Eur Spine J DOI 10.1007/s00586-014-3214-1

ORIGINAL ARTICLE

The potential role of heat shock proteins in acute spinal cord injury Yijun Zhou • Leilei Xu • Xinghua Song • Liwen Ding • Jiangtao Chen • Chong Wang • Yuling Gan • Xiaomeng Zhu Yipin Yu • Qiuzhen Liang

•

Received: 4 October 2013 / Revised: 26 November 2013 / Accepted: 8 January 2014 Ó Springer-Verlag Berlin Heidelberg 2014

Abstract Purpose This study aims to investigate the differential expression proteins profile of spinal cord tissues after acute spinal cord injury (ASCI), provide preliminary results for further study and explore the secondary injury mechanisms underlying ASCI. Methods Using Allen’s frame to establish ASCI model of Sprague–Dawley rats, then a stable isotope-labelled strategy using isobaric tags for relative and absolute quantitation (iTRAQ) coupled with two-dimensional (2D) liquid chromatography tandem mass spectrometry (2D LC–MS/ MS) was performed to separate and identify differentially expressed proteins. Results A total of 220 differentially expressed proteins were identified in the spinal cord tissues of H-8 group (acute spinal cord injury after 8 h) compared with H-0 group (acute spinal cord injury after 0 h); Up to 116 proteins were up-regulated, whereas 104 proteins were downregulated in the spinal cord tissues. Three of the

Y. Zhou and L. Xu contributed equally to this paper and are also cofirst authors for this article. Y. Zhou XinJiang Medical University, New Medical Road, ¨ ru¨mqi 830054, People’s Republic of China U e-mail: [email protected] L. Xu
X. Song (&)
L. Ding
J. Chen
C. Wang
Y. Gan
X. Zhu
Y. Yu
Q. Liang Orthopaedic Department, The First Affiliated Hospital of XinJiang Medical University, New Medical Road, ¨ ru¨mqi 830054, People’s Republic of China U e-mail: [email protected] L. Xu e-mail: [email protected]

differentially expressed Heat shock proteins (HSPs) namely, Hsp90ab1, Hspa4 and Hspe1 were downregulated. Conclusion The differentially expressed proteins of spinal cord tissues after ASCI will provide scientific foundation for further study to explore the secondary injury mechanism of ASCI. Keywords

HSPs
ASCI
iTRAQ
2D LC–MS/MS

Introduction Spinal cord injury (SCI) has serious implications on the quality of life of affected individuals. The initial trauma, which causes inevitably loss of neurons by necrosis, is followed by a chain of events (secondary injury response) characterised by haemorrhage, excitotoxicity, increase in free radicals, intracellular calcium and fluid-electrolyte disturbances, which lead to further neuronal loss, glial scarring and cavity formation [1]. In animal models, secondary injury mechanisms result in apoptotic cell death and contribute to cell loss after trauma in the acute spinal cord injury (ASCI) [2, 3]. SCI-associated genes and proteins have been reported in a few studies wherein SCI models were used in most cases [4]. However, the use of iTRAQ technology to identify the differentially expressed proteins associated ASCI has not been reported to date. HSPs are normal intracellular proteins produced in greater amounts when cells are subjected to stress or injury [5]. These proteins have a key function in the modulation of secondary injury that occurs after the initial ASCI [6]. HSPs normally act as molecular chaperones and are called protein guardians because they can repair partially damaged proteins. Normally intracellular, HSPs can also be

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liberated into the systemic circulation to act as important inflammatory mediators [7, 8]. In the context of ASCI, HSP induction is beneficial. These proteins are liberated primarily by acutely stressed microglial, endothelial and ependymal cells. HSPs also assist in the protection of motor neurons and prevent chronic inflammation after SCI. In animal models, several experimental drugs have exhibited neuroprotective effects on the spinal cord and appear to function by modulating HSPs [9]. This study aims to explore the mechanism of sequential ASCI on the Sprague–Dawley (SD) rats and has been designed to add further knowledge to previous findings. This study is based on a rather limited number of protein spots showing alterations in intensity on two-dimensional (2D) gels. For this purpose, we used a novel proteomic method called iTRAQ. Aside from 2D differential in-gel electrophoresis (2D-DIGE) or proteomic methods based on stable isotope labelling (e.g., isotope-coded affinity tag, ICAT), the iTRAQ technique is very suitable, especially for comparative studies involving eight samples (the samples should be evaluated in parallel) [10, 11]. This approach is more sensitive than 2D-DIGE and ICAT [12]. In contrast to methods that make use of stable isotope labelling, iTRAQ enables all samples to be processed simultaneously, thereby reducing analysis time [13]. The major advantage of iTRAQ over 2D electrophoresis, which is the most commonly used method in proteomics, lies in the possibility of being able to detect low-level expression of proteins and integral membrane proteins [14]. Membrane proteins must first be solubilised by detergents before 2D electrophoresis, which can be a difficult task [15, 16]. iTRAQ is a powerful proteomic approach based on the use of four amine-specific isobaric reagents that label the primary amines of peptides from four different biological samples. These isobaric mass labels are attached to the N-termini and lysine side chains of peptides and produce abundant MS/MS signature ions. Their relative peak areas are determined by the relative proportions of the labelled peptides [12]. In this study, we succeeded in identifying 2,432 proteins in spinal cord tissue after ASCI using the iTRAQ proteomics technique.

Materials and methods At present, time window limit (6 to 8 h after ASCI) for the treatment of ASCI has still no significant breakthrough. Studies found that the timely and effective treatment can obviously improve the patient’s motor and sensory function after injured 6–8 h [52]. Therefore, this study applied iTRAQ technology to explore the differential expression proteins of SD rats after injured 0 and 8 h.

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Ethics statement All animal procedures were performed in accordance to the guidelines of the China Council for Animal Care and approved by the XinJiang Medical University animal care committee (Protocol No: 20120220005). Animals were housed in specific pathogen-free facilities at the XinJiang Medical University. All surgery and imaging were performed under anaesthesia using isofluorane, and all efforts were made to minimise suffering. Humane euthanasia of mice was performed by carbon dioxide with a flow rate of 20 % of chamber volume/min following the guidelines of the China Council for Animal Care. Animals Ten male SD rats (XinJiang Medical University Laboratory; 8–10 weeks old; weighing 300–350 g) were used for the study. All animals were maintained in a standard housing environment with ad libitum access to food and water. The animals were used for spinal cord tissue sample collection to search for biomarkers by iTRAQ coupled with 2D LC–MS/MS. In all experiments, the animals were randomly selected and equally divided into two experimental groups: H-0 groups (acute spinal cord injury after 0 h; n = 5) and H-8 groups (acute spinal cord injury after 8 h; n = 5). Surgery Animals were anaesthetized with an intraperitoneal (i.p.) injection of ketamine (100 mg/kg), atropine (10 mg/kg) and diazepam (5 mg/kg). After inducing a surgical plane of anaesthesia (end point of anaesthesia was assessed by toe pinch and eye blink reflex), the surgical area was shaved and disinfected. The animals were placed in a stereotaxic apparatus, and a dorsal midline incision was made from T6 to T12. After carefully exposing the thoracic vertebrae, laminectomy was performed with fine rongeur at the T8/10 level to expose the spinal cord. Following laminectomy, the spine was stabilised by clamping the transverse processes of one segment above and below the lesion site [17]. The contusion was generated using the displacement-controlled Allen’s frame [18]. The impactor tip was focused over the centre of the T9. The contusion was done with displacement in the two groups. After surgery, the animals were kept in a temperature-controlled incubator until they were completely awake. Subcutaneous administration of 10 ml of normal saline solution and morphine (0.03 mg/kg) was performed to prevent postoperative pain.

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Spinal cord tissue collection The spinal cords were collected at 0 or 8 h after injury. The T8/10 spinal cords (about 1-cm long and 500 mg) were dissected, meninges were carefully removed, and the tissues were thoroughly rinsed with saline to remove residual blood. The spinal cords were immediately frozen in liquid nitrogen and preserved at -80 °C until use. iTRAQ labelling and SCX fractionation Spinal cord tissues were seeded in five-well plates and transfected with 5-pmol miR-451 mimics using Lipofectamine 2000 (Invitrogen). After 48-h transfection, cells were harvested, and then protein preparation and digestion were conducted. Digested peptides were labelled with different iTRAQ reagents (Applied Biosystems), including 114 and 116. The samples of the miR-451 mimic transfected and control groups were labelled with 114 and 116, respectively. Subsequently, the labelled peptides were mixed equally and were fractionated using a polysulphoethyl ATM SCX column (200 9 4.6 mm, 5 lm particle ˚ pore size) by an HPLC system (Shimadzu, size, 200 A Japan). The HPLC gradient consisted of 100 % buffer A (10 mM KH2PO4, 25 % acetonitrile, pH 2.85) for 5 min; 0–20 % buffer B (10 mM KH2PO4, 25 % ACN, 500 mM KCL, pH 3.0) for 15 min; 20–40 % buffer B for 10 min; 40–100 % buffer B for 5 min followed by 100 % buffer A for 10 min. The chromatograms were recorded at 218 nm. A total of 12 fractions were collected. The collected fractions were desalted with Sep-PakÒ Vac C18 cartridges (Waters, Milford, Massachusetts) concentrated to dryness using a vacuum centrifuge and reconstituted in 0.1 % formic acid for LC–MS/MS analysis. LC–MS/MS LC–MS/MS analysis of the samples was performed using an AB SCIEX TripleTOFTM 5600 mass spectrometer (AB SCIEX, Framingham, MA, USA) coupled with an online microflow HPLC system (Shimadzu, Japan). The HPLC system consisted of a trap column (200 lm 9 2 cm, C18 ˚ ) and an analytical column material 5–10 lm, 120 A ˚ ). The pep(75 lm 9 10 cm, C18 material 5 lm, 120 A tides were separated using nanobored C18 column with a picofrit nanospray tip (75 lm ID 9 15 cm, 5 lm particles) (New Objectives, Wubrun, MA). The separation was implemented at a constant flow rate of 20 ll min-1, with a splitter to achieve an effective flow rate of 0.2 ll min-1. The MS spectra were acquired in the positive ion mode, targeting the peptides with ?2 to ?4 ions in the survey scan, with a selected mass range of 300 m/z to 2,000 m/ z. The three most abundantly charged peptides above a

count threshold were selected for MS/MS and dynamically excluded for 30 s with ±30 mDa mass tolerance. The fragment intensity multiplier was set to 20 and the maximum accumulation time was 2 s. The peak areas of the iTRAQ reporter ions reflect the relative abundance of the proteins in the samples. Bioinformatics analysis The 2.3.02 version of the Mascot software (Matrix Science) was used to simultaneously identify and quantify proteins. In this version, only unique peptides used for protein quantification can be chosen: thus, it is a more precise method for quantifying proteins. Spectra were combined into one Mascot generic format (MGF) file after loading the raw data, and the MGF files were searched. The search parameters were as follows: trypsin was chosen as the enzyme with one missed cleavage allowed; fixed modifications of carbamidomethylation at Cys, variable modifications of oxidation at Met; peptide tolerance was set at 10 ppm and MS/MS tolerance was set at 0.05 Da. Peptide charge was set as Mr, and monoisotopic mass was chosen. iTRAQ 8plex was chosen for simultaneous quantification during the search. The search results were passed through additional filters before exporting the data. For protein identification, the filters were set as follows: significance threshold of p \ 0.05 (with 95 % confidence) and ion score or expected cutoff\0.05 (with 95 % confidence). For protein quantitation, the filters were set as follows: ‘‘median’’ was chosen for protein ratio type (http://www.matrixscience.com/help/ quant_config_help.html); minimum precursor charge was set to 2? and minimum peptide was set to 2; only unique peptides were used to quantify proteins. Median intensities were set as normalisation, and outliers were removed automatically. The peptide threshold was set as above for Identity. Proteins with 1.2-fold or more change between treatment and control samples and p \ 0.05 were considered as differentially expressed. Quantitation was performed at the peptide level by following the procedures described in http://www.matrixscience.com/help/quant_statistics_help. html. Student t test was performed using the Mascot 2.3.02 software. Metabolic pathway analysis of the identified proteins was conducted according to the KEGG Pathway Database. Cluster of Orthologous Groups of proteins (COG) analysis (http://www.geneontology.org) was conducted according to the literature.

Results The iTRAQ analysis of proteins in the spinal cord tissues was performed. In the spinal cord, a total of 349,035 MS/

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Ostc Oligosaccharyltransferase complex subunit OSTC

Gspt1 Uncharacterized protein

Olr1121 olfactory receptor Olr1121

Mt-atp8 ATP synthase protein 8

LOC362795 Ig gamma-2C chain C region

Tmem160 Uncharacterized protein

ceb2 Transcription elongation factor B polypeptide 2

Svip Small VCP/p97interacting protein Sypl1 Uncharacterized protein

IPI00373430

IPI00368457

IPI00187805

IPI00195772

IPI00782787

IPI00206354

IPI00210012

IPI00561657

IPI00231434

Fkbp1a Peptidyl-prolyl cis– trans isomerase FKBP1A

Sfxn1 Uncharacterized protein

IPI00464440

IPI00471762

Description

Accession

14,710

33,211

11,399

15,652

20,297

43,754

9,462

40,069

83,804

18,743

40,109

Mass

13

4.3

14.5

7.6

6.9

3

11.9

2.2

1.6

8.1

3.4

Cov

1.355

0.553

1.359

1.636

1.815

2.288

0.615

0.698

1.064

1.44

2.276

iTRAQ ratio

*

*

*

*

*

*

*

*

*

*

*

Sig

Regulation of protein phosphorylation; muscle system process; activin receptor signalling pathway; cardiac muscle tissue morphogenesis; regulation of immune system process; protein ubiquitination; peptidyl-amino acid modification; regulation of ion transmembrane transporter activity; regulation of protein binding; regulation of protein phosphatase type 2B activity; extracellular matrix organisation; response to metal ion; negative regulation of sequestering of calcium ion; I-kappaB kinase/NF-kappaB cascade; T cell activation; protein folding; response to purine-containing compound; amyloid precursor protein catabolic process; trabecula formation

Establishment of localization; cell–cell signalling

–

Translation; regulation of transcription from RNA polymerase II promoter; protein modification by small protein conjugation; macromolecular complex assembly; positive regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolic process; transcription elongation, DNA dependent

–

–

Hydrogen transport; ATP biosynthetic process

Cell surface receptor-linked signalling pathway; sensory perception of chemical stimulus

Translation; poptosis; protein modification by small protein conjugation; protein alkylation; purine ribonucleoside triphosphate catabolic process; nuclear-transcribed mRNA catabolic process

Protein N-linked glycosylation

Myeloid cell differentiation; transition metal ion transport

GO biological process

Axon part; sarcoplasmic reticulum; vesicular fraction; endoplasmic reticulum membrane

Clathrin-coated vesicle; vesicle membrane; pigment granule; integral to membrane

–

Cytoplasmic ubiquitin ligase complex; nuclear lumen

Intrinsic to membrane; intracellular membranebounded organelle

Membrane

Intrinsic to membrane; mitochondrial protontransporting ATP synthase complex; protontransporting two-sector ATPase complex, proton-transporting domain

Intrinsic to membrane

Cell part

Endoplasmic reticulum membrane; intrinsic to membrane

Organelle inner membrane; mitochondrial envelope; intrinsic to membrane

GO cellular component

Table 1 The proteins have one peptide, which comprises a part of the 220 differentially expressed proteins (H-0 vs. H-8) in the spinal cord tissue after ASCI

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Description

Vps4a Vacuolar protein sorting-associated protein 4A

LOC100360822 Uncharacterized protein (Fragment)

Mrps36 28S ribosomal protein S36, mitochondrial

Bsg Isoform 2 of Basigin

Txn1 Thioredoxin

Slc7a10 Uncharacterized protein

Rpl11 60S ribosomal protein L11

Accession

IPI00204016

IPI00556952

IPI00372007

IPI00193425

IPI00231368

IPI00421427

IPI00359103

Table 1 continued

25,639

62,273

15,962

34,719

14,448

15,121

61,634

Mass

7.9

4.2

12.4

4.8

12.6

13

2.7

Cov

0.543

1.59

0.65

0.885

1.282

1.438

1.341

iTRAQ ratio

*

*

*

*

*

*

*

Sig

Induction of programmed cell death; translation; viral genome expression; pancreas development; protein localization to organelle; ribosome biogenesis; intracellular protein transport

Immune system process; alanine transport; nitrogen compound metabolic process; hemostasis; serine transport; L-amino acid transport

Positive regulation of binding; regulation of transcription from RNA polymerase II promoter; nucleobase, nucleoside and nucleotide metabolic process; organic ether metabolic process; generation of precursor metabolites and energy; signalling; cell communication; cellular homeostasis; protein import into nucleus, translocation; response to abiotic stimulus; negative regulation of nucleocytoplasmic transport

Odontogenesis; response to metal ion; response to hormone stimulus; monocarboxylic acid metabolic process; organic anion transport; immune system process; hemostasis; signal transduction; developmental process involved in reproduction

Gene expression

–

Vesicle-mediated transport

GO biological process

Nuclear lumen; large ribosomal subunit

Neuron projection; integral to membrane

Nuclear lumen; cytoplasmic part

Intrinsic to membrane; endomembrane system; pigment granule; plasma membrane

Organellar small ribosomal subunit; intracellular membrane-bounded organelle

–

Late endosome; endosome membrane

GO cellular component

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MS spectrum were acquired from all iTRAQ runs, among which 20,577 spectrum were utilised to assign 8,237 unique peptides, representing 2,432 proteins. All spectrums were searched against a decoy database with all Swiss-Prot sequences reversed. Only proteins that were present at a CI value of 95 % or greater and identified by at least two peptides were considered. The foregoing are defined as the differential expression proteins whose difference multiples are 1.2-fold or more and whose statistical test p value is \0.05. A total of 220 differential expression proteins were identified in the spinal cord tissues of H-8 group (acute spinal cord injury after 8 h) compared with H-0 (Acute spinal cord injury after 0 h), among which 116 proteins were up-regulated and 104 proteins were down-regulated. Among them, we selected several differential expression proteins with only one peptides (Table 1). Twelve HSPs (including Hsp90ab1, HSP 90-beta; Hspa4, heat shock 70-kDa protein 4; Hspe1, 10-kDa HSP, mitochondrial; Hsph1, HSP 105 kDa; Trap1, HSP 75 kDa, mitochondrial; Hspd1, 60-kDa HSP, mitochondrial; Hsp90aa1, HSP 90-alpha; Hspa8, heat shock cognate 71-kDa protein; Hspa2, Heat shock-related 70-kDa protein 2; Hspa13, heat shock 70-kDa protein 13; Hsbp1, heat shock factor-binding protein 1; Hspb1, HSP beta-1) were expressed in the spinal cord tissue after ASCI (Table 2). Three proteins were up-regulated and nine proteins were down-regulated in HSPs of spinal cord tissue. Among them, the three differentially expressed proteins were identified to be Hsp90ab1, Hspa4 and Hspe1, and all three were down-regulated. The three differentially expressed proteins (Hsp90ab1, Hspa4 and Hspe1) have 115 MS/MS spectrum. We selected one MS/MS spectra of Hsp90ab1 (Fig. 1). COG is a database used for classifying orthologous proteins. All of the members of COG proteins are assumed to come from the same ancestral of a protein. Not only do ‘‘orthologs’’ or ‘‘paralog orthologs’’ come from different species of proteins that evolved from vertical home system (speciation), these proteins also keep the same specific function as the original protein. Paralogs are derived from genetic replication proteins in certain species and may evolve new functions that are related to original proteins. The purpose of this analysis is to compare the identified proteins with the COG database and explore these proteins’ functions or their functional classifications (Fig. 2). In this study, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of our selected dataset suggests the involvement of several pathways, with the most significant pathway involving HSPs (Hsp90ab1 and Hspa4) being ‘‘antigen processing and presentation’’ (Fig. 3). To gain novel insights into the ASCI pathology from these expression proteomics studies, we performed a KEGG pathway analysis of all HSP proteins that were found to be differentially expressed

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(difference multiple is 1.2-fold and p B 0.05) in ASCI animals from the current iTRAQ analysis of SD rat spinal cords.

Discussion Numerous successful explorations about the molecular mechanisms of sequential ASCI on gene and transcription have been reported. Compared with genomes and transcriptomes, which are relatively static and will be transcribed into a variety of functional distinct proteins, proteomes may provide a more realistic picture of functional aberrations in ASCI. Proteomics has become the frontier in the post-genomic era; new proteomic methods and a large number of achievements are emerging. Traditional 2-DE technique has been used extensively for comparative proteomics with a significant achievement, but it poses several disadvantages. iTRAQ quantitative proteomics offers sensitivity, which can overcome these deficiencies (such as being able to accurately identify membrane proteins) and increase the number and types of differentially expressed proteins identified. In contrast to 2-DE, iTRAQ technology has better repeatability and quantification accuracy [19]. In this study, a stable isotope-labelled strategy using iTRAQ coupled with 2D LC–MS/MS mass spectrometry was performed to separate and identify the differentially expressed proteins. A total of 12 HSPs (including the three differential expression proteins Hsp90ab1, Hspa4, Hspe1) were found in the spinal cord tissue. HSPs are the most highly expressed cellular proteins across all species [20]. These proteins account for 1–2 % of total proteins in unstressed cells. However, when cells are heated or subjected to other stressors, the fraction of HSPs increases to 4–6 % of all cellular proteins expressed [20]. The molecular weights of HSPs can range from 10 to 150 kDa and they are named according to their molecular weights, for example: Hsp10, Hsp27, Hsp60, Hsp70, Hsp75, Hsp90 and Hsp105. The HSP structure across various species has been described in several previous studies. HSP 90 is the most abundant nonribosomal protein and is also the most abundant protein in the endoplasmic reticulum [21– 23]. However, HSP 70 is the most widely studied [21, 24]. Significant studies have been made regarding the action of HSPs in ASCI of animal models [9]. Based on these animal studies, HSPs are clearly important modulators of ASCI. In ASCI, the primary injury produces a state of hyperexcitability caused by the production of excitatory neurotransmitters (including glutamate, lactate, pyruvate and aspartate), liberation of free radicals and intracellular entry of calcium. Put together, these events trigger secondary injury cascade [25]. The role of HSPs is complex: they can be pro-inflammatory but they also have significant

Eur Spine J Table 2 iTRAQ ratio–value [1.0 indicates up-regulation; iTRAQ ratio–value \1.0 indicates down-regulation Accession

Description

Mass (Da)

Coverage (%)

iTRAQ ratio

Sig

GO cellular component

Peptide

IPI00471584

Hsp90ab1 Heat shock protein HSP 90-beta

106,691

36.6

0.852

*

Cell fraction; pigment granule; brush border; plasma membrane part

22

IPI00387868

Hspa4 Heat shock 70-kDa protein 4

119,436

35.1

0.755

*

Nuclear lumen; cytoplasmic part

20

IPI00326433

Hspe1 10-kDa heat shock protein, mitochondrial

14,545

43.1

0.55

*

Mitochondrial part

IPI00471835

Hsph1 Heat shock protein 105 kDa

121,359

16.6

1.123

–

Intracellular membrane-bounded organelle

10

IPI00369217

Trap1 Heat shock protein 75 kDa, mitochondrial

94,936

8.5

1.334

–

Intracellular membrane-bounded organelle

5

IPI00339148

Hspd1 60-kDa heat shock protein, mitochondrial

77,820

45.9

0.916

–

Endoplasmic reticulum; stored secretory granule; endomembrane system; receptor complex; membrane part; mitochondrial inner membrane; endosome; extracellular region part

18

IPI00210566

Hsp90aa1 Heat shock protein HSP 90-alpha

109,802

31.5

0.881

–

Cell fraction; pigment granule; brush border; extracellular region part; plasma membrane part

21

IPI00208205

Hspa8 Heat shock cognate 71-kDa protein

87,787

42

0.875

–

Macromolecular complex; cell fraction; pigment granule; clathrin-coated vesicle membrane

21

IPI00207355

Hspa2 Heat shock-related 70-kDa protein 2

86,007

18

1.286

–

Germ cell nucleus; condensed nuclear chromosome

8

IPI00206300

Hspa13 Heat shock 70-kDa protein 13

60,338

4.7

0.746

–

Vesicular fraction; intracellular membranebounded organelle

2

IPI00202706

Hsbp1 Heat shock factorbinding protein 1

10,404

39.5

0.922

–

Intracellular membrane-bounded organelle; intracellular non-membrane-bounded organelle

2

IPI00201586

Hspb1 Heat shock protein beta-1

25,065

27.2

0.99

–

Intracellular membrane-bounded organelle; protein complex; microtubule cytoskeleton; I band; cell fraction; membrane

3

4

Twelve HSPs were expressed in the spinal cord after ASCI, among which the three proteins Hsp90ab1, Hspa4 and Hspe1 were differentially expressed (the Sig is *) which expressed in the spinal cord after ASCI, among them, three proteins are differential expression proteins (The Sig is *) which are Hsp90ab1, Hspa4 and Hspe1

anti-inflammatory and cytoprotective properties [7, 8]. Studies have shown that HSPs have a multifaceted modulatory role in inflammation. These proteins initially promote inflammation and then exert an anti-inflammatory effect, thereby limiting chronic sequelae. Other studies suggest that extracellular HSPs in the cerebrospinal fluid inhibit the development of chronic inflammatory disease after acute injury to the central nervous system [6]. One of the differentially expressed proteins in spinal cord tissue during ASCI is Hsp90ab1 (HSP 90-beta), which is a calcium-binding protein and a member of the Hsp90 family [26]. This protein family has essential functions in the folding, maturation and activity of various proteins involved in signal transduction and transcriptional regulation. Hsp90 found on the cell surface could be a target for immune response; this protein has been described as an antigen in several diseases, such as rheumatoid arthritis [27] and breast cancer [28]. Hsp90ab1 is expressed specifically on the surface of oligodendrocyte precursor cells (OPCs) [29]. In cell culture

studies [30], anti-Hsp90ab1 antibody-dependent, complement-mediated cytotoxicity against these cells causes a significant decrease in the number of pre-oligodendrocytes, including adult pre-oligodendrocytes. The inhibition of complement by physical treatment, chemical agents or the inhibitor C1-Inh prevents this phenomenon. C1-Inh is a physiological inhibitor of complement activation, therefore this glycoprotein may have a function in the mechanism of OPC death induced by anti-Hsp90ab1 antibodies. These findings suggest a mechanism for pre-oligodendrocyte exhaustion that depends on anti-Hsp90b antibody, complement and C1-Inh levels [30]. Anti-Hsp90ab1 antibodies recognise only OPCs, and anti-Hsp90ab1 antibody-dependent cell death seems to occur only in these cells. Previous studies reinforce the idea that Hsp90ab1 is a specific target on OPCs including adult OPCs [29, 30]. C1-Inh prevents anti-Hsp90ab1 antibody-induced and complement-mediated OPC death, which are the only physiological C1r and C1s inhibitors. C1-Inh is a key regulator of the complement system and could, therefore, be used to specifically inhibit

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damage resulting from complement activation in inflammatory reactions [31]. However, the role of Hsp90ab1 in ASCI has not been reported. In the present study, Hsp90ab1 is a member of the plasma membrane proteins. Hsp90ab1 is involved in the regulation of several pathways. Such as the NOD-like

receptor signalling pathway, pathways in cancer, protein processing in the endoplasmic reticulum, prostate cancer, progesterone-mediated oocyte maturation, as well as antigen processing and presentation (Fig. 3). Gene ontology (GO) analysis shows that Hsp90ab1 actively participates in several biological processes including regulation of the

Fig. 1 A MS/MS spectra of Hsp90ab1, which have five MS/MS spectrum (peptide_sequence: EGLELPEDEEEK). a Black lines indicate mismatch with the ion; red lines indicate matching a-type and b-type ions; blue lines indicate the matching y-type ions; solid line height indicates the intensities of the ion; dotted lines indicate the

annotation of corresponding ions. b The ion relative strength chart of peptides in the report. This experiment marked 114 and 116. The figure only shows two kinds of markers related to ionic strength; green solid line heights indicate the relative size of the intensities; the data indicate the abscissa of the m/z

Fig. 2 Statistics of different functional proteins in the samples. Abscissa for COG classification items; ordinate for functional classification related to the number of proteins

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Fig. 3 Pathway of antigen processing and presentation. The green box indicates down-regulation

cytokine-mediated signalling pathway (GO: 0001959), positive regulation of cellular biosynthetic process (GO: 0031328), cellular protein metabolic process (GO: 0044267), negative regulation of cellular catabolic process (GO: 0031330), organ development (GO: 0048513), response to osmotic stress (GO: 0006970), response to organic cyclic compound (GO: 0014070) and chemotaxis (GO: 0006935). Hspa4 (heat shock 70-kDa protein 4) is a member of the stress protein superfamily of multifunctional proteins that are induced by a variety of stresses and injuries [32–34]. Induction of these stress proteins may be a critical feature in the proper functioning of these cells in a hostile microenvironment by acting as molecular chaperones or polypeptide binding proteins. They prevent improper proteins folding and prevent improper interactions among proteins before their synthesis is completed, thereby maintaining their translocation competence [35]. Numerous studies on the brain have suggested a relationship between hsp70 expression and sensitivity of particular brain regions to ischemic injuries [36–38]. In the present

study, Hspa4 is a cytoplasmic protein involved in the regulation of the antigen processing and presentation pathway (Fig. 3). After GO analysis, Hspa4 is found to be involved in several biological processes, including regulation of protein binding (GO: 0043393), protein targeting to membrane (GO: 0006612), chaperone-mediated protein complex assembly (GO: 0051131) and response to stress (GO: 0006950). Hspe1 (10-kDa heat shock protein, mitochondrial) was first identified in capacitated mouse spermatozoa using an immunoprecipitation strategy with HSPD1 as the bait. Although eukaryotic Hspe1 has primarily been described as a mitochondrial chaperone [39, 40], more recent reports suggest that it may also function in signal transduction, cell cycle regulation, nucleocytoplasmic transport and metabolism [41]. Hspe1 families of molecular chaperones interact to fulfil a well-characterised role in mediating the correct folding of a variety of protein substrates [42, 43]. In our study, Hspe1 was identified as a mitochondrial protein. However, consistent with the findings of this study, an emerging body of the literature suggests that these proteins

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are also present on the cell surface and in the extracellular fluid [44, 45]. GO analysis found that Hspe1 is mainly involved in two biological processes, namely, de novo posttranslational protein folding (GO: 0051084) and response to stress (GO: 0006950). Interestingly, the down-regulation of Hsp90ab1 and Hspa4 was prominent in the MHC-I antigen processing and presentation pathways. The antigen bound on MHC-I and presented to the T cell receptors of CD8? cells is normally produced by proteasomes or immunoproteasomes, which do not have completely overlapping antigen proteolytic activities [46]. This finding indicates that the secondary injuries of ASCI may be induced by immunisation with auto-antigens, and that the sequential SCI may be a disease with autoimmune components. Auto-reactive T cells are parts of mediators of the sequential SCI pathology [47–49]. Both CD4? and CD8? T cells reportedly contribute to nervous system disease initiation and progression [50]. Auto antigen-reactive CD8? T cells, typically clonally expanded and specific for myelin antigens, are found in patients early in the course of the disease [51]. In this study, Hsph1, Trap1, Hspd1, Hsp90aa1, Hspa8, Hspa2, Hspa13, Hsbp1 and Hspb1 were not differentially expressed in the spinal cord tissue. However, GO analysis found that these proteins are involved in several biological processes relating to ASCI, including processes related to inflammation response such as positive regulation of leukocyte activation, response to molecule of bacterial origin and response to other organism; processes-related immunoreactions such as T cell-mediated immune response to tumour cell, cytokine production involved in immune response, regulation of T cell activation, positive regulation of T cell differentiation and chaperone-mediated protein complex assembly; processes related to signal transmission such as toll-like receptor signalling pathway, isotype switching, signalling, protein targeting to membrane, translational initiation and regulation of neurotransmitter levels; processes related to stress such as response to stress, response to stimulus, response to steroid hormone stimulus, detection of chemical stimulus, response to reactive oxygen species, positive regulation of type I interferon production, regulation of anti-apoptosis, response to oxidative stress and negative regulation of apoptosis; processes related to metabolic process such as purine ribonucleoside triphosphate catabolic process, cellular nitrogen compound biosynthetic process, regulation of monooxygenase activity, purine ribonucleoside triphosphate catabolic process, cellular protein metabolic process and RNA metabolic process; processes related to protein synthesis: sequencespecific DNA binding, regulation of transcription from RNA polymerase II promoter, Golgi vesicle transport, de novo’ posttranslational protein folding, cellular protein complex assembly, protein folding and regulation of

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protein stability; and other functions such as response to tropane, cell migration, cell cycle process, regulation of lamellipodium assembly, chemotaxis, response to osmotic stress, positive regulation of cellular biosynthetic process, male gamete generation, germ cell development, meiosis I, cell cycle, regulation of cyclin-dependent protein kinase activity involved in G2/M, protein dimerization activity, multi-organism process, synaptonemal complex organisation and muscle system process. This study does not cover all the biological processes related to HSP functions based on protein expression in the spinal cord tissue during ASCI. iTRAQ analysis of spinal cord tissues is an effective method for the proteomic investigation of neurological disease in animal models. This study aimed to identify differentially expressed proteins that can be used to monitor these sequential ASCI process changes under the assumption that morphologic changes are accompanied by abnormal expression of proteins. Findings indicated that HSPs are important modulators of secondary injury mechanism of ASCI. Thus, additional research using a larger number of ASCI in animal studies is needed to confirm our findings. Future studies will be conducted to further elucidate the precise molecular mechanisms through which the proteins act on sequential ASCI. Conflict of interest

None.

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