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

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

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]