutamate neurotoxicity and ROS, and the identification of glutamate transporter member 7 was very promising for the elucidation of another possible mechanism of MT-3's participation in glutamate regulation. Furthermore, several identified proteins either contained zinc finger structure or utilized zinc for biological activity. This reminded us that MT-3 might have close relationship with zinc for exerting some of most distinct effects in CNS. Although a few of proteins of interest have been netted that are promising for deciphering the mechanism of MT-3's multifunctions by employing comparative proteome analysis, further studies with other biochemical, genetic and cell methods were still necessary for elucidating the whole story of MT-3.

Acknowledgements The authors would like to thank Professor Zhen-Quan Guo for his generous help in cell culture and fluorescent microscopic observation.

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Received: December 30, 2002 Accepted: March 18, 2003

*Corresponding author：

RU Bing-Gen： Tel, 86-10-62751842; Fax, 86-10-62751842; e-mail, [email protected];

JI Jian-Guo： Tel, 86-10-62753115; e-mail, [email protected]