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Http://www.abbs.info e-mail:[email protected] ISSN 0582-9879
ACTA BIOCHIMICA et BIOPHYSICA SINICA 2001, 33(4):
373-378
CN 31-1300/Q |
Preliminary
Function Study of NAG7 Using Two-dimensional Electrophoresis and Mass
Spectrometry
( Cancer Research Institute, Xiang Ya
Medical College, Central South University, Changsha 410078, China; 1College
of Life Science, Hunan Normal University, Changsha 410081, China )
1.1 Chemicals
and materials
Immobilized
pH gradient (IPG) strips were purchased from Amersham Pharmacia Biotechnology
(Uppsala, Sweden). Zinc-imidazole staining kit, pI calibration markers,
Acrylamide and other reagents for the polyacrylamide gel preparation were from
Bio-Rad (Richmond, CA, USA), as well as PDQuest software. Urea (ultrapure),
CHAPS and Noidet P-40 (NP-40) were from Sigma (St. Louis, MO, USA). Agarose was
from Gibco BRL (Grand Island, NY, USA).
1.2 Cell
lines and cell culture
HNE1
cell line was established by our lab[10]. HNE1 cells and cells
transfected with NAG7 were cultured in RPMI 1640 media supplemented with
10% fetal bovine serum (FBS), 10 u/ml of penicillin and 100 mg/L of
streptomycin in a water-saturated, 5% CO2 atmosphere at 37 ℃
in 50 ml flasks. Cells were collected during the exponential growth phase.
1.2.1 Construction of vectors To observe the function of NAG7 gene on
the NPC cells, NAG7 gene was introduced into NAG7 down-expressed
HNE1 cells. A 466 bp cDNA fragment containing open reading frame from cDNA
biopsies was obtained by PCR, and cloned into pGEM-T easy vector(Life
Technologies). The vector was digested by EcoRI, and the resulting
fragments were separated by agarose gel electrophoresis. A pcDNA3.1(+) vector
(Invitrogen, Carlsbad, Calif) was digested by EcoRI in the multiple
cloning site, and the resulting fragment was cloned into it by T4 DNA ligase.
The directional cloning of NAG7 cDNA insert fragment was confirmed by
restriction mapping.
1.2.2 Gene transfection HNE1 cells were transfected with the pcDNA3.1(+)
vector containing NAG7 cDNA fragment using Lipofectin (Life
Technologies) according to the supplier's instructions. Geneticin (G418
sulfate) (Life Technologies) at a concentration of 0.5 g/L was used to select
for cells that neomycin-resistant, indicating that the vector was present in
the cells. Several (n＝16)
individual cell clones were isolated from the population of cells carrying the
pcDNA3.1(+) vector. All of the transfected cells and cell clones were
maintained in RPMI 1640 with 10% FBS and G418 (0.25 g/L).
1.3
PCR
Genomic
DNA was isolated from HNE1 cells as well as cells transfected with pcDNA3.1(+)
vector according to the protocol of genomic DNA isolation kit (Promega,
Madison, Wis). PCR reaction mixtures(50 ml) contained gDNA, 0.1 mmol/L sense
and anitsense oligonucleotide primers, 200 mmol/L dNTP, 1.5 mmol/L MgCl2,
50 mmol/L KCl, 10 mmol/L Tris buffer (pH 8.3), two units Taq polymerase
were added and this mixture was covered with equal mineral oil. Following
preincubation at 95 ℃
for 5 min, this mixture was cycled 30 times at 94 ℃
for 1 min, 57 ℃
for 80 s, and 72 ℃
for 1 min; this was followed by 10
min at 72 ℃.
The PCR-amplifed DNA fragments were electrophoretically fractionated on agarose
gels.
1.4
2-D Electrophoresis
1.4.1 Protein
extraction Cells
from the HNE1 and pcDNA3.1(+)/NAG7/HNE1 were harvested and centrifuged
at 1 500 r/min, washed in ice-cold PBS, resuspended in PBS, and then counted.
The number of cells was adjusted to 3×108
cells/ml. Soluble proteins were extracted with buffer containing 50 mmol/L
Tris-HCl, pH 7.4, 10 mmol/L EDTA, 65 mmol/L DTT, 1.5 mmol/L
phenylmethylsulfonyl fluoride (PMSF), and one tablet of anti-proteases for 10
ml of buffer as previously described. After centrifugation, the supernatant
containing soluble proteins was supplemented with 7 mol/L urea, 2 mol/L
thiourea and 4% CHAPS. Aliquots were stored at -20 ℃
until use.
1.4.2 2-D
electrophoresis with immobilized pH gradient strips 2-D electrophoresis
was performed as described[11], using precast immobilized pH gradient
(IPG) strips (pH 3-10),
linear(Pharmacia, Uppsala, Sweden) in the first dimension (IEF). Samples were
applied via rehydration of IPG strips in sample solution overnight. Before
application, samples were diluted to a total volume of 350 ml with 8 mol/L urea,
2% CHAPS, 2% IPG buffer (pH 3-10,
linear), 0.3% DTT and a trace of bromophenol blue. Typically, 500 mg protein
were loaded on each IPG strip and focusing was carried out for 45 500 Vh. After
IEF separation, the strips were immediately equilibrated 2×15
min with 50 mmol/L Tris-HCl, pH 6.8, 6 mol/L urea, 30% glycerol and 2% SDS. In
the first equilibration solution, DTT (2%) was included, and 2.5% iodoacetamide
was added in the second equilibration step to alkylate thiols. SDS-PAGE was
performed using 0.75 mm thick, 12.5% SDS-polyacrylamide gels with piperizine
diacrylamide as cross-linker. The strips were held in place with 0.5% agarose
dissolved in SDS-Tris running buffer and electrophoresis was carried out at
constant current (40 mA/gel) and temperature (20 ℃).
After electrophoresis, gels were stained with silver nitrate.
1.4.3 Image
analysis and spot identification Image
analysis was performed using the PDQuest system according to the protocol
provided by manufacturer. To account for experimental variations, three gels
were prepared for each cell line. The gel spot pattern of each gel was
summarized in a standard after spot matching. Thus, we obtained one standard
gel for each cell line. These standards were then matched to yield information
about new spots related to the gene transfection (up- or down-regulation of
spots).
1.4.4 In-gel
protein digestion The
stained protein spots were excised from preparative gels using biospy punches.
Proteins were in-gel digested as previously described[11]. Briefly,
the spots were washed several times with 50% acetonitrile, which was then
removed. Gel pieces were dried in a vacuum centrifuge. The cysteine reduction
and alkylation steps consisted of incubation first in 10 mmol/L DTT, 100 mmol/L
NH4HCO3 for 45 min in the dark at room temperature. The
gel pieces were then dried again and rehydrated in 30 ml of 50 mmol/L NH4HCO3
containing trypsin for 45 min in ice. The concentration of trypsin used was 0.1
mg/L. The excess liquid was removed and the pieces of gel were immersed
overnight in 50 mmol/L NH4HCO3 at 37 ℃.
The resulting peptide mixture was extracted from the gel by centrifugation.
Desalting of peptides was performed using Ziptips following the manufacture's
instructions.
1.5 MALDI-TOF-MS
analysis
Peptide
mass maps were generated by matrix-assisted laser desorption/ionizaton
time-of-flight (MALDI-TOF) mass spectrometry (ProFLEXTM III, Bruker Co., USA)
with a reflection and delayed extraction. DBA was used as matrix. A volume of
0.5 ml was mixed with the same volume of the sample. The TOF was measured using
the following parameters: 21 kV
accelerating voltage, 74% grid voltage. 0% guide wire voltage, 200 ns delay,
and low mass gate of 500. External calibration was preformed using
desArg1-bradykinin (M+H+, 904.46) and ACTH (clip 18-39 M+H+,
2465.20) in the same series as the samples to be measured. Internal calibration
was also performed using autodigestion peaks of trypsin (M+H+,
906.50 and 2163.06)[12,13].
2.1
PCR analysis
PCR
was used to confirm whether HNE1 cells were transfected with pcDNA3.1(+) vector
containing NAG7 cDNA fragment.One set of primers
[5'-AATAATGACGTATGTTCCCATAG-3' and 5'-GAGGAAATGTACCACCCTACA-3'] was designed to
amplify an approximately 800 bp gDNA fragment containing partial of vector
fragment and NAG7 cDNA fragment. The result showed that only three
clones have the 800 bp gDNA fragment, which suggested these three clones
containing the pcDNA3.1(+) vector and NAG7 cDNA fragment, so we named
these three clones as pcDNA3.1(+)/NAG7/HNE1(Fig.1).
Fig.1 Agarose gel electrophoresis of
PCR products
(A) M, PCR marker; C, pcDNA3.1(+)/NAG7;
1-8,
HNE1 cells transfected with pcDNA3.1(+)/NAG7, respectively. (B) M, PCR
marker; 9-16,
HNE1 cells transfected with pcDNA3.1(+)/NAG7, respectively.
HNE1
and pcDNA3.1(+)/NAG7/HEN1 cell extracts were separated by 2-D
electrophoresis and the protein spots were visualized following silver
staining. Three pairs of gels from different batches of control and transfected
cells were analyzed for the purpose of quantitative spot comparisons with the
PDQuest analysis software. Figure 2 shows a representative example of the cell
proteins separated on a 2-D gel, where 0.5 mg of total protein were applied.
Approximately 500 protein spots were detected on the silver-stained gel by the
software(516±19
spots in HNE1 2-D map and 554±37
spots in NAG7/HNE1 map on average, respectively). The most spots
distributed on the map from pI 4.0 to 7.0 and molecular weight from 24 kD to 70
kD. After matching analysis, twelve protein spots, their abundance not related
to transfection, were up-regulated in pcDNA3.1(+)/NAG7/HNE1 cells. These
spots were marked with arrows at the corresponding site in figure 2.
Fig.2 2-D maps of HNE1 cell proteins
(left) and NAG7/HNE1 cell proteins (right)
The proteins from cells were extracted
and separated on a pH 3-10
nonlinear IPG strip, followed by a 12.5% SDS-polyacrylamide gel. These gels
were stained with silver stain kit. The spots marked with arrow were analyzed
by MALDI-MS.
These
twelve proteins were identified by MALDI-TOF-MS on the basis of peptide mass
matching[14], following in-gel digestion with trypsin. The peptide
masses were matched with the theoretical peptide masses of all proteins from
the human species of the SWISS-PROT database. Figure 3 showed the spectrum of
the trypsin digest of protein spot 10. Nine of these proteins were successfully
identified by MALDI-TOF-MS. The other three digests produced no spectrum. The
table 1 lists the identified proteins.
3 Discussion
Using
2-D electrophoresis and mass spectrometry, we could show that these proteins
may play important roles in the transfected cells caused by NAG7
involved in the cell cycling, transcription regulation, and apoptosis in
vitro. It is thus of interest to examine the properties of these proteins.
3.1 Growth
arrest specific protein
Growth
arrest specific protein is an integral membrane protein. As a specific growth
arrest protein in growth suppression, it can block cell entry to S phase of
normal and transformed cells. There are two restriction points or checkpoint in
the cycle at which a decision may be taken on whether to proceed. The first one
is called START, which permits the cell entry to S phase from G1
phase. Growth arrest specific protein may have the effect on the START and
block cells entry to S phase[15]. In tumor, there are more cells in
cycle, so this protein may play an important role in tumor growth suppression. NAG7
may exert the function by up-regulating the expression of this protein.
3.2 DNA
binding protein
There
are many types of DNA binding domains that regulate transcription by using
particular motifs to bind DNA, and lead to differential expression of some
proteins known to be involved in the proliferation of cells[16].
This DNA binding protein containing a zinc finger motif suggests it may has the
effect on the regulation of gene expression by recognizing and binding to
specific DNA sequence[17].
3.3 c-myc
promoter-binding protein
The
cellular oncogene c-myc is expressed in many different tissues and
cultured cell lines. The human c-myc contains four regulatory sequences
similar to ISRE that was found in many interferon-dependent genes. These
ISRE-like sequences were assumed to be involved in the regulation of
transcription of human c-myc gene[18]. C-myc promoter-binding
protein may play a role in transcription regulation by recognizing an ISRE-like
motif and binding to ISRE-like sequence of the p2 promoter region of c-myc.
The NAG7, a tumor suppression candidate gene, may cause this protein in
high abundance expression and bind to the ISRE-like sequences in c-myc,
and play a role in the inhibition of the gene transcription initiating intron I[19].
3.4 Caspase
6
Cytoplasmic
caspase 6 (ICE 6), a new member of the apototic Ced/Ice cysteine protease gene
family. This protein is composed of heterodimer of a 18 kD (p18) and a 11 kD
(p11) subunit. The function of the caspase 6 is involved in the activation
cascade of caspases responsible for apoptosis execution by cleaving the poly
(ADP-ribose) polymerase in vitro, as well as lamins. Its over-expression
promotes programmed cell death. Apoptotic cell death is essential for normal
development and maintenance of normal tissue size homeostasis in multicellular
organism. There is growing evidence that dysregulation of apoptosis may lead to
many kinds of cancers[20].
Even
if the relationship of the other proteins to NAG7 were not clear at
present, they may still play an important role on cell in vitro. Our
results suggested that the changes of the protein profile of cells might be
correlated with NAG7. In summary, MS analysis is a very effective and
sufficient method for large-scale protein identification. It is still a
bottleneck that how to combine the bioinformatics with the need for parallel
computers to deal with such huge amounts of data. Once the breakthrough is made
in proteomics, the understanding of mechanism of NPC pathogenesis is ultimately
to progress.
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Received: January 16, 2001 Accepted: March 5, 2001
This work was supported by the state 863
High Technology R & D Project of China(No.102-10-01-05, No.Z10-01-01-03),
the Special Funds for Major State Basic Research of China (No.G1998051008), and
the National Natural Science Foundation of China (No.39900163)
*Corresponding author
LI Gui-Yuan: Tel, 86-731-4805446; Fax, 86-731-4805383; e-mail,
[email protected];
LIANG Song-Ping: Tel, 86-731-8872556;
Fax, 86-731-8861304; e-mail, [email protected]