Med. Weter. 2013, 69 (10) 612
Praca oryginalna Original paper
The waters of South Africa and the Asian countries bordering the Mediterranean Sea are the natural habitat of the African catfish (Clarias gariepinus). However, the farming of this fish species in Europe began in the early 1980s (12). Since Clarias gariepinus is an omni-vore, it tolerates a variable diet containing both plant and animal foods. Its other attributes, such as a high growth-rate, resistance to changing environmental conditions, and the fact that is not easily susceptible to diseases, make the African catfish a good candidate for aquaculture (1).
Proteomics can be defined as the analysis of the entire set of proteins expressed in a cell, tissue type, or biological fluid at a defined time and under specific conditions (5). Proteins perform many cellular func-tions, and in contradistinction to the genome, the pro-teome changes dynamically in response to different factors (e.g. physiological, patophysiological, environ-mental) (4). A variety of proteomic tools have been developed, but one of the most frequently used is the combination of two-dimnesional electophoresis (2-DE) and mass spectrometry (MS).
Recently, many attempts have been made to imple-ment proteomic techniques in order to gain insight into the catfish physiology (8) and to assess the influence of environmental contaminants (11) or bacterial infec-tions (2). For example, Pierrard et al. (11), using 2D-DIGE (two-dimensional difference gel electropho-resis) and LC-MS/MS (liquid chromatography-tandem mass spectrometry), detected 116 differently expres-sed proteins in peripheral blood mononuclear cells (PBMC) from Asian catfish (Pangasianodon hypo-phthalmus) exposed to malachite green (an antiseptic chemical). The authors divided the above-mentioned proteins into the following functional classes: energetic metabolism (e.g. enolase 1, glyceraldehyde-3-phosphate dehydrogenase), cytoskeleton proteins (e.g. tropo-myosin 1, tubulin), molecular chaperones (e.g. heat shock cognate protein 70 (HSC 70), heat shock protein 60 (HSP 60)), ubiquitin proteasome system (e.g. pro-teasome subunit alpha type, 26S protease regulatory subunit 6A), nucleic acid binding (e.g. pre-mRNAspli-cing factor SPF27).
Plasma proteomic profiling of African catfish
(Clarias gariepinus)
AGNIESZKA HEROSIMCZYK, MA£GORZATA O¯GO, ADAM LEPCZYÑSKI, ANDRZEJ CIECHANOWICZ, ADELINE COLUSSI*, PAULINA SENIUTA,
WIES£AW FRANCISZEK SKRZYPCZAK
Department of Physiology, Cytobiology and Proteomics, Faculty of Biotechnology and Animal Husbandry, West Pomeranian University of Technology, Doktora Judyma Str. 6, 71-466 Szczecin, Poland
*Department of Biology, Faculty of Natural Sciences and Mathematics, Swiss Federal Institute of Technology ETH Zurich, Wolfgang-Pauli Str. 27, CH-8093 Zurich, Switzerland
Herosimczyk A., O¿go M., Lepczyñski A., Ciechanowicz A., Colussi A., Seniuta P., Skrzypczak W. F.
Plasma proteomic profiling of African catfish (Clarias gariepinus) Summary
This study was aimed at profiling plasma proteins of healthy 8-month-old African catfish. Plasma proteins within the isoelectric point ranging from 3.0 to 10.0 were separated by high resolution two-dimensional electrophoresis (2-DE). In total, 8 different gene products were identified with a matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometer. These included carrier proteins (fatty acid-binding protein 1, metallothionein, mitoferrin-2), a structural protein (vimentin), proteins involved in the regulation of transcription (thyroid hormone receptor alpha A, endothelial differentiation-related factor 1 homolog), and others (B-cell lymphoma 6 protein homolog, FGFR1 oncogene partner 2 homolog). This is the first study attempting to map the plasma proteome of Clarias gariepinus. Owing to the lack of information concerning catfish protein sequences in the relevant databases, protein spots were identified by matching peptide data to interspecies homology. The results of this preliminary study may encourage other authors to undertake further research on the plasma proteome of the African catfish.
Med. Weter. 2013, 69 (10) 613 However, proteomic studies of catfish blood plasma/
serum are sparse. Previously, Osman et al. (10) used SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) to compare the influence of water pollution from the El-Madapigh canal and the river Nile at Assiut on serum protein band changes in African catfish. The authors reported a substantial reduction in the number of protein bands in fish collected from the El-Madapigh canal compared to those in fish col-lected from the Nile. Osman et al. (10) suggest that these changes may be attributed to reduced protein synthesis in response to increased water pollution. The majority of the fish species used in plasma/serum pro-teomic studied to date have been those of great impor-tance in global aquaculture production. These fish species include Atlantic salmon (Salmo salar) (17), Gilthead sea bream (Sparus aurata) (7), and Rainbow trout (Oncorhynchus mykiss) (3, 13). Moreover, it is well known that there are interspecies differences in blood plasma protein composition. Previously, De Smet et al. (16) compared the electrophoretic patterns of plasma proteins in Brown trout (Salmo trutta) and Common carp (Cyprinus carpio). According to the authors, these three 2-D profiles differ considerably, especially in protein composition and physicochemical parameters (16).
Up to date, no proteomic studies of blood plasma proteins in African catfish have been reported. There-fore, our research was aimed at establishing plasma protein composition characteristic of healthy adult Clarias gariepinus.
Material and methods
Fish. A total of five healthy 8-month-old male African catfish (Clarias gariepinus) were used. The fish were kept in an artificial breeding pool in rain water at 24-27°C without access to light throughout the whole experiment.
During the first two weeks of life, alevins were fed crustacean larvae (Artemia salina) every two hours at 10% of the body weight. From the third week onwards, the fish were fed CatCo CRUMBLE Excellent (Coppens Inter-national) every two hours with the addition of â-glucans. Subsequently, from the second month until the end of the experimental period, they were fed CatCo SELECT-13 EF twice a day at 1% of the body weight. The use and handling of the animals in this experiment was approved by the Local Ethical Committee (no. 11/2012 of 23.05.2012).
Sample collection. The fish were anaesthetized (benzo-caine, Sigma) prior to blood sampling. Blood samples were collected directly from the heart ventricle into tubes pre-coated with K3EDTA. The samples were subsequently centrifuged at 3000 rpm for 10 minutes at 4°C. Plasma was then stored at 80°C until analysis.
Two-dimensional electrophoresis (2-DE). Before analysis, plasma samples were completely thawed at 0°C and processed by the protein equalizer technology with a ProteoMiner Protein enrichment Large-Capacity kit (Bio-Rad) to decrease the concentration of abundant pro-teins and to increase the concentration of scarce propro-teins.
The samples were then precipitated with four volumes of cold acetone (80°C) for two hours, and thus achieved protein pellets were dissolved in the lysis buffer (5 M urea, 2 M thiourea, 4% w/v CHAPS, 40 mM Tris, 0.2% w/v 3-10 ampholytes, 2 mM TBP). Total proteins (1.0 mg) were resoluabilized with rehydratation solution (9 M urea, 4% w/v CHAPS, 100 mM DTT, 0.2% w/v 3-10 ampholytes) to a total volume of 350 µl and applied to 3-10, 17 cm L (linear) ReadyStrip IPG Strips (Bio-Rad). The isoelec-trofocusing (IEF) was run (Protean® IEF Cell, Bio-Rad) in total 75 000 Vh. After IEF, the IPG strips were reduced with DTT in equilibration buffer (6 M urea, 0.5 M Tris/ HCl, pH 6.8, 2% w/v SDS, 305% w/v glycerol and 1% w/v DTT) for 15 minutes and then alkylated with iodoacetamide (2.5% w/v) for 20 minutes at ambient temperature. The second dimension was performed (Protean Plus Dodeca Cell electrophoretic chamber, Bio-Rad) on 12% SDS polyacrylamide gels (20 × 25 cm) at 40 V for 1 hour and subsequently at 120 V for 16 hours at 10°C. After 2-DE separation, the gels were stained for 72 hours with colloidal Coomassie Brilliant Blue G-250.
Image analysis. All gels were scanned with a GS-800 calibrated densitometer (Bio-Rad). The 2-D image compu-ter analysis was performed by means of PDQuest Analysis software version 8.0. Advanced (Bio-Rad). Analytical pro-cedures performed on each gel included spot background substraction, spot detection, and matching. The parameters used for inter-gel comparison were the size of the faintest spot, the smallest spot, and the size of the largest spot. The selected master gel represented the highest number of spots and the best protein pattern. Normalization of each indivi-dual spot was performed using the local regression model (LOESS).
MALDI-TOF MS. Protein spots were manually excised from Coomassie-stained gels and decolorized by washing with buffer (25 mM NH4HCO3 in 5% v/v ACN), followed by two washes with a solution containing 25 mM NH4HCO3 in 50% v/v ACN. The excised gel pieces were subsequently dehydrated with 100% ACN and vacuum dried (Concen-trator 5301, Eppendorf). The samples were then incubated with trypsin (20 µl/spot of 12.5 µg/ml in 25 mM NH4HCO3; Sigma-Aldrich, St. Louis, MO) for 16 h at 37°C. After incubation, the obtained peptides were extracted with 100% ACN, combined with an equal volume of matrix solution (5 mg/ml CHCA, 0,1% v/v TFA, 50% v/v ACN), and loaded onto a MALDI-MSP AnchorChip 600/96 plate
(Bruker Daltonics, Germany). For calibrating, mass scale Peptide mass standard II (Bruker Daltonics, Germany within mass range 700-4000 Da) was used. Mass spectra were acquired in the positive-ion reflector mode with a Micro-flex MALDI TOF mass spectrometer (Bruker Daltonics,
Germany). The PMF (peptide mass fingerprinting) data were compared with fish and mammalian databases (SWISS--PROT; http://us.expasy.org/uniprot/) by means of the MASCOT search engine (http://www.matrixscience.com/). Search parameters were trypsin as an enzyme, carbamido-methylation of cystein as fixed modification, methionine oxidation as variable modification, mass tolerance to 150 ppm, a maximum of one missed cleavage site. On this basis, the achieved results were further validated by MASCOT
Med. Weter. 2013, 69 (10) 614
score (only statistically relevant hits were applied) and sequence coverage.
Results and discussion Fig. 1 shows a representative 2-D gel of African catfish blood plasma proteins, on which protein spots in a range of pH 3-10 were detected. The plasma protein pattern was obtained by loading samples containing 1.0 mg of proteins and then stained with Coomassie. The number of spots presented on the gels varied between the five gels from 150 to 180. In order to measure the reproducibility of the gels, PDQuest 8.0 Advanced software was used. By means of this pro-gram the following analyses were performed: spot background substraction, spot detection, matching, and spot volume nor-malization (LOESS). As a result, we showed that spot locations and stain intensities on 2-D gels from different samples were similar. On this basis, 96 protein spots were considered as reproducible (pre-sented on each gel analysed).
Ninety-six protein spots were excised for identification by pep-tide mass fingerprinting (PMF)
using MALDI-TOF MS. The reproducibility of these 96 protein spots was based on the calculation of inter-sample variability. The analysis showed an average coefficient of variation (CV) of 47.28%.
In total, 13 spots were successfully identified, which cor-respond to 9 distinct gene products. Deta-iled information on the identified proteins is presented in Tab. 1. Among the 83 spots that remained uniden-tified, 38 were not named because of a low protein content. The failed identifica-tion of the remaining 45 protein spots was attributed to the limi-ted number of entries in the available pro-tein databases for fish species. Owing to the
lack of information on catfish protein sequences in the databases, the protein spots were identified by matching peptide data to interspecies homology (Tab. 1).
Fig. 1. 2D protein map of blood plasma proteins of eight month old male Clarias gariepinus. Two dimensional gel presents coomassie stained plasma protein pattern (1.0 mg of proteins, 3-10 L IPG, 12% SDS-PAGE). Spot numbers correspond to those in Tab. 1.
Tab. 1. Summary of the identified proteins by MALDI-TOF MS
t o p S Proteinname A(cScweissssi-oPnrono)t. Scoevqeureangcee Species 1 Fattyacid-bindingprotein1 O42494 61% Takfiugurubirpes 2 Metallothionein P25128 31% Noemacheliusbarbatulus 3 Vimenitn P48674 28% Oncorhynchusmykiss 4 27% 5 B-celllymphoma6proteinhomolog Q5ZM39 23% Gallusgallus 6 22% 7 26% 8 Mtioferirn-2 Q7T292 18% Danioreiro 9 26% 0 1 FGFR1oncogenepatrner2homolog Q7T338 38% Danioreiro 1 1 28% 2 1 Endotheilaldifferenitaitonr-elatedfactor1homolog Q6PBY3 47% Danioreiro 3 1 ThyroidhormonereceptoralphaA Q91241 20% Parailchthysoilvaceus
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Explanation: a) If not stated in the available literature, according to SWISS-PROT and NCBI databases e m a n n i e t o r P Funcitonsa) 1 n i e t o r p g n i d n i b -d i c a y tt a F ) P B A F ( Ith'tsataFhAiBghPlsyrcoolensseinrvceludd,ecyfatottpylaascmidicupptraokteei,nrttahanstpboinrd,talonndg-mcheatainbofailsttmy.acidsandotherhydrophobic ilgands. tIisthought ) T M ( n i e n o i h t o ll a t e M Ipr'tostceyins.teitInpela(aybsovuatir3o0u%sfoufntcheitontostainlcrleusdidinugesm)aainntdenhaenacveyomfhetoamlse(o7s-t1a0siesqoufivthaeleenstssepneritamloo,lilgdoeepleemndeinntgsoiznncthaenmdectoap)lpeirc,rh .) 4 1 ( s n o it i d n o c s s e rt s m o rf n o it c e t o r p d n a y r u c r e m d n a m u i m d a c e k il s l a t e m c i x o t f o s t c e ff e l u f m r a h e h t t s n i a g a e s n e f e d 2 -n ir r e f o ti M aThndisFper-oStecinlusisterresapsosnesmibblleyifnornmonti-oecrhytohnrodidiralceriollsn(a1c5cu.)mulaiton.Probablyrequriedforhemesynthesisofhemoproteins n it n e m i V iVsimaettancithnedistcolathsesI-nuIIcinleteursm,eednidaoteplfalisammicenrtefitocuunldumin,vaandiromustioncohno-nedptiirhae,eilatilhceerlllas,teersaplleycoiarllteyrmmiensaenllych(6ym.)alcells.Vimenitn r o t p e c e r e n o m r o h d i o r y h T A a h p l a Hpliagyhaafreifngutiylarteocreyprtoolrefoinrthrteiiogdrootwhythronaninder.eTphryordouidcithoonrmofosnoemseareifsihmsppoectraiensti(n9e.)alrydevelopmentandmetamorphosisand -n o it a it n e r e ff i d l a il e h t o d n E g o l o m o h 1 r o t c a f d e t a l e r- Probable rtanscirpitonalcoacitvato.r n i e t o r p 6 a m o h p m y l ll e c -B g o l o m o h iTmrapnostrcairnptritoonlealinrelpyrmesphsoormraegqeunrieesdisf.orgerminalcenterformaitonandanitbodyafifntiymaturaiton.Probablyplaysan 2 r e n tr a p e n e g o c n o 1 R F G F g o l o m o h Maybeinvolvedinwoundheailngpathway.
Tab. 2. Detailed functions of the identified proteins
The proteins identified in the present study were sorted into six groups according to their role in various biological functions: carrier proteins (fatty acid-binding protein 1, metallothionein, mitoferrin-2), a structural protein (vimentin), proteins involved in the regulation of transcription (thyroid hormone receptor alpha A, endothelial differentiation-related factor 1 homolog), and others (B-cell lymphoma 6 protein homolog, FGFR1 oncogene partner 2 homolog). Detailed func-tions of the identified proteins are listed in Tab. 2.
This is the first study attempting to map the blood plasma proteome of African catfish (Clarias gariepi-nus). Unfortunately, a substantial number of proteins remained unidentified mainly because of a limited number of entries in the available protein databases for fish species. The map presented here may therefore be considered as preliminary. It awaits further refine-ments through the implementation of much more sophi-sticated proteomic tools (e.g. LC-MS/MS) in order to characterize the complete plasma proteome of Clarias gariepinus. Limited, as they are, the data demonstrate that two-dimensional electrophoresis coupled with MALDI-TOF MS has the potential to become a power-ful tool for fish plasma protein analysis. These results may be considered as a point of reference for other scientist interested in the proteomic analysis of the African catfish.
References
1.Appelbaum S., McGeer J. C.: Effect of diet and light regime on growth and survival of African catfish (Clarias gariepinus) larvae and early juveniles. Aquacult. Nutr. 1998, 3, 157-164.
2.Booth N. J., Bilodeau-Bourgeois A.: Proteomic analysis of head kidney tissue from high and low susceptibility families of channel catfish following challenge with Edwardsiella ictaluri. Fish Shellfish Immun. 2009, 26, 193--196.
3.Brunt J., Hansen R., Jamieson D. J., Austin B.: Proteomic analysis of rainbow trout (Oncorhynchus mykiss, Walbaum) serum after administration of probiotics in diets. Vet. Immunol. Immunopathol. 2008, 121, 199-205.
4.Dowd W. W., Renshaw G. M. C., Cech J. J., Kültz D.: Compensatory proteome adjustments imply tissue-specific structural and metabolic reorganization following episodic hypoxia or anoxia in the epaulette shark (Hemiscyllium ocellatum). Physiol. Genomics 2010, 42, 93-114.
5.Herosimczyk A., Dejeans N., Sayd T., O¿go M., Skrzypczak W. F., Mazur A.: Plasma proteome analysis: 2D gels and chips. J. Physiol. Pharmacol. 2006, 57 (Supp. 7), 81-93.
6.Herrmann H., Münick M. D., Brettel M., Fouquet B., Markl J.: Vimentin in a cold-water fish, the rainbow trout: highly conserved primary structure but unique assembly properties. J. Cell Sci. 1996, 109, 569-578.
7.Isani G., Andreani G., Carpenè E., Di Molfetta S., Eletto D., Spisni E.: Effects of waterborne Cu exposure in gilthead sea bream (Sparus aurata): A proteomic approach. Fish Shellfish Immun. 2011, 31, 1051-1058. 8.Mohanty B. P., Bhattacharjee S., Das M. K.: Lens proteome map and
á-crystallin profile of the catfish Rita rita. Indian J. Biochem. Biophys. 2011, 48, 35-41.
9.Nelson E. R., Habibi H. R.: Thyroid receptor subtypes: structure and function in fish. Gen. Comp. Endocr. 2009, 161, 90-96.
10.Osman A. G. M., Al-Awadhi R. M., Harabawy A. S. A., Mahmoud U. M.: Evaluation of the use of protein electrophoresis of the African catfish Clarias gariepinus (Burchell, 1822) for biomonitoring aquatic pollution. Environ. Res. J. 2010, 4, 235-243.
11.Pierrard M. A., Kestemont P., Phuong N. T., Tran M. P., Delaive E., Thezenas M. L., Dieu M., Raes M., Silvestre F.: Proteomic analysis of blood cells in fish exposed to chemotherapeutics: Evidence for long term effects. J. Proteomics 2012, 75, 2454-2467.
12.Polak-Juszczak L.: Chemical characteristics of fishes new to the Polish market. Acta Sci. Pol., Piscaria 2007, 6, 23-32.
13.Russell S., Hayes M. A., Simko E., Lumsden J. S.: Plasma proteomic analysis of the acute phase response of rainbow trout (Oncorhynchus mykiss) to intraperitoneal inflammation and LPS injection. Dev. Comp. Immunol. 2006, 30, 393-406.
14.Scudiero R., Temussi P. A., Parisid E.: Fish and mammalian metallothioneins: a comparative study. Gene 2005, 345, 21-26.
15.Shaw G. C., Cope J. J., Li L., Corson K., Hersey C., Ackermann G. E., Gwynn B., Lambert A. J., Wingert R. A., Traver D., Trede N. S., Barut B. A., Zhou Y., Minet E., Donovan A., Brownlie A., Balzan R., Weiss M. J., Peters L. L., Kaplan J., Zon L. I., Paw B. H.: Mitoferrin is essential for erythroid iron assimilation. Nature 2006, 440, 96-100.
16.Smet H. de, Blust R., Moens L.: Absence of albumin in the plasma of the common carp Cyprinus carpio: binding of fatty acids to high density lipo-protein. Fish Physiol. Biochem. 1998, 19, 71-81.
17.Veiseth-Kent E., Grove H., Færgestad E. M., Fjæra S. O.: Changes in muscle and blood plasma proteomes of Atlantic salmon (Salmo salar) induced by crowding. Aquaculture 2010, 309, 272-279.
Corresponding author: Prof. Assistant Ma³gorzata O¿go, Department of Physiology, Cytobiology and Proteomics, Doktora Judyma Str. 6, 71-466 Szczecin, Poland; e-mail: malgorzata.ozgo@zut.edu.pl
