Revised Manuscript No: FSIM-D-10 – 00398R1 1
Molecular cloning and functional analysis of an orange-spotted grouper (Epinephelus 2
coioides) secreted protein acidic and rich in cysteine (SPARC) and characterization of 3
its expression response to nodavirus 4
Young-Mao Chena, b, c, 1, Cham-En Kuod, 1, Yi-Ling Huanga , Pei-Shiuan Shiea, Jhong-Jian 5
Liaoa, Yuan-Chih Yanga and Tzong-Yueh Chena, b, c, * 6
7
aLaboratory of Molecular Genetics, Institute of Biotechnology, College of Bioscience and 8
Biotechnology; bResearch Center of Ocean Environment and Technology, and cAgriculture 9
Biotechnology Research Center,National Cheng Kung University, Tainan 70101, Taiwan 10
dDepartment of Nursing, Tzu Hui Institute of Technology, Pingtung 926, Taiwan 11
12
Running title: Nodavirus decreases SPARC production 13
* Correspondence to: Dr. Tzong-Yueh Chen, Laboratory of Molecular Genetics, Institute of 14
Biotechnology, College of Bioscience and Biotechnology, National Cheng Kung University, 15
Tainan 70101, Taiwan; Phone: 886-6-2757575 ext 65622 ext 610; Fax: 886-6-2766505; E- 16
mail: ibcty@mail.ncku.edu.tw 17
18
1 These authors contributed equally to this work.
19
Source:Fish & Shellfish Immunology, Vol. 31, No. 2, pp. 232-242 Year of Publication:2011
ISSN:1050-4648 Publisher:Elsevier
DOI:10.1016/j.fsi.2011.05.008
© 2011 Elsevier Ltd. All rights reserved
Abstract 21
Mammalian secreted protein acidic and rich in cysteine (SPARC) is the primary regulator of 22
cell shape and cell adhesion to fibronectin. We, for the first time, report the complete 23
sequencing of SPARC cDNA from orange-spotted grouper. Despite the difference in the 24
lengths of the SPARC transcripts, all of the SPARC molecules encoded a signal peptide, 25
follistain-like copper binding sequence (KGHK) domain, and extracellular domain. The 26
grouper SPARC gene was differentially expressed in vivo and contributed differently to high- 27
level expression of SPARC in muscle. Immunohistochemical staining demonstrated a 28
decreased level of SPARC in nodavirus-infected grouper compared with healthy grouper.
29
Comparative real-time polymerase chain reaction analyses of eye tissues of viral nervous 30
necrosis grouper and healthy grouper were performed. Recombinant SPARC produced 31
changes in grouper cell shape 24 h after treatment. The results provide new insight into the 32
pathogenesis of nodavirus, and demonstrate an experimental rationale for SPARC 33
characterization in nodavirus-infected grouper.
34 35
Keywords: Nodavirus, secreted protein acidic and rich in cysteine (SPARC) 36
37
1. Introduction 38
SPARC (Secreted Protein Acidic and Rich in Cysteine), also termed osteonectin and BM-40, 39
is a secreted, multifunctional, matricelluar glycoprotein that participates in physiological 40
functions especially cell adhesion [1] and proliferation [2], and which regulates a range of 41
biological processes such as development [3], tissue repair [4] and remodeling [5]. SPARC 42
deficient fibroblasts and endothelial cells arrest in the G1 phase of the cell cycle [6].
43
Pulmonary tissue fibroblast cells can display reduced cell adhesion to integrin-ligand coated 44
surfaces as the result of disruption of focal adhesion complexes, giving rise to inhibition of 45
cell spreading [7]. In the case of several growth factors and matrix metalloproteinases, 46
SPARC interacts with several extracellular matrix components to associate with, and regulate, 47
their expression and activity [8]. These activities of SPARC cause antiangiogenesis, likely 48
through blockade of a specific set of vascular endothelial growth factor (VEGF) proteins that 49
induce endothelial cell proliferation, or inhibit tumor stromal cell growth through blocking of 50
platelet-derived growth factor (PDGF) activity [9–11].
51
SPARC of teleost species consists of three distinct domains: an acidic domain in the N- 52
terminus of the polypeptide; a follistatin-like domain, which is a cysteine-rich region that 53
contains a copper binding lysine-glycine-histidine-lysine [(K)GHK] sequence, and a 54
extracellular Ca2+ -binding (EC) domain that contains two EF-hand motifs located in the C- 55
terminus of SPARC [12,13]. The SPARC gene has been cloned in representatives of several 56
teleost fish including zebrafish [14], medaka [15], and gilthead seabream [16]. Nevertheless, 57
at the present time, betanodavirus is neuropathogenic and inflicts conspicuous damage that is 58
characterized by vacuolation and degeneration of neurons throughout the central nervous 59
system [17]. Piscine nodavirus, a member of the Betanodavirdae family, is the causative 60
agent of viral nervous necrosis or fish encephalitis that produces high mortalities in hatchery- 61
reared larvae and juveniles of marine fishes in Taiwan, Japan, Australia, and Europe [18].
62
However, little is known on the role of SPARC in nodavirus infection. In previous microarray 63
studies, piscine SPARC was shown to be downregulated in infectious salmon anemia virus 64
(ISAV)-infected Atlantic salmon [19] and upregulated with integrin receptor family member 65
CD9, a well-known cell motility and migration marker [19, 20]. Just as a role for SPARC in 66
modulating cell migration, the SPARC function not only induces loss of focal adhesion but 67
also regulates the expression of metalloproteinases (MMPs) [21] and plasminogen-activator 68
factor (PAF) [22]. The observations rely either on SPARC expressed by migrating cells or on 69
structural SPARC, which is composed of part of basal membranes and extracellular matrix 70
(ECM) [23]. In SPARC-deficient mice, the mechanism behind this influence on increasing 71
neutrophil and leukocyte infiltration presents in bleomycin-induced peritonitis [24] and TPA- 72
induced skin inflammation [25]. In tissue remodeling, although the role of SPARC has not 73
been linked directly to cell migration, through its binding to collagen type IV in basal 74
membrane, and to collagen type I in the dermis [26]. Similarly, the lack of SPARC in the 75
environment accelerates the onset of T-cell priming by hastening Langerhans cells 76
(LCs)/dendritic-cells (DCs) migration and in turn, SPARC also contributes to antigen- 77
specific immune responses by conditioning DC migration [27]. On the other hand, medaka 78
SPARC represents a marker of early sclerotome development, and has been used to analyze 79
early skeletal development and formation of extracellular bone matrix in this vertebrate 80
model [15]. In contrast, zebrafish SPARC is required for otolith (ear stones) growth by 81
interacting with otolin-1 to influence the assembly of the otolith framework and 82
mineralization [28]. Changes in SPARC expression levels have been linked to the extent of 83
the size and number of mice adipocytes [29]. The expression of sea bream SPARC is 84
downregulated by the hypercalcemic hormone [16]. On the other hand, the overexpression of 85
SPARC in response to 5-fluorouracil (FU)-based chemotherapy decreases hepatocellular 86
carcinoma (HCC) cell viability [30]. Thus, altered expression of SPARC in different cancer 87
cells seems to be associated with good prognosis [31]. In nodavirus-infected grouper, the 88
activation of the host immune response and direct invasion of cells are believed to contribute 89
to this pathogenesis [32, 33], which includes induction of inflammatory cells and the host 90
immune response. For example, ubiquitin conjugating enzyme 7 interacting protein, which 91
functions in apoptosis, and interferon induced with helicase C domain protein 1, which 92
contributes to apoptosis and mediates type I interferon production, are differentially 93
expressed in infected and control cells [34]. Piscine SPARC is a rarely studied gene; the 94
significant function of SPARC in nodavirus-infected grouper remains unclear. Furthermore, 95
no studies have reported the relationship between changes in SPARC expression levels and 96
nodavirus-infected grouper.
97
The purpose of the present study was 1) clone and molecularly characterize SPARC 98
cDNA from the orange-spotted grouper Epinephelus coioides, 2) to compare its sequences 99
and conduct phylogenetic analyses with other SPARC, 3) to examine the transcription and 100
translation of SPARC from different tissues, 4) to evaluate the changes of SPARC expression 101
in uninfected and nodavirus infected grouper, and 5) to evaluate the effect on the regulation 102
of cell shape after recombinant SPARC treatment of the GF-1 grouper cells.
103 104 105
2. Materials and methods 106
2.1. Cell culture and reagents 107
The grouper cell line GF-1 [35] was grown at 28 °C in Leibovitz's L-15 medium (GibcoBRL, 108
Gaithersburg, MD, USA) supplemented with 5% fetal bovine serum (FBS). GF-1 cells, which 109
are susceptible to nodavirus infection and nodavirus replication, were obtained from the 110
Bioresources Collection and Research Center in Taiwan BCRC960094. Transient transfection 111
was performed by introducing to 2 g of plasmids encoding grouper SPARC into cells by 112
using Lipofectamine (Invitrogen, Carlsbad, CA, USA). After transfection, cells were grown 113
for 24 to 30 h. Intracellular localization of SPARC proteins was examined by using an 114
Olympus IX70 microscope. An alkaline phosphatase-conjugated substrate Western blotting 115
detection system kit was purchased from Bio-Rad (Hercules, CA, USA). Alkaline 116
phosphatase-conjugated anti-mouse, anti-rabbit, and or anti-goat IgG antibodies (Santa Cruz 117
Biotechnology, Santa Cruz, CA, USA) were diluted 1:5000 prior to use.
118
2.2. RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR) 119
Total RNA was isolated from grouper at 40 to 45 days post-hatching using a previously 120
described single-step acid guanidinium thiocyanate-phenol-chloroform extraction method 121
[36]. Extracted cellular total RNA (5 g) as template was incubated at 42 oC for 60 min in 20 122
L of 1X reaction buffer containing 2 U Moloney murine leukemia virus (M-MLV) reverse 123
transcriptase (Promega, Madison, WI, USA), 0.25 mM dNTP and 4 M oligo(dT)15 primer, 124
and lastly 0.4 U RNase (Boehringer Mannheim Biochemicals, Mannheim, Germany) was 125
added. Putative SPARC sequences were examined for homogeneity by comparison with 126
published SPARC sequences using Blast (http://www.ncbi.nlm.nih.gov/BLAST). In the first 127
experiment, grouper that were naturally infected with nodavirus were obtained at 40 to 45 128
days post-hatching from hatchery farms. Eyes were excised from healthy and nodavirus 129
naturally-infected grouper, and total RNA was extracted for study of SPARC expression.
130
First-strand cDNA was synthesized using total RNA (5 g) and cDNA (250 ng) as a template 131
for PCR. The grouper SPARC nucleotide sequences of forward and reverse primers were:
132
SPARC-RT-S and SPARC-RT-A (Table 1). The sequences were designed to specifically 133
amplify a 911-bp PCR fragment. Grouper -actin gene expression was analyzed as an 134
internal marker using the following primers: -actin-RT-S436 and -actin-RT-A1170 (Table 135
1). PCR was conducted under the following conditions: 94 oC for 1 min, 60 oC for 1 min, and 136
72 oC for 1 min, with 25 cycles; the last cycle was followed by extension for 5 min at 72 oC.
137
The total amount of cDNA was calibrated based on the amplification of -actin cDNA from 138
the same template, and PCR products were analyzed by agarose gel electrophoresis. In the 139
second experiment, real-time RT-PCR was used to quantify the expression of mRNA for 140
SPARC with -actin as control. First-strand cDNA was synthesized using 2 g total RNA and 141
the SuperScript First Strand cDNA synthesis kit (Invitrogen). The amplification was 142
performed using a quantitative PCR (qPCR) core kit for SYBR Green (Qiagen, Valencia, CA, 143
USA) and Step-One™ Real Time PCR system (Applied Biosystems, Foster City, CA, USA).
144
Typical profile times used were an initial step of 95 °C for 15 min, followed by a second step 145
at 94 °C for 15 sec, 60 °C for 30 sec and 72 °C for 30 sec for 40 cycles with melting curve 146
analysis. After the PCR program, fluorescent real-time PCR data from three replicate samples 147
were analyzed.
148
2.3. Suppression subtractive hybridization 149
cDNA subtraction was performed to generate a subtracted cDNA library between 40 and 45 150
days post-hatching for nodavirus naturally-infected grouper (tester) and healthy grouper 151
(driver) using PCR-Select cDNA subtraction Kit (Clontech, Mountain View, CA, USA) 152
according to the manufacturer’s protocol. Briefly, poly(A+) RNA was extracted using the 153
FastTrack mRNA isolation kit (Clontech). cDNA was synthesized using the SMART
154
PCR cDNA Synthesis Kit (Clontech) according to the manufacturer’s instruction. Tester and 155
driver cDNAs were purified by ethanol precipitation, and then digested with Rsa1 at 37 oC 156
overnight to obtain shorter blunt-ended molecules. Two different adaptors, adaptor 1 and 157
adaptor 2R, were ligated to the 5-end of each strand of tester cDNA, both of which were 158
separately hybridized at 68 oC for 8 h with an excess of driver cDNA after denaturation at 98 159
oC for 90 s. After the first hybridization, the two samples were mixed together without 160
denaturation and hybridized again with freshly heat-denatured driver cDNAs for 18 h at 68 161
oC. The resulting mixture was diluted 1:50, and then amplified by two rounds of PCR to 162
enrich desired cDNAs containing both adaptors by exponential amplification of these 163
products [37]. Nested PCR amplicons were subcloned into a pGEM-T easy Cloning Kit 164
(Promega). Finally, the efficiency was evaluated by PCR with -actin forward and reverse 165
primers performed on tester (unsubtracted) and subtracted cDNAs for 25 cycles.
166
2.4. Rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR) 167
RACE-PCR was conducted as previously described [38]. Based on the verified sequence of 168
the 1344 bps fragment of SPARC cDNA from the subtracted plasmid library, SPARC- 169
5RACE and SPARC-3RACE primers (Table 1) were applied in PCR amplification and 170
cloning of the cDNA 5’ end and 3’ end, respectively. Two adapter primers for both ends were 171
provided in the Marathon cDNA Amplication Kit (Clontech). The RACE-PCR thermal cycle 172
profile was as follows: 94 oC for 1 min; 94 oC for 30 sec, 72 oC for 4 min, 72 oC for 10 min 173
with 30 cycles; and extension at 72 oC for 10 min. The amplified fragment was verified with 174
subcloning into pCR II vectors for sequencing. To minimize the polymerization errors during 175
PCR, a proofreading polymerase was employed in PCR reactions.
176
2.5. Phylogenetic analysis 177
BLAST searches for amino acid sequence similarities were conducted using programs at the 178
National Center for Biotechnology Information (http://www.ncbi.nlm.gov/BLAST/).
179
Sequence alignments were carried out and the sequence identities were calculated by the 180
MegAlign v7.2.1 program from the LASERGENE software suite (DNASTAR, Madison, WI, 181
USA). To elucidate the evolutionary history of SPARC, novel SPARC sequences were 182
identified in zebrafish and mammalian species, and used to explore the phylogenetic 183
relationships of the SPARC gene family. A neighbor-joining phylogenetic tree was produced 184
by the MEGA3.1 program [39] on the basis of a ClustalW alignment of the nucleotide 185
sequences of the open-reading frames along with all known complete sequences of SPARC 186
cDNA with the complete deletion of gaps for 1000 bootstrap replications. Numbers indicated 187
bootstrap confidence values through 1000 replications; only bootstrap values over 70% are 188
exhibited.
189
2.6. Real Time RT-PCR 190
Real time RT-PCR was used to quantify the expression of mRNA for SPARC with expression 191
of elongation factor 1 (EF-1) as the reference gene. The grouper SPARC nucleotide 192
sequences of forward and reverse primers were: SPARC-QRT-S, and SPARC-QRT-A, 193
(Table 1). The primers were designed to specifically amplify a 173-bp PCR fragment. The 194
SPARC primer pair used in this experiment was designed to cross intron/exon boundaries as 195
inferred from sequence of grouper SPARC structure. Grouper EF-1 gene expression was 196
analyzed as an internal marker using the following primers: EF-1-QRT-S and EF-1-QRT- 197
A, as shown in Table 1, were designed to specifically amplify a 184-bp PCR fragment. Total 198
RNA was separated from healthy grouper following the single-step acid guanidinium 199
thiocyanate-phenol-chlorofrom extraction method. First-strand cDNA was synthesized using 200
2 g total RNA and the SuperScript First Strand cDNA synthesis kit (Invitrogen). The 201
amplification was performed using the qPCR core kit for SYBR Green (Qiagen) and 202
StepOne Real Time PCR system (Applied Biosystems). Typical profile times used were an 203
initial step of 95 °C for 15 min, followed by a second step at 94 °C for 15 sec, 60 °C for 30 204
sec and 72 °C for 30 sec for 40 cycles with melting curve analysis. The level of target mRNA 205
was normalized to the level of EF-1 and is expressed as relative to controls (healthy grouper) 206
by the 2-CT method.
207
2.7. Production of purification of SPARC protein 208
The cDNA fragments encoding the putative mature peptide were amplified by PCR, followed 209
by primer pairs SPARC-EX-S and SPARC-EX-A (see Table 1). The fragments were 210
separated on a 1% agarose gel, purified using a gel extraction kit (Qiagen), and digested with 211
restriction enzymes B and H. The digested fragment were inserted into the pET29 expression 212
vector at the EcoRI and XhoI restriction enzymes sites, and the resultant plasmid, pET29- 213
SPARC18-303, was sequenced for confirmation of the SPARC gene sequence. The resultant 214
recombinant protein was named r-SPARC. To allow expression of soluble proteins, the 215
plasmids were transformed into Escherichia coli BL21 (DE3) cells. Induction and 216
purification of the recombinant protein were performed as described previously (40). To 217
eliminate the potential contamination of bacterial endotoxins such as lipopolysaccharide (LPS) 218
during protein preparation, the purified recombinant protein was loaded onto a polymyxin B 219
column (Sigma-Aldrich, St. Louis, MO, USA) and the collected samples were stored at -80 220
ºC before use. The LPS concentration in the purified proteins was measured using a Pyrogent 221
5000 kit (Cambrex, East Rutherford, NJ, USA), with LPS standards being replaced by a 222
series of dilutions of known LPS concentration. Purity of the rSPARC was checked on by 10- 223
12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel stained 224
with Brilliant Blue, and concentration was measured by comparing the protein band density 225
with a standard protein in the same gel using an Ultra gel imaging system. Western blot 226
analysis was performed to confirm the identity of the r-SPARC using a chromogenic Western 227
blot immunodetection kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s 228
instructions. Penta-His antibody (Ab) (Invitrogen Life Technologies) was used as the primary 229
Ab for Western blot detection.
230
2.8. Production of polyclonal rabbit anti-grouper SPARC antiserum 231
The cDNA for the mature SPARC from amino acids 1–304 of orange-spotted grouper was 232
cloned into pGEM-Tvector (Promega) and subcloned into the pET-29 vector (Novagen, 233
Madison, WI, USA) between the EcoRI and XhoI sites to obtain pET29-SPARC. The 234
resulting expression vector encoded SPARC with a (His)6 and several extra amino acids at the 235
N-terminus. The expression vector pET29-SPARC was transformed into the bacterial host, E.
236
coli BL21 (DE3), for expression driven by T7 polymerase. Induction by 0.5 mM isopropyl-- 237
thiogalactopyranoside was carried out 28 ºC for 3 h. After undergoing freezing and thawing 238
once, cells were sonicated on ice, and the cleared lysate was obtained by centrifugation at 239
12,000 rpm for 15 min. The (His)6-tagged SPARC was bound to a nickel-charged HisTrap 240
column (HisTrap HP, 5 mL bed volume; Amersham Biosciences, Piscataway, NJ, USA) 241
which was pre-equilibrated with binding buffer, and washed with binding buffer containing 242
50 mM imidazole. The bound SPARC was eluted with binding buffer containing imidazole in 243
a step-gradient manner (100–500 mM). The SPARC protein peaks, eluted with 250–350 mM 244
imidazole, were combined and dialyzed against buffer A (50 mM Tris-HCl, pH 8.0, 1 mM 245
dithiothreitol, 50 mM NaCl, 5 mM MgCl2, 10% glycerol), followed by freezing at -70 ºC in a 246
minimal aliquot. Protein concentrations were determined using a Bio-Rad protein assay kit 247
with bovine serum albumin as standard. To obtain anti-grouper SPARC rabbit polyclonal 248
antibody, (His)6-tagged SPARC was used to immunize two New Zealand white rabbits with a 249
primary injection emulsified in Freund’s incomplete adjuvant at 1 mg ml-1, and 1 ml was 250
injected subcutaneously into two rabbits. The rabbits were boosted after 4, 8, and 12 weeks 251
with the same amount of antigen in the adjuvant. The SPARC antibody was obtained after 252
clotting overnight at 4 ºC followed by centrifugation at 1200 rpm [41].
253
2.9. Immunohistochemical studies of SPARC tissue distribution 254
To study SPARC distribution on grouper tissues, immunohistochemical studies were 255
performed. Grouper at 40-45 days post-hatching were fixed in 10% formalin and embedded 256
in paraffin following a routine procedure. Each 5 μm-thick section was mounted on a 257
polylysin-coated slide, deparaffinized in xylene, and rehydrated in descending grades (100%–
258
70%) of ethanol. Endogenous peroxidase activity was blocked by 10 min incubation at room 259
temperature with absolute methanol containing 3% hydrogen peroxide. The sections were 260
sequentially blocked with power block solution (Bio Genex, San Ramon, CA, USA), washed 261
with phosphate-buffered saline (PBS) and incubated with polyclonal rabbit anti-grouper 262
SPARC or polyclonal rabbit anti-coat protein Ab (1:500 dilution) at 4 oC overnight. The 263
sections were washed twice with PBS, incubated with secondary Ab (Super Sensitive 264
Polymer-HRP IHC; Bio Genex) for 30 min at room temperature. The peroxidase activity was 265
developed with 3,3’-diaminobenzidine (used as chromogen) for 10 min. The sections were 266
counterstained with Harris hematoxylin for nuclei, dehydrated and mounted. Negative 267
controls were performed with preimmune rabbit serum and incubation with PBS instead of 268
the anti-grouper SPARC Abs. The sections were observed using an Axiovert 40 microscope 269
(Carl Zeiss, Gottingen, Germany). The images were obtained with an SPOT RT3 camera 270
(Diagnostic, Sterling Heights, MI, USA).
271
2.10. Adhesion assay in vitro 272
Grouper GF-1 cells were seeded in a 24-well plate at a density of 104 cells/well. Recombinant 273
SPARC was added immediately to cells in suspension and the cells were monitored hourly by 274
phase contrast microscopy, up to 24 hours. Quantification of cell spreading was performed by 275
calculation of a Rounding Index, as described by Lane and Sage [42]. A score of 3 indicates 276
rounded cells that have not initiated spreading, whereas a score of 1 indicates fully spread 277
cells.
278 279
3. Results 280
3.1. Molecular cloning and nucleotide sequencing of grouper SPARC gene 281
From the PCR-based subtracted library, recombinant clones were randomly selected and 282
sequenced. A partial cDNA fragment (approximately 0.8 kb) was identified by sequence 283
analysis and was considered likely to be SPARC. The deduced amino acid sequence showed 284
homology with that of medaka SPARC at the C-terminus [15]. SPARC full-length cDNA was 285
generated by RACE, which was conducted to determine the nucleotide sequence of the 286
grouper SPARC cDNA (Fig. 1A). The complete SPARC cDNA is shown in Fig. 1A. Based on 287
the putative SPARC cDNA sequences obtained by the PCR-based subtracted library, primers 288
for 5 and 3 RACE were designed. The PCR-amplified 5 and 3 UTR cDNAs were used for 289
direct DNA sequencing and/or cloned into a pGEM T Easy vector. Several 5-termini were 290
found, corresponding to alternative transcription sites. A comparison of the cDNA sequence 291
of grouper SPARC with those of the fish SPARC indicated several motifs in grouper SPARC.
292
Analysis of the predicted translation revealed a protein of 52 kDa, with a theoretical signal 293
peptide (residues 1–17). The predicted mature peptide of grouper SPARC contained three 294
functional domains representing conserved human SPARC domains. Domain I was an acidic 295
rich domain (residues 18–73) that represented an acidic region rich in aspartic acid and 296
glutamic acid. The horizontal bar line amino acid sequence (74–154) showed a follistatin-like 297
domain, the characteristic KGHR motif (137–140) was fully conserved in grouper. A 298
potential glycosylation site (NXT) was found at amino acid position 116–118. Lastly, a 299
potential extracellular calcium binding domain (amino acids 155–303) displayed the highest 300
similarity within teleost species [8, 12]. Transient transfections were performed by 301
introducing g of plasmid encoding grouper SPARC-green fluorescent protein (GFP) 302
fusion protein into cells. Strong fluorescence probably resulting from overexpression was 303
evident in the cytoplasm and also accumulated in the nucleus, with an anti-adhesive effect of 304
SPARC (Fig. 1B). Expression of embryonic chicken SPARC is a negative regulator of 305
cellular adhesion activity [43]. The result was consistent with a common ancestor of all 306
vertebrate SPARCs. Additionally, among the GF-1 cells that had been tested for the 307
production of grouper SPARC-GFP, some recovered supernatant medium contained 308
detectable levels of mature SPARC-GFP when examined by immunoblotting, representing 309
overexpression of grouper SPARC-GFP in grouper cells. Lysates of SPARC-GFP producing 310
grouper cells yielded an immunoreactive band of 60.8 kDa on Western blotting. On the other 311
hand, another immunoreactive band of recombinant grouper SPARC-GFP migrated slightly 312
slower than the 67 kDa band characteristic of cell lysates, a possible consequence of cellular 313
production of N-glycoproteins containing a modification of many carbohydrate chains of 314
SPARC-GFP protein (Fig. 1C). The results provided evidence that grouper SPARC is a 315
secreted protein with high homology between fish and mammalian molecules, suggesting that 316
all SPARC molecules have a similar modular structure, which includes a signal peptide, 317
acidic rich domain, follistatin-like domain and extracellular calcium binding domain.
318
3.2. Phylogenetic tree analysis 319
SPARC is a secreted protein acid and rich in cysteine that plays important roles in signal 320
transduction networks, cell cycle inhibition and support of cell-matrix interaction [12].
321
SPARC expression is also associated with mineralization and bone remodeling [14]. To 322
elucidate the evolutionary history of SPARC, we identified novel SPARC sequences of 20 323
species across all kingdoms of organisms and all sequences available in GenBank, and 324
explored the phylogenetic relationships of the SPARC gene family.Phylogenetic analysis was 325
conducted by the CLUSTAL X method from a multiple sequence alignment of orange- 326
spotted grouper SPARC gene, and alignment of the amino acid sequences of the open-reading 327
frames along with all known complete sequences of SPARC cDNA (Fig. 2A). As expected, 328
the results indicated the presence of teleost SPARC forming a well-supported clade and 329
separating clades. The phylogenetic tree revealed the relationship between the full-length 330
orange-spotted grouper SPARC amino acid sequence, with other known SPARC orthologous 331
teleost SPARC family members (Fig. 2B).
332
3.3. Grouper SPARC transcript expression in different organs 333
Real time quantitative RT-PCR using SYBR Green dye demonstrated levels of SPARC 334
expression at similar levels in all tissues (Fig. 3A). The SPARC transcript was ubiquitously 335
expressed in all organs. Staining was visible as a brown granular reaction product with 3, 3’- 336
diaminobenzidine in chromogen solution (DAB) in the muscle and fin. The muscle produced 337
a patchy, focal pattern of SPARC expression. An immunohistochemical (IHC) study 338
indicated that grouper SPARC was highly expressed in liver and bone matrix. However, the 339
IHC-evident expression of SPARC protein decreased during nodavirus infection of grouper 340
(Fig. 3B).
341
3.4. Influence of SPARC expression in response to nodavirus 342
To analyze SPARC expression during nodavirus naturally-infected grouper, total RNA from 343
grouper eye was reverse transcribed and amplified for 25 cycles with the grouper SPARC 344
primers SPARC-RT-S and SPARC-RT-A. Amplification of total eye RNA produced a PCR 345
product of approximately 450 bp (Fig. 4A). The differential expression of SPARC was also 346
examined by real-time qRT-PCR during nodavirus infection (Fig. 4B). Grouper SPARC 347
mRNA levels decreased about 1.5 times that of control levels in eye tissue. The expression of 348
coat protein (CP) was monitored in a nodavirus-infected sample using Western-blot analysis.
349
IHC imaging detected SPARC to a significantly higher degree in the muscle region of healthy 350
grouper rather than the muscle of nodavirus-infected grouper (Figs. 5A and 5B). In contrast 351
to grouper SPARC levels, the levels of myostatin did not change significantly in response to 352
nodavirus infection (Figs. 5C and 5D). Nodavirus CP was demonstrated by IHC in the brain 353
from nodavirus naturally-infected grouper, but not in healthy grouper (Figs. 5E and 5F).
354
Vacuolization was found in the brain area of nodavirus-infected grouper during the 355
examination of H&E stained sections (Figs. 5G and 5H).
356
3.5. Recombinant grouper SPARC possesses de-adhesive bioactivity for GF-1 in vitro 357
To determine the biological activities of grouper SPARC, the putative mature peptide was 358
expressed as an N-terminal 6-histidine-tagged fusion protein in E. coli cells and purified 359
under native condition by affinity chromatography (Fig. 6A). The recombinant SPARC 360
protein was confirmed by Western blot analysis using a monoclonal Ab against the histidine 361
tag in which a single band was detected (Fig. 6B). The results indicated that expression of 362
SPARC may provide a feasible host-derived factor that regulates cell-matrix interaction.
363
There was a close link between SPARC expression and de-adhesive activity [44]. Presently, 364
recombinant grouper SPARC added to GF-1 cells in suspension, rSPARC (0.5 µg mL-1; Fig.
365
7A-d), rSPARC (1.0 µg mL-1; Fig. 7A-e) and rSPARC (1.5 µg mL-1; Fig. 7A-f), inhibited the 366
spreading on culture plastic in the presence of 5% FBS. Comparing to non-treated samples 367
(Fig. 7A-a), 50 mM imidazole-treated samples (Fig. 7A-b), or bovine serum albumin-treated 368
controls (Fig. 7A-c), this effect was apparent up to 24 h after plating, after which rSPARC 369
treated GF-1 completed de-adhesion. As shown in Fig. 7B, we have confirmed the results that 370
have been mentioned previously and used Rounding Indices to quantified anti-adhesive 371
activity. Cell viability was determined under all conditions shown in Fig. 7C.
372 373
4. Discussion 375
Expression of SPARC genes has been reported in zebrafish, medaka and gilthead seabream 376
[14–16]. In the present study, the SPARC gene was cloned from orange-spotted grouper (E.
377
coioides) and determined to encode a protein 303 amino acids in length. The cloned grouper 378
SPARC was similar in structure to that of zebrafish, medaka and gilthead seabream. Indeed, 379
the grouper SPARC gene was found to show high homology with teleosts. The phylogenetic 380
tree also indicated that grouper, medaka, fugu and gilthead seabream all belonged to the same 381
cluster, but was distant from mammalian and avian versions. Kawasaki et al [45] previously 382
reported that SPARC may be critical for initial mineralization, a finding that was likely due to 383
phylogenetic analysis of SPARC and SPARCL1 (SPARC-like 1), which provided evidence 384
that secretory calcium-binding phosphoprotein (SCPP) genes arose after the divergence of 385
cartilaginous fish and bony fish, indicating that early vertebrate mineralization did not use 386
SCPPs. Developmental tissue mineralization associated with nodavirus infection is poorly 387
understood, especially pertaining to infection-mediated brain injury.
388
The presently studied SPARC protein has the ability to modulate cell shape and cell 389
adhesion to different substrates, such as fibronectin [46]. The protein possesses highly 390
conserved sequence alignment of grouper SPARC with other known SPARC proteins, 391
especially in the regions of extracellular calcium binding domain. Interestingly, grouper 392
SPARC-GFP fusion protein was evenly spread out across the cytoplasm and nucleus (Fig.
393
1B). Moreover, homology analysis revealed that it was possible to indicate potential 394
structural and functional features of grouper SPARCs depending on conserved features of the 395
extant SPARC types. Gooden et al. [43] reported that, in embryonic chicken cells, SPARC is 396
expressed at the nuclear matrix during interphase, indicating a potential role of SPARC in the 397
regulation of mitosis during development. Further investigations need to be carried out to 398
determine whether the characteristics of intracellular SPARC protein observed in nodavirus- 399
infected grouper acts on the nuclear translocation of SPARC or on the regulatory network 400
associated with nuclear substrates.
401
When groupers become infected by nodavirus, the virus accumulates in the brain and 402
eyes, and undergoes replication at these two sites. These two sites are, therefore, very 403
important in the study of virus-host interactions. The eye is a very important tissue in the 404
study of virus-induced stress. Our real-time PCR analysis indicated that when groupers were 405
naturally-infected with nodavirus a faint decrease in SPARC transcripts could be discerned in 406
eye tissue. The SPARC expression was consistent with the significant increase in the 407
expression level of grouper SPARC at 12, 24 and 48 h post nodavirus infection when 408
compared to the control results (Fig. 3). The molecular mechanism of SPARC reduction and 409
the role of grouper SPARC during nodavirus infection remain to be elucidated. In humans, 410
the ability of virus to generate reactive oxygen species (ROS) or reactive nitrogen species 411
from phagocytes are evidence of the considerable host cell stress inflicted by viral infection 412
[47]. It is possible that generation of ROS has an antiviral effect on cells, but that this is also 413
damaging to the host cells, giving rise to a large amount of denatured protein. In view of 414
nodavirus infection and subsequent ROS production, aggregation of misfolding proteins in 415
the host cell may lead to stress status. The SPARC expression is considered to be associated 416
with a protective mechanism in the lens, whereby SPARCs possess the necessary chaperone- 417
like machinery to refold into their original structure under physiologically relevant stress 418
conditions including down-regulation of focal adhesions and reduced phosphorylation of 419
focal adhesion protein [48].
420
Experiments in vitro provided evidences that SPARC could disrupt cell adhesion [49] and 421
inhibit cell spreading [50] through a possible tyrosine phosphorylation-dependent pathway 422
[51]. SPARC also induces extracellular matrix and matrix metalloprotease production, 423
therefore, it regulates the connection of cell and matrix and finally, it makes changes in cell 424
morphology and cell migration [21, 22]. In addition, alignment of deduced grouper SPARC 425
amino acid sequence with those in various species, it is highly conserved among them (Fig.
426
2A). Therefore, recombinant grouper SPARC has been successfully produced previously 427
using either eukaryotic system or in vitro translation. It has been proven that recombinant 428
human SPARC expressed in E. coli exhibits biological characteristics including cellular anti- 429
adhesion assays [52]. Thus, protein glycosylation may be not essential for the biological 430
activity of grouper SPARC. In the current study, we produced recombinant grouper SPARC 431
with an E. coli expression system, and the results corroborated the anti-adhesion activity. In 432
addition, the expression level of SPARC in the fish tissue that contacts external environments 433
such as eye, gill and intestine is higher than other tissues because those tissues are more 434
prone to injury and require more SPARC protein to repair the damage. Socha et al [53]
435
demonstrated that the increased fibroblasts SPARC levels have been involved in the 436
pathogenesis of fibrosis and chronic tubulointerstitial disease. In addition, SPARC not only 437
binds to collagen, but also regulates extracellular matrix deposition through mediation 438
cellular matrix metalloproteinases [54]. The investigation explains about the precise grouper 439
SPARC mediates a change in cell shape from a spread to rounded morphology during 440
nodavirus-infected grouper biologically. Moreover, little is known about the role of grouper 441
SPARC in influencing leukocyte migration, injury or the inflammatory response. SPARC 442
shares some functions with matricellular protein with respect to collagen fibrillogenesis [46].
443
Matricellular protein is with anti-adhesive properties and with a pattern of expression similar 444
to that grouper SPARC. Sangaletti et al. [25] demonstrated that the absence of SPARC is 445
associated with a defect in collagen type IV deposition in newly formed basement membrane- 446
like structures which present lobular carcinoma. In general, newly formed or remodeled 447
matrix is less structured and then easier to degrade and more accessible to leukocyte 448
migration in the absence of SPARC. Additionally, in SPARC null-mice, neutrophil 449
accumulation has been associated with acute pulmonary inflammation [24]. Hence, grouper 450
SPARC may play a causal role in invading pathogens infection from repairing immune 451
system damage.
452 453
Acknowledgements 454
We thank Dr. Brian D. Hoyle for editing the manuscript. This research was supported by the 455
National Science and Technology Program for Agricultural Biotechnology, Council 456
of Agriculture (97AS-1.2.1-ST-a3(23)), and National Science Council (NSC96-2317-B-006- 457
002), and the Landmark Project (B0127) of National Cheng Kung University, the plan of 458
University Advancement, Ministry of Education.
459 460
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605 606
Figure Legends 607
Fig. 1. Characterization of a grouper SPARC. (A) The complete nucleotide and deduced 608
amino acid sequences of the grouper E. coioides SPARC gene. The dotted line indicates a 609
potential signal peptide sequence (amino acids 1–17). The underlined amino acid sequence 610
(18–73) represents an acidic region rich in aspartic acid and glutamic acid. The horizontal bar 611
line depicts the amino acid sequence (74–154) showing a follistatin-like domain. Potential 612
extracellular calcium binding domain is denoted by the marked line (amino acids 155–303).
613
Conserved cysteine residues on grouper SPARC are in boxes. The stop (TGA) codon is 614
indicated with a dot. The positions of deduced amino acids are indicated in bold numbers.
615
This nucleotide sequence data reported in this paper have been submitted to the GenBank 616
nucleotide sequence databases and have been assigned the accession no. HM855206. (B) 617
Detection of the transiently expressed grouper SPARC protein in grouper cells. Visualized 618
fluorescence of cells after transfection with pcDNA3.1/SPARC-EGFP expression plasmid.
619
Intracellular localization of GFP-fusion protein was examined by microscopy. (C) The 620
detection of secreted SPARC protein expression of grouper cells by Western blotting. Cell 621
lysates from GF-1 grouper cells were transiently transfected with pcDNA3.1/EGFP, lane1, 622
and pcDNA3.1/SPARC-EGFP, lane3, respectively. After 3–4 days of incubation, the culture 623
supernatant from transfected cells, lane 4 pcDNA3.1/SPARC-EGFP (or from control, lane2 624
pcDNA3.1/EGFP) was centrifuged and the supernatant was frozen at -80 ºC. Protein from 625
cell lysates and culture supernatants were separated by SDS-PAGE and secreted SPARC- 626
EGFP fusion protein detected by anti-SPARC antibody of Western blotting. The arrows 627
indicate the full-length fusion protein and the main glycoprotein products. Molecular mass 628
standards are shown on the left in kDa.
629 630
Fig. 2. Functional domains in human SPARC are conserved in grouper. (A) Sequence 631
alignment of SPARC proteins. The domains used for prediction according to the human 632
SPARC protein [12]. Identical amino-acids are represented by asterisks. The conservative 633
amino-acids are indicated by colons and semi-conservative substitution amino-acids are 634
depicted by dots. (B) Phylogenetic tree showing the relationships among SPARC family of 635
different organisms. The tree is based on an alignment corresponding to the open-reading 636
frames of the SPARC sequences. The numbers on the branches are bootstrap values. The 637
GenBank accession codes of the sequence designations. Homo sapiens, NM_003118; Bos 638
taurus, J03233; Sus scrofa, AY963262; Rattus norvegicus, Y13714; Mus musculus, 639
NM_009242; Gallus gallus, NM_204410; Coturnix japonica, M61908; Coturnix coturnix, 640
AF077327; Rana catesbeiana, AB116365; Xenopus laevis, BC054150; Xenopus tropicalis, 641
AY575077; Danio rerio, AY575072; Oncorhynchus mykiss, U25721; Oryzias latipes, 642
AY575076; Epinephelus coioides, HM855206; Takifugu rubripes, AY575073; Sparus 643
aurata, AJ564190; Caenorhabditis elegans, L21758; Drosophila melanogaster, NM_143252;
644
and Artemia franciscana, AB052961; Only bootstrap values higher than 70% was used on 645
each branch. The scale for branch length (0.1 substitutions/site) is shown below the tree.
646 647
Fig. 3. Expression levels of SPARC transcript in different organs of grouper. (A) 648
Relative expression levels of SPARC in different tissues from orange-spotted grouper, 649
Epinephelus coioides. (B) Representative IHC image of the orange-spotted grouper, 40–45 650
days post-hatching healthy grouper and nodavirus experimentally-infected grouper section 651
following IHC staining using an anti-grouper SPARC Ab (original magnification 10).
652 653
Fig. 4. Analysis of grouper SPARC expression during nodavirus infection. (A) Ethidium 654
bromide-stained gels demonstrate amounts of amplified grouper SPARC in nodavirus 655
naturally-infected eye tissue and a comparison with healthy grouper eye tissue at the 656
indicated number of cycles (three fish per group). Ethidium bromide-stained gel showing 657
amplification of grouper -actin gene expression was as an internal control to ensure that 658
equal amounts of mRNA were present in each experiment. Nodavirus RNA2 served as a 659
positive control since it is present in nodavirus naturally-infected grouper. The primers 5' 660
TTCAGCCAATGTGCCCCGC 3' and 5' TACACRGCACGGTCRACRTC 3' were used to 661
detect nodavirus RNA2. (B) The level of expression was calculated relative to the ef-1 alpha 662
expression level. Data represent the mean ± standard deviation of results for three fish.
663
664
Fig. 5. The expression pattern of SPARC, myostatin, and nodavirus coat protein in 665
nodavirus-infected grouper compared with healthy grouper. Histological sections of 666
muscle tissues from grouper with pathological changes that could be attributed to nodavirus, 667
immunohistochemistry for SPARC (A, B), Myostatin (C, D), nodavirus coat protein (E, F), 668
and staining with H&E (G, H).
669 670
Fig. 6. Production and purification of grouper recombinant SPARC (rSPARC) in E. coli.
671
The plasmid pET29-SPARC expressing the putative mature SPARC was transformed into E.
672
coli BL21(DE3) cells, and production of rSPARC was induced by isopropyl β–D- 673
thiogalactoside. Bacterial lysate or purified rSPARC was separated by 10-12% SDS-PAGE 674
under denaturing condition and confirmed by Western blot analysis. (A) Lanes 1–3, lysate 675
from uninduced cells, lysate from isopropyl β–D-thiogalactoside-induced cells, and purified 676
rSPARC. (B) Lane 1, 100 ng of purified rSPARC detected by Western blot analysis using a 677
mAb against the histidine tag.
678 679
Fig. 7. Soluble recombinant SPARC inhibits spreading for GF-1 in vitro. (A) Soluble 680
recombinant SPARC is anti-adhesive for GF-1 in vitro. Recombinant SPARC (rSPARC) (a) 681
0 µg mL-1, (b) 50 mM imidazole, (c) Bovine serum albumin 0.5 µg µL-1 (Control cells plated 682
without exogenous r-SPARC), (d) rSPARC 0.5 µg mL-1, (e) rSPARC 1.0 µg mL-1, (f) 683
rSPARC 1.5 µg mL-1. The purified rSPARC was added immediately to cells in suspension, 684
and the cells were monitored 24 hours by phase-contrast microscopy (Magnification×200). (B) 685
Inhibition of cell adhesion by rSPARC. Cells were scored for degree of cell spreading based 686
on Lane and Sage (42). (C) Cells viability was determined using the MTT assay.
687
Table 1. Position and sequence of synthetic oligonucleotide primers.
Name Location Sequence 5’ to 3’ Direction
SPARC-RT-S 531–553 CCTGCGTGGACTCTGAGCTCAA Forward SPARC -RT-A 981–958 TCAGATGATGAGGTCTTTGTCCA Reverse SPARC -5RACE-SP1 535–515 GCAGGGCTCGATGAATTTGCA Reverse
SPARC -5RACE-SP2 500–477 CCAGGTGCAGCTTGTGGCCCTTCT Reverse SPARC -3RACE-SP1 738–761 ACTACAACATGTACATCTTCCCCG Forward SPARC -3RACE-SP2 781–800 AGCTCGACCAGCACCCCGTTG Forward SPARC -QRT-S 71–93 ATGAGGGTGTGGATTGTCTTCCT Forward SPARC -QRT-A 243–221 TCGATGGCCTCGTCAAACTCTCC Reverse
EF-1-QRT-S ACCGCTCACATTGCTTGCA Forward
EF-1-QRT-A CAGCGAAGCGACCCAGTGGA Reverse
-ACTIN-QRT-S 527–554 TGCCTCTGGTCGTACCACTGGTATTGTC Forward
-ACTIN-QRT-A 791–768 GGCAGCAGTGCCCATCTCCTGCTC Reverse
-ACTIN-RT-S436 437–459 AGCCAACAGGGAGAAGATGACCC Forward
-ACTN-RT-A1170 1170–1151 TGATCCACATCTGCTGGAAG Reverse SPARC-EX-S 122–142 GCGCCGGAATTCCGCTCCTACTGAGGAGGAGCCC Forward
SPARC-EX-A 979–957 GCGCCGCTCGAGGATGATGAGGTCTTTGTCCACGT Reverse
The orientation is indicated as sense (S) and antisense (A). Nucleotide location of the SPARC and -ACTIN primers in DNA is according to Fig.
1. and GenBank nucleotide sequence databases FJ644278, respectively.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
