Edited by: Pung P. Hwang, Academia Sinica, Taiwan
Reviewed by: Alberto Cuesta, University of Murcia, Spain; Mathilakath Vijayan, University of Calgary, Canada
*Correspondence: Jaume Pérez-Sánchez
This article was submitted to Aquatic Physiology, a section of the journal Frontiers in Physiology
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
The skin mucus of gilthead sea bream was mapped by one-dimensional gel electrophoresis followed by liquid chromatography coupled to high resolution mass spectrometry using a quadrupole time-of-flight mass analyzer. More than 2,000 proteins were identified with a protein score filter of 30. The identified proteins were represented in 418 canonical pathways of the Ingenuity Pathway software. After filtering by canonical pathway overlapping, the retained proteins were clustered in three groups. The mitochondrial cluster contained 59 proteins related to oxidative phosphorylation and mitochondrial dysfunction. The second cluster contained 79 proteins related to antigen presentation and protein ubiquitination pathways. The third cluster contained 257 proteins where proteins related to protein synthesis, cellular assembly, and epithelial integrity were over-represented. The latter group also included acute phase response signaling. In parallel, two-dimensional gel electrophoresis methodology identified six proteins spots of different protein abundance when comparing unstressed fish with chronically stressed fish in an experimental model that mimicked daily farming activities. The major changes were associated with a higher abundance of cytokeratin 8 in the skin mucus proteome of stressed fish, which was confirmed by immunoblotting. Thus, the increased abundance of markers of skin epithelial turnover results in a promising indicator of chronic stress in fish.
A keratinized multi-sheet cellular layer (stratum corneum) covers the epidermis of amphibian adults, reptiles, birds and mammals, whereas skin mucus constitutes the outermost epidermal barrier in fish and aquatic amphibian larvae (Schempp et al.,
In teleost fish, stress activates the hypothalamus-pituitary-interrenal axis, leading to a rapid release of the glucocorticoid hormone cortisol by the interrenal tissue, the tissue analogous to the adrenal cortex of mammals (Pottinger,
Recently, important research efforts have also been invested in mapping the skin mucus proteome of warm-water marine fish, such as gilthead sea bream (Jurado et al.,
Two-year old gilthead sea bream (average body weight of 320 g) coming from the study of Bermejo-Nogales et al. (
The protein composition of mucus was first analyzed by one-dimensional electrophoresis (1-DE). Initially, mucus samples from all animals (CTRL and M-ST fish) were pooled, and triplicate samples (54-56 μg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a TGX Any kD precast gel (Bio-Rad, Hercules, CA, USA) run at 200V for 25 min and stained overnight with colloidal Coomassie (Bio-Rad). The gel was then divided into 10 slices (0.65 cm) that were analyzed independently. Proteins in the gel were digested with protein-grade trypsin (Promega, Madison, WI, USA) and concentrated by speed vacuum at a final volume of 12 μL for mass spectrometry.
Individual samples of CTRL and M-ST fish (
About 150 μg of protein (incubated in 65 mM DTT and 1% ampholytes) were loaded into Immobiline DryStrips (pH 3-11 NL, 24 cm), rehydrated overnight in 8 M urea, 4% w/v CHAPS, 12 μL/mL DeStreak reagent, 1% ampholytes. After focusing at 32 kVh at 20°C, strips were equilibrated first for 15 min in reducing solution (6 M urea, 50 mM Tris-HCl, 30% v/v glycerol, 2% w/v SDS, 2% w/v DTT) and then in alkylating solution (6 M urea, 50 mM Tris-HCl, 30% v/v glycerol, 2% w/v SDS, 2.5% w/v iodoacetamide) for 15 min. The second dimension (12.5% polyacrylamide, 25 × 21 cm) was run at 20°C at a constant power of 2 W for 60 min followed by 15 W until the bromophenol blue tracking front had run off the end of the gel (6 h). Fluorescence images were obtained on a Typhoon 9,400 scanner (GE HealthCare Life Sciences). Cy2, Cy3, and Cy5 images were scanned at excitation/emission wavelengths of 488/520 nm, 532/580 nm, and 633/670 nm, respectively, at a resolution of 100 μm. Image analysis was performed using DeCyder v.6.5 software (GE HealthCare Life Sciences). Protein spots displaying a statistically significant difference between groups were manually excised from analytical gels and digested with sequencing-grade trypsin prior to mass spectrometry analysis.
Samples (5 μl) from 1-DE and two-dimensional electrophoresis (2-DE) were analyzed by liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) using a quadrupole time-of-flight mass analyzer (qQTOF). Briefly, samples were loaded onto a trap column (NanoLC Column, 3 μ C18-CL, 350 μm × 0.5 mm, Nikkyo Technos Co. Ltd., Tokyo, Japan) desalted with 0.1% TFA at 3 μL/min for 10 min. Peptide mixtures were then loaded onto an analytical column (LC Column, 3 μ C18-CL, 75 μm × 12 cm, Nikkyo Technos Co. Ltd.) equilibrated in 5% acetonitrile and 0.1% formic acid. Separation was carried out with a linear gradient of 5–40% acetonitrile gradient with 0.1% formic acid at a flow rate of 300 nL/min. Peptides were analyzed in a high resolution nanoESI (qQ) TOF mass spectrometer (AB SCIEX TripleTOF 5,600 System, Applied Biosystems/MDS Sciex, Foster City, CA). The (qQ) TOF was operated in information-dependent acquisition mode, in which a 0.25-s TOF MS scan from 350 to 1,250 m/z, was performed, followed by 0.05 s product-ion scans from 100 to 1,500 m/z on the 50 most intensely 2–5 charged ions. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD004115 and PXD004116.
Protein identity was determined using ProteinPilot v4.5 (AB SCIEX, Applied Biosystems/MDS Sciex), which incorporated the Mascot search algorithm (v2.2, Matrix Science, London, UK). ProteinPilot default parameters were used to generate peak list directly from 5600 TripleTOF wiff files. Mascot was used to search the Expasy protein database or the IATS-CSIC gilthead sea bream database (
In order to validate the results of 2-DE analysis, the increased abundance of keratin type II cytoskeletal 8 in M-ST compared to CTRL group was assessed by means of a Western blot analysis using an antibody directed to human cytokeratin 8. Total protein concentration from mucus samples of CTRL and M-ST fish was determined using the Bradford protein assay (Bio-Rad). The quantified protein analyzed remained almost equal in both experimental groups (1 μg/μl) and equal amounts from the two different groups were mixed with 2 × SDS sample buffer (1.5 M Tris, pH 8.8, 0.2% glycerol, 0.4% SDS, 0.1% 2-mercaptoethanol and 0.05% bromophenol blue), heated for 5 min at 50°C and separated by SDS-PAGE. After electrophoresis, proteins were transferred to polyvinylidenedifluoride (PVDF) membranes (Invitrogen, Gaithersburg, MD, USA) at 15 V for 1 h at room temperature. The membranes were then blocked in 5% nonfat dry milk prepared in TBS (20 Mm Tris pH 7.5, 500 mM NaCl) overnight at 4°C. After blocking, membranes were incubated with rabbit anti-human cytokeratin 8 antibody (PA5-29607, Thermo Scientific, Wilgminton, DE, USA) in antibody buffer (0.1% Tween 20, 1% bovine serum albumin), using a 1:2000 dilution of the supplied antibody concentration. The peptide immunogen (252 amino acids in length) of this primary antibody shared 81% identity (93% homology) with the gilthead sea bream sequence of cytokeratin 8. After primary antibody incubation, membranes were washed four times for 10 min each in T-TBS (TBS with 0.1% Tween 20), incubated with HRP-conjugated goat anti-rabbit IgG at 1:9000 dilution in antibody buffer for 2 h at room temperature, and washed four times for 10 min each in T-TBS. Immunodetection was performed using a chemiluminescent system (Western Blotting Luminol Reagent, Santa Cruz Biotechnologies, CA, USA) and the image on the membrane was captured by VersaDoc Imaging system model 5,000.
Quantification of relative protein levels in 2-DE electrophoresis was performed using Decyder v.6.5 software. Statistical significance was assessed using Student's
The current study analyzed the skin mucus of gilthead sea bream, combining 1-DE and 2-DE MS-based proteomic approaches. The primary finding was the large number of proteins that were identified by 1-DE followed by LC-HRMS in comparison to previous proteomic studies in this fish species, in which attention was focused on the most abundant proteins with an over-representation of structural and immune-related proteins. Hence, in the first reference proteome map of gilthead sea bream epidermal mucus (Sanahuja and Ibarz,
Among the final number of mucus proteins (2,060), more than 89% (1,848 proteins) were eligible for functional pathway analysis using the IPA software. These proteins were represented in 418 canonical pathways out of 644. To easy identify the more relevant pathways and biological processes, an overlapping analysis was performed with a filter of six common proteins among related pathways. From this integrative approach, 17 canonical pathways with significant
Aconitase 2, mitochondrial | ACO2 | 1 | |
Apoptosis-inducing factor 1, mitochondrial | AIFM1 | 1 | |
ATP synthase subunit alpha, mitochondrial | ATP5A1 | 1,2 | |
ATP synthase subunit beta, mitochondrial | ATP5B | 1,2 | |
ATP synthase subunit gamma, mitochondrial | ATP5C1 | 1,2 | |
ATP synthase subunit delta, mitochondrial | ATP5D | 1,2 | |
ATP synthase subunit epsilon, mitochondrial | ATP5E | 1,2 | |
ATP synthase subunit b, mitochondrial | ATP5F1 | 1,2 | |
ATP synthase lipid-binding protein, mitochondrial | ATP5G1 | 1,2 | |
ATP synthase subunit d, mitochondrial | ATP5H | 1,2 | |
ATP synthase subunit e, mitochondrial | ATP5I | 1,2 | |
ATP synthase subunit f, mitochondrial | ATP5J2 | 1,2 | |
ATP synthase subunit g, mitochondrial | ATP5L | 1,2 | |
ATP synthase subunit O, mitochondrial | ATP5O | 1,2 | |
Caspase 3 | CASP3 | 1 | |
Cytochrome c oxidase subunit 4 isoform 1, mitochondrial | COX4I1 | 1,2 | |
Cytochrome c oxidase subunit 4 isoform 2, mitochondrial | COX4I2 | 1,2 | |
Cytochrome c oxidase subunit 5A, mitochondrial | COX5A | 1,2 | |
Cytochrome c oxidase subunit 6A, mitochondrial | COX6A1 | 1,2 | |
Cytochrome c oxidase subunit 6B1 | COX6B1 | 1,2 | |
Cytochrome c oxidase subunit 7B, mitochondrial | COX7B | 1,2 | |
Carnitine O-palmitoyltransferase 1, liver isoform | CPT1A | 1 | |
NADH-cytochrome b5 reductase 3 | CYB5R3 | 1 | |
Cytochrome c1, heme protein, mitochondrial | CYC1 | 1,2 | |
Mitochondrial fission 1 protein | FIS1 | 1 | |
Glutathione reductase, mitochondrial | GSR | 1 | |
3-hydroxyacyl-CoA dehydratase 2 | HSD17B10 | 1 | |
ATP synthase subunit a | MT-ATP6 | 1,2 | |
Cytochrome c oxidase subunit 3 | MT-CO3 | 1,2 | |
Nicastrin | NCSTN | 1 | |
NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 1 | NDUFA1 | 1,2 | |
NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 12 | NDUFA12 | 1,2 | |
NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 2 | NDUFA2 | 1,2 | |
NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 4 | NDUFA4 | 1,2 | |
NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 6 | NDUFA6 | 1,2 | |
NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9, mitochondrial | NDUFA9 | 1,2 | |
Acyl carrier protein, mitochondrial | NDUFAB1 | 1,2 | |
NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 10 | NDUFB10 | 1,2 | |
NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 4 | NDUFB4 | 1,2 | |
NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 6 | NDUFB6 | 1,2 | |
NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 7 | NDUFB7 | 1,2 | |
NADH-ubiquinone oxidoreductase 75 kDa subunit, mitochondrial | NDUFS1 | 1,2 | |
NADH dehydrogenase [ubiquinone] iron-sulfur protein 3, mitochondrial | NDUFS3 | 1,2 | |
NADH dehydrogenase [ubiquinone] iron-sulfur protein 6, mitochondrial | NDUFS6 | 1,2 | |
NADH dehydrogenase [ubiquinone] iron-sulfur protein 7, mitochondrial | NDUFS7 | 1,2 | |
NADH dehydrogenase [ubiquinone] iron-sulfur protein 8, mitochondrial | NDUFS8 | 1,2 | |
NADH dehydrogenase [ubiquinone] flavoprotein 2, mitochondrial | NDUFV2 | 1,2 | |
Parkinson protein 7 | PARK7 | 1 | |
Pyruvate dehydrogenase E1 component subunit alpha, somatic form, mitochondrial | PDHA1 | 1 | |
Peroxiredoxin 3 | PRDX3 | 1 | |
Peroxiredoxin 5 | PRDX5 | 1 | |
Succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial | SDHA | 1,2 | |
Succinate dehydrogenase [ubiquinone] iron-sulfur subunit, mitochondrial | SDHB | 1,2 | |
Superoxide dismutase 2, mitochondrial | SOD2 | 1 | |
Ubiquinol-cytochrome c reductase, complex III subunit X | UQCR10 | 1,2 | |
Ubiquinol-cytochrome c reductase binding protein | UQCRB | 1,2 | |
Ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1 | UQCRFS1 | 1,2 | |
Ubiquinol-cytochrome c reductase hinge protein | UQCRH | 1,2 | |
Ubiquinol-cytochrome c reductase, complex III subunit VII, 9.5kDa | UQCRQ | 1,2 | |
Voltage-dependent anion-selective channel protein 1 | VDAC1 | 1 |
As pointed out by Sanahuja and Ibarz (
The second node of interconnected skin proteins was composed of 79 proteins involved in protein ubiquitination and antigen presentation pathways with a high representation of major histocompatibility complex, proteasome subunits, ubiquitin enzymes and molecular chaperones, including calnexin, calreticulin and heat shock proteins representative of the six major HSP families based on molecular mass (small HSPs, HSP40, HSP60, HSP70, HSP90 and HSP100) with either cytoplasmic, nuclear plasma membrane or extracellular locations (Table
E3 ubiquitin-protein ligase AMFR | AMFR | 4 | |
Anaphase-promoting complex subunit 11 | ANAPC11 | 4 | |
Anaphase-promoting complex subunit 4 | ANAPC4 | 4 | |
Beta-2-microglobulin | B2M | 3,4 | |
Calreticulin | CALR | 3 | |
Calnexin | CANX | 3 | |
DnaJ homolog subfamily A member 1 | DNAJA1 | 4 | |
DnaJ homolog subfamily C member 17 | DNAJC17 | 4 | |
DnaJ homolog subfamily C member 22 | DNAJC22 | 4 | |
HLA class II histocompatibility antigen, DP beta 1 chain | HLA-DPB1 | 3 | |
H-2 class II histocompatibility antigen, A-R alpha chain | HLA-DQA1 | 3 | |
DLA class II histocompatibility antigen, DR-1 beta chain | HLA-DR1 | 3 | |
H-2 class II histocompatibility antigen, E-D alpha chain | HLA-DRA | 3 | |
HLA class II histocompatibility antigen, DRB1-4 beta chain | HLA-DRB4 | 3 | |
Heat shock protein HSP 90-alpha 1 | HSP90AA1 | 4 | |
Heat shock protein HSP 90-beta | HSP90AB1 | 4 | |
Endoplasmin (GRP-94) | HSP90B1 | 4 | |
Heat shock 70 kDa protein 4 | HSPA4 | 4 | |
78 kDa glucose-regulated protein | HSPA5 | 4 | |
Heat shock cognate 71 kDa protein | HSPA8 | 4 | |
Stress-70 protein, mitochondrial | HSPA9 | 4 | |
Heat shock protein beta-11 | HSPB11 | 4 | |
60 kDa heat shock protein, mitochondrial | HSPD1 | 4 | |
10 kDa heat shock protein, mitochondrial | HSPE1 | 4 | |
Heat shock protein 105 kDa | HSPH1 | 4 | |
Major histocompatibility complex class I-related gene protein | MR1 | 3 | |
Protein disulfide-isomerase A3 | PDIA3 | 3 | |
Proteasome subunit alpha type-1 | PSMA1 | 4 | |
Proteasome subunit alpha type-2 | PSMA2 | 4 | |
Proteasome subunit alpha type-3 | PSMA3 | 4 | |
Proteasome subunit alpha type-5 | PSMA5 | 4 | |
Proteasome subunit alpha type-6 | PSMA6 | 4 | |
Proteasome subunit alpha type-7 | PSMA7 | 4 | |
Proteasome subunit beta type-1-B | PSMB1 | 4 | |
Proteasome subunit beta type-10 | PSMB10 | 4 | |
Proteasome subunit beta type-2 | PSMB2 | 4 | |
Proteasome subunit beta type-3 | PSMB3 | 4 | |
Proteasome subunit beta type-4 (Fragment) | PSMB4 | 4 | |
Proteasome subunit beta type-5 | PSMB5 | 3,4 | |
Proteasome subunit beta type-6-B like protein | PSMB6 | 3,4 | |
Proteasome subunit beta type-9 | PSMB9 | 3,4 | |
26S protease regulatory subunit 4 | PSMC1 | 4 | |
26S protease regulatory subunit 7 | PSMC2 | 4 | |
26S protease regulatory subunit 6A | PSMC3 | 4 | |
26S protease regulatory subunit 6B | PSMC4 | 4 | |
26S protease regulatory subunit 8 | PSMC5 | 4 | |
26S protease regulatory subunit 10B | PSMC6 | 4 | |
26S proteasome non-ATPase regulatory subunit 1 | PSMD1 | 4 | |
26S proteasome non-ATPase regulatory subunit 11 | PSMD11 | 4 | |
26S proteasome non-ATPase regulatory subunit 12 | PSMD12 | 4 | |
26S proteasome non-ATPase regulatory subunit 13 | PSMD13 | 4 | |
26S proteasome non-ATPase regulatory subunit 14 | PSMD14 | 4 | |
26S proteasome non-ATPase regulatory subunit 2 | PSMD2 | 4 | |
26S proteasome non-ATPase regulatory subunit 3 | PSMD3 | 4 | |
26S proteasome non-ATPase regulatory subunit 6 | PSMD6 | 4 | |
26S proteasome non-ATPase regulatory subunit 7 | PSMD7 | 4 | |
26S proteasome non-ATPase regulatory subunit 8 | PSMD8 | 4 | |
Proteasome activator complex subunit 1 | PSME1 | 4 | |
Proteasome activator complex subunit 2 | PSME2 | 4 | |
S-phase kinase-associated protein 1 | SKP1 | 4 | |
Antigen peptide transporter 1 | TAP1 | 3,4 | |
Antigen peptide transporter 2 | TAP2 | 3,4 | |
Tapasin | TAPBP | 3 | |
Transcription elongation factor B polypeptide 1 | TCEB1 | 4 | |
Transcription elongation factor B polypeptide 2 | TCEB2 | 4 | |
Thimet oligopeptidase | THOP1 | 4 | |
Ubiquitin-like modifier-activating enzyme 1 | UBA1 | 4 | |
Ubiquitin-conjugating enzyme E2 D2 | UBE2D2 | 4 | |
Ubiquitin-conjugating enzyme E2 D3 | UBE2D3 | 4 | |
Ubiquitin-conjugating enzyme E2 N | UBE2N | 4 | |
Ubiquitin-conjugating enzyme E2 variant 1C | UBE2V1 | 4 | |
Ubiquitin-protein ligase E3A | UBE3A | 4 | |
Ubiquitin carboxyl-terminal hydrolase isozyme L1 | UCHL1 | 4 | |
Ubiquitin carboxyl-terminal hydrolase isozyme L3 | UCHL3 | 4 | |
Ubiquitin carboxyl-terminal hydrolase 14 | USP14 | 4 | |
Ubiquitin carboxyl-terminal hydrolase 22 | USP22 | 4 | |
Ubiquitin carboxyl-terminal hydrolase 37 | USP37 | 4 | |
Ubiquitin carboxyl-terminal hydrolase 8 | USP8 | 4 | |
Probable ubiquitin carboxyl-terminal hydrolase FAF-X | USP9X | 4 |
The third cluster was the most populated one with 257 proteins in 13 interconnected canonical pathways (Table
Alpha-2-macroglobulin | A2M | 6 | |
Actin, cytoplasmic 1 | ACTB | 5,7,9,10,12,14,15,16,17 | |
Actin, alpha cardiac | ACTC1 | 5,7,9,10,12,14,15,16,17 | |
Actin, cytoplasmic 2 | ACTG1 | 5,7,9,10,14,15,16,17 | |
Alpha-actinin-3 | ACTN3 | 5,9,10,16 | |
Alpha-actinin-4 | ACTN4 | 5,9,10,16 | |
Actin-related protein 2-A | ACTR2 | 5,7,9,10,12,14,15,16,17 | |
Actin-related protein 3 | ACTR3 | 5,7,9,10,12,14,15,16,17 | |
Protein argonaute-2 | AGO2 | 8,13 | |
Angiotensinogen | AGT | 6 | |
Alpha-2-HS-glycoprotein | AHSG | 6 | |
Protein AMBP | AMBP | 6 | |
AP-1 complex subunit beta-1 | AP1B1 | 7 | |
AP-2 complex subunit beta | AP2B1 | 7 | |
AP-2 complex subunit mu-1-A | AP2M1 | 7 | |
Serum amyloid P-component | APCS | 6 | |
Apolipoprotein A-I | APOA1 | 6,7 | |
Apolipoprotein B-100 | APOB | 7 | |
Apolipoprotein Eb | APOE | 7 | |
ADP-ribosylation factor 1-like 2 | ARF1 | 10 | |
ADP-ribosylation factor 4 | ARF4 | 10 | |
ADP-ribosylation factor 6 | ARF6 | 7,10,16 | |
Rho GDP-dissociation inhibitor 1 | ARHGDIA | 12,15 | |
Rho guanine nucleotide exchange factor 5 | ARHGEF5 | 15,17 | |
Actin-related protein 2/3 complex subunit 1A | ARPC1A | 5,7,9,10,12,14,15,16,17 | |
Actin-related protein 2/3 complex subunit 1B | ARPC1B | 5,7,9,10,12,14,15,16,17 | |
Actin-related protein 2/3 complex subunit 2 | ARPC2 | 5,7,9,10,12,14,15,16,17 | |
Actin-related protein 2/3 complex subunit 3 | ARPC3 | 5,7,9,10,12,14,15,16,17 | |
Actin-related protein 2/3 complex subunit 4 | ARPC4 | 5,7,9,10,12,14,15,16,17 | |
Actin-related protein 2/3 complex subunit 5 | ARPC5 | 5,7,9,10,12,14,15,16,17 | |
Arf-GAP with SH3 domain, ANK repeat and PH domain-containing protein 1 | ASAP1 | 10 | |
Complement C2 | C2 | 6 | |
Complement C3 | C3 | 6 | |
Complement C5 | C5 | 6 | |
Complement component C9 | C9 | 6 | |
Calpain-1 catalytic subunit | CAPN1 | 10 | |
Calpain-5 | CAPN5 | 10 | |
Calpain-8 | CAPN8 | 10 | |
Calpain small subunit 1 | CAPNS1 | 10 | |
CD2-associated protein | CD2AP | 7 | |
Cdc42 effector protein 2 | CDC42EP2 | 14,17 | |
Cadherin-1 | CDH1 | 9,15,16,17 | |
Cadherin-2 | CDH2 | 9,15,17 | |
Complement factor B | CFB | 6 | |
Cofilin-2 | CFL2 | 5,14,15,17 | |
Calcium-binding protein p22 | CHP1 | 7 | |
CAP-Gly domain-containing linker protein 1 | CLIP1 | 9,16,17 | |
Ceruloplasmin | CP | 6 | |
Casein kinase II subunit alpha | CSNK2A1 | 7 | |
Casein kinase II subunit beta | CSNK2B | 7 | |
Catenin alpha-2 | CTNNA2 | 9,16 | |
Catenin delta-1 | CTNND1 | 9,16 | |
Cytoplasmic FMR1-interacting protein 2 | CYFIP2 | 5 | |
Dynamin-2 | DNM2 | 7,16 | |
Eukaryotic translation initiation factor 1A, X-chromosomal | EIF1AX | 8,13 | |
Eukaryotic translation initiation factor 2 subunit 1 | EIF2S1 | 8,13 | |
Eukaryotic translation initiation factor 2 subunit 2 | EIF2S2 | 8,13 | |
Eukaryotic translation initiation factor 2 subunit 3 | EIF2S3 | 8,13 | |
Eukaryotic translation initiation factor 3 subunit A | EIF3A | 8,11,13 | |
Eukaryotic translation initiation factor 3 subunit B | EIF3B | 8,11,13 | |
Eukaryotic translation initiation factor 3 subunit E | EIF3E | 8,11,13 | |
Eukaryotic translation initiation factor 3 subunit F | EIF3F | 8,11,13 | |
Eukaryotic translation initiation factor 3 subunit G | EIF3G | 8,11,13 | |
Eukaryotic translation initiation factor 3 subunit I | EIF3I | 8,11,13 | |
Eukaryotic translation initiation factor 3 subunit K | EIF3K | 8,11,13 | |
Eukaryotic translation initiation factor 3 subunit L | EIF3L | 8,11,13 | |
Eukaryotic translation initiation factor 3 subunit M | EIF3M | 8,11,13 | |
Eukaryotic initiation factor 4A-I | EIF4A1 | 8,11,13 | |
Eukaryotic initiation factor 4A-II | EIF4A2 | 8,11,13 | |
Eukaryotic initiation factor 4A-III | EIF4A3 | 8,11,13 | |
Eukaryotic translation initiation factor 4E | EIF4E | 8,11,13 | |
Eukaryotic translation initiation factor 4E-binding protein 2 | EIF4EBP2 | 13 | |
Eukaryotic translation initiation factor 4 gamma 1 | EIF4G1 | 8,11,13 | |
Prothrombin | F2 | 5,6,7 | |
Fibrinogen beta chain | FGB | 6 | |
Fibrinogen gamma chain | FGG | 6 | |
Formin-binding protein 1 homolog | FNBP1 | 10,11,12,15,17 | |
Ferritin light chain, oocyte isoform | FTL | 6 | |
Rab GDP dissociation inhibitor alpha | GDI1 | 15 | |
Rab GDP dissociation inhibitor beta | GDI2 | 15 | |
Guanine nucleotide-binding protein subunit alpha-11 | GNA11 | 15,17 | |
Guanine nucleotide-binding protein subunit alpha-13 | GNA13 | 5,14,15,17 | |
Guanine nucleotide-binding protein G(i) subunit alpha-1 | GNAI1 | 15,17 | |
Guanine nucleotide-binding protein G(k) subunit alpha | GNAI3 | 15,17 | |
Guanine nucleotide-binding protein G(t) subunit alpha-2 | GNAT2 | 15,17 | |
Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 | GNB1 | 15,17 | |
Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-2 | GNB2 | 15,17 | |
Guanine nucleotide-binding protein subunit beta-2-like 1 | GNB2L1 | 15,17 | |
Guanine nucleotide-binding protein subunit beta-4 | GNB4 | 15,17 | |
Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-12 | GNG12 | 1,15,17 | |
Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 | GNG2 | 15,17 | |
Growth factor receptor-bound protein 2 | GRB2 | 5,6,7,8,10,13 | |
Gelsolin | GSN | 5,12 | |
Heme oxygenase | HMOX1 | 6,11 | |
Heterogeneous nuclear ribonucleoprotein K | HNRNPK | 6 | |
Hemopexin | HPX | 6 | |
Heat shock cognate 71 kDa protein | HSPA8 | 7 | |
Inhibitor of nuclear factor kappa-B kinase subunit epsilon | IKBKE | 6 | |
Interleukin-6 receptor subunit alpha | IL6R | 6 | |
Ras GTPase-activating-like protein IQGAP1 | IQGAP1 | 5,9,16,17 | |
Ras GTPase-activating-like protein IQGAP2 | IQGAP2 | 5 | |
Inter-alpha-trypsin inhibitor heavy chain H2 | ITIH2 | 6 | |
Inter-alpha-trypsin inhibitor heavy chain H3 | ITIH3 | 6 | |
Junction plakoglobin | JUP | 9 | |
Kininogen (Fragments) | KNG1 | 5 | |
GTPase KRas | KRAS | 5,6,8,9,10,11,13 | |
Dual specificity mitogen-activated protein kinase kinase 6 | MAP2K6 | 6 | |
Mitogen-activated protein kinase 1 | MAPK1 | 5,6,8,10,11,13,17 | |
Microtubule-associated protein RP/EB family member 1 | MAPRE1 | 16 | |
Moesin | MSN | 5,14,15,17 | |
Myosin-11 | MYH11 | 5,9 | |
Myosin-6 | MYH6 | 5,9 | |
Myosin-9 | MYH9 | 5,9 | |
Myosin light chain 1, skeletal muscle isoform | MYL1 | 5,9,12,14,15,17 | |
Myosin light chain 3, skeletal muscle isoform | MYL3 | 5,9,12,14,15,17 | |
Myosin light polypeptide 6 | MYL6 | 5,9,12,14,15,17 | |
Myosin regulatory light polypeptide 9 | MYL9 | 5,9,10,12,14,15,17 | |
Myosin regulatory light chain 2, smooth muscle minor isoform | MYLPF | 5,12,14,15,17 | |
Myosin-Ie | MYO1E | 7 | |
Myosin-VI | MYO6 | 7 | |
Nuclear factor NF-kappa-B p105 subunit | NFKB1 | 6,17 | |
Ephexin-1 | NGEF | 14 | |
Nucleoside diphosphate kinase A1 | NME1 | 16 | |
Glucocorticoid receptor | NR3C1 | 6 | |
Polyadenylate-binding protein 1 | PABPC1 | 8,13 | |
Serine/threonine-protein kinase PAK 2 | PAK2 | 5,10,12,15,17 | |
3-phosphoinositide-dependent protein kinase 1 | PDPK1 | 6,8,11,13 | |
Profilin-1 | PFN1 | 5,12,14 | |
1-phosphatidylinositol-3-phosphate 5-kinase | PIKFYVE | 5,12,14,15,17 | |
1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase gamma-1 | PLCG1 | 10 | |
1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase gamma-2 | PLCG2 | 10 | |
Serine/threonine-protein phosphatase PP1-beta catalytic subunit | PPP1CB | 5,8,10,12,14 | |
Serine/threonine-protein phosphatase PP1-gamma catalytic subunit | PPP1CC | 8 | |
Serine/threonine-protein phosphatase 2A catalytic subunit alpha isoform | PPP2CA | 11,13 | |
Serine/threonine-protein phosphatase 2A catalytic subunit beta isoform | PPP2CB | 11,13 | |
Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A beta isoform | PPP2R1B | 11,13 | |
Serine/threonine-protein phosphatase 2B catalytic subunit alpha isoform | PPP3CA | 7 | |
Calcineurin subunit B type 1 | PPP3R1 | 7 | |
Protein kinase C beta type | PRKCB | 11 | |
Ras-related protein Rab-11A | RAB11A | 7 | |
Ras-related protein Rab-11B | RAB11B | 7 | |
Ras-related protein Rab-4B | RAB4B | 7 | |
Ras-related protein Rab-5A | RAB5A | 7,16 | |
Ras-related protein Rab-5B | RAB5B | 7,16 | |
Ras-related protein Rab-5C | RAB5C | 7,16 | |
Ras-related protein Rab7 | RAB7A | 7,16 | |
Ras-related C3 botulinum toxin substrate 1 | RAC1 | 5,7,9,10,11,12,15,17 | |
Ras-related C3 botulinum toxin substrate 2 | RAC2 | 5,10,12 | |
Ras-related protein Ral-B | RALB | 10 | |
Ras-related protein Rap-1b | RAP1B | 9,1 | |
Radixin | RDX | 5,14,15,17 | |
Transforming protein RhoA | RHOA | 5,9,10,11,12,14,15,17 | |
Rho-related GTP-binding protein RhoC | RHOC | 10,11,12,15,17 | |
Rho-related GTP-binding protein RhoG | RHOG | 10,11,12,15,17 | |
60S ribosomal protein L10 | RPL10 | 8 | |
60S ribosomal protein L10a | RPL10A | 8 | |
60S ribosomal protein L11 | RPL11 | 8 | |
60S ribosomal protein L12 | RPL12 | 8 | |
60S ribosomal protein L13 | RPL13 | 8 | |
60S ribosomal protein L13a | RPL13A | 8 | |
60S ribosomal protein L14 | RPL14 | 8 | |
60S ribosomal protein L15 | RPL15 | 8 | |
60S ribosomal protein L17 | RPL17 | 8 | |
60S ribosomal protein L18 | RPL18 | 8 | |
60S ribosomal protein L18a | RPL18A | 8 | |
60S ribosomal protein L19 | RPL19 | 8 | |
60S ribosomal protein L21 | RPL21 | 8 | |
60S ribosomal protein L22 | RPL22 | 8 | |
60S ribosomal protein L22-like 1 | RPL22L1 | 8 | |
60S ribosomal protein L23 | RPL23 | 8 | |
60S ribosomal protein L23a | RPL23A | 8 | |
60S ribosomal protein L24 | RPL24 | 8 | |
60S ribosomal protein L26 | RPL26 | 8 | |
60S ribosomal protein L27 | RPL27 | 8 | |
60S ribosomal protein L27a | RPL27A | 8 | |
60S ribosomal protein L28 | RPL28 | 8 | |
60S ribosomal protein L3 | RPL3 | 8 | |
60S ribosomal protein L30 | RPL30 | 8 | |
60S ribosomal protein L31 | RPL31 | 8 | |
60S ribosomal protein L34 | RPL34 | 8 | |
60S ribosomal protein L35 | RPL35 | 8 | |
60S ribosomal protein L35a | RPL35A | 8 | |
60S ribosomal protein L36 | RPL36 | 8 | |
60S ribosomal protein L36a | Rpl36a | 8 | |
60S ribosomal protein L37 | RPL37 | 8 | |
60S ribosomal protein L38 | RPL38 | 8 | |
60S ribosomal protein L4 | RPL4 | 8 | |
60S ribosomal protein L5 | RPL5 | 8 | |
60S ribosomal protein L6 | RPL6 | 8 | |
60S ribosomal protein L7 | RPL7 | 8 | |
60S ribosomal protein L7a | RPL7A | 8 | |
60S ribosomal protein L8 | RPL8 | 8 | |
60S ribosomal protein L9 | RPL9 | 8 | |
60S acidic ribosomal protein P0 | RPLP0 | 8 | |
60S acidic ribosomal protein P1 | RPLP1 | 8 | |
60S acidic ribosomal protein P2 | RPLP2 | 8 | |
40S ribosomal protein S10 | RPS10 | 8,11,13 | |
40S ribosomal protein S11 | RPS11 | 8,11,13 | |
40S ribosomal protein S12 | RPS12 | 8,11,13 | |
40S ribosomal protein S14 | RPS14 | 8,11,13 | |
40S ribosomal protein S15 | RPS15 | 8,11,13 | |
40S ribosomal protein S15a | RPS15A | 8,11,13 | |
40S ribosomal protein S16 | RPS16 | 8,11,13 | |
40S ribosomal protein S17 | RPS17 | 8,11,13 | |
40S ribosomal protein S18 | RPS18 | 8,11,13 | |
40S ribosomal protein S19 | RPS19 | 8,11,13 | |
40S ribosomal protein S2 | RPS2 | 8,11,13 | |
40S ribosomal protein S20 | RPS20 | 8,11,13 | |
40S ribosomal protein S21 | RPS21 | 8,11,13 | |
40S ribosomal protein S23 | RPS23 | 8,11,13 | |
40S ribosomal protein S24 | RPS24 | 8,11,13 | |
40S ribosomal protein S25 | RPS25 | 8,11,13 | |
40S ribosomal protein S26 | RPS26 | 8,11,13 | |
Ubiquitin-40S ribosomal protein S27a | RPS27A | 8,11,13 | |
40S ribosomal protein S28 | RPS28 | 8,11,13 | |
40S ribosomal protein S29 | RPS29 | 8,11,13 | |
40S ribosomal protein S3 | RPS3 | 8,11,13 | |
40S ribosomal protein S4 | RPS4 | 8,11,13 | |
40S ribosomal protein S5 | RPS5 | 8,11,13 | |
40S ribosomal protein S6 | RPS6 | 8,11,13 | |
Ribosomal protein S6 kinase 2 alpha | RPS6KA1 | 11 | |
Ribosomal protein S6 kinase alpha-3 | RPS6KA3 | 11 | |
40S ribosomal protein S7 | RPS7 | 8,11,13 | |
40S ribosomal protein S8 | RPS8 | 8,11,13 | |
40S ribosomal protein S9 | RPS9 | 8,11,13 | |
40S ribosomal protein SA | RPSA | 8,11,13 | |
Ras-related protein R-Ras | RRAS | 5,6,8,9,10,11,13 | |
Ras-related protein R-Ras2 | RRAS2 | 5,6,8,9,10,11,13 | |
Septin-10 | SEPT10 | 14,17 | |
Septin-2 | SEPT2 | 14,17 | |
Septin-6 | SEPT6 | 14,17 | |
Septin-7 | SEPT7 | 14,17 | |
Septin-8-A | SEPT8 | 14,17 | |
Serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 1 | SERPINA1 | 6,7 | |
Serpin peptidase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor), member 2 | SERPINF2 | 6 | |
Endophilin-A1 | SH3GL2 | 7 | |
SHC-transforming protein 1 | SHC1 | 5,6,8,10,13 | |
Superoxide dismutase [Mn], mitochondrial | SOD2 | 6 | |
Protein phosphatase Slingshot homolog | SSH1 | 5 | |
Signal transducer and activator of transcription 3 | STAT3 | 6 | |
Transcription factor 7-like 2 | TCF7L2 | 9 | |
Transferrin | TF | 6,7 | |
Talin-1 | TLN1 | 5,1 | |
Tumor necrosis factor receptor superfamily member 1B | TNFRSF1B | 6 | |
Activated CDC42 kinase 1 | TNK2 | 10 | |
Tumor necrosis factor receptor type 1-associated DEATH domain protein | TRADD | 6 | |
Titin | TTN | 5,10,14 | |
Transthyretin | TTR | 6 | |
Tubulin alpha-1A chain | TUBA1A | 9,16 | |
Tubulin alpha-4A chain | TUBA4A | 9,16 | |
Tubulin beta chain | TUBB | 9,16 | |
Tubulin beta-1 chain | TUBB1 | 9,16 | |
Tubulin beta-2C chain | TUBB4B | 9,16 | |
Ubiquitin-60S ribosomal protein L40 | UBA52 | 8 | |
Probable ubiquitin carboxyl-terminal hydrolase FAF-X | USP9X | 7 | |
Vinculin | VCL | 5,9,10,16 | |
Vimentin | VIM | 17 |
Principal components analysis from image processing of 2-DE of the mucus proteins from control CTRL vs. M-ST did not clearly separate individuals from both groups (Figure
743 | Elongation factor 2 | 0.026 | 0.71 | APLMVYISK/CDLLYEGPPDDEAAMGIK/EGVLCEENMR/FSVSPVVR/GGG |
|
815 | Keratin type II cytoskeletal 8 | 0.014 | 2.71 | ANLEAQIAEAEER/AQYEDIANR/FASFIDKVR/IRDLEDALQR/NLDMDSIVAEVK | |
1,321 | Keratin type II cytoskeletal 8 | 0.024 | 1.78 | DTSVIVEMDNSR/FASFIDKVR/FLEQQNK/IRDLEDALQR/LALDIEIATYRK/NM |
|
1,549 | Actin, cytoplasmic 1 | 0.019 | 0.71 | AGFAGDDAPR/AVFPSIVGRPR/DLTDYLMK/IIAPPERK/LAPSTMKIK/SYELP |
|
1,816 | Actin, cytoplasmic 1 | 0.026 | 0.63 | DLYANTVLSGGTTMYPGIADR/GYSFTTTAER/SYELPDGQVITIGNER/VAPEE |
|
2,181 | Cytochrome c1, heme protein mitochondrial | 0.018 | 1.64 | LSDYFPKPYPNPESAR/NLVGVSHTEAEVK |
Clear evidence for the prominent mechanical function of keratins comes from multiple human diseases and murine knockouts. However, distinct keratins emerge as highly dynamic scaffolds contributing to cell size determination, translation control, proliferation, malignant transformation and various stress responses (Magin et al.,
A high resolution mass spectrometry-based proteomic approach was able to identify 2,062 proteins in the skin mucus of gilthead bream after matching in a homologous protein database. Three major clusters with more than 350 proteins were retained after filtering by canonical pathway overlapping. Among them, proteins of oxidative phosphorylation, mitochondrial dysfunction, protein ubiquitination, immune response, epithelial remodeling, and cellular assembly were highly represented. This was reinforced by the observation that major changes related to the abundance of cytokeratin 8 in the skin mucus of stressed fish under our experimental model of chronic stress were found by means of 2-DE methodology and confirmed by immunoblotting. All this information will be useful in developing more targeted approaches that address specific changes in the skin mucus proteome of farmed fish, with special emphasis on markers of skin epithelial cell turnover.
JP, RO, and AS conceived and designed the study. OF supervised animal handling and sampling. JP, GT, PS, SR, and JC performed protein identification and functional characterization of mucus proteome and stress-regulated proteins. JP and PS conducted Western blot analysis. JP, GT, AS, and JC wrote the manuscript. All authors read and approved the final manuscript.
This study was funded by the European Union (AQUAEXCEL, FP7/2007/2013; grant agreement No. 262336, Aquaculture infrastructures for excellence in European fish research) project. The views expressed in this work are the sole responsibility of the authors and do not necessarily reflect the views of the European Commission. Additional funding was obtained from the Spanish Ministerio de Economía y Competitividad (MI2-Fish, AGL2013-48560) and from Generalitat Valenciana (PROMETEO FASE II-2014/085). Proteomics study was done at Proteomics laboratory of University of Valencia, Spain (SCSIE). This laboratory is a member of Proteored, PRB2-ISCIII and is supported by grant PT13/0001, of the PE I+D+i 2013–2016, funded by Instituto de Salud Carlos III and Fondo Europeo de Desarrollo Regional (FEDER).
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The Supplementary Material for this article can be found online at: