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Comparison of the biochemical composition of normal epidermal mucus and extruded slime of hagfish (Myxine glutinosa L.)
Fish & Shellfish Immunology 25 (2008) 625–632
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Comparison of the biochemical composition of normal epidermal mucus and extruded slime of hagfish (Myxine glutinosa L.) S. Subramanian a, N.W. Ross b, S.L. MacKinnon b, * a b
Department of Biology, Dalhousie University, Halifax, Nova Scotia B3H 4J1, Canada National Research Council, Institute for Marine Biosciences, Halifax, Nova Scotia B3H 3Z1, Canada
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 April 2008 Received in revised form 21 August 2008 Accepted 26 August 2008 Available online 6 September 2008
Hagfish (Myxine glutinosa) secrete normal epidermal mucus and extruded slime. The epidermal mucus is produced continuously to prevent pathogen adherence while the extruded slime is observed predominantly during feeding, provocation or stress. To date little is known about the involvement of extruded slime in the physiological functions of hagfish. In this preliminary study, innate immune enzymes and the protein composition of hagfish normal epidermal mucus and extruded slime were analysed and compared. The lysozyme specific activity of hagfish was observed approximately two-fold higher in extruded slime than that of epidermal mucus. The extruded slime had approximately 3.5–5.0 fold increased levels of alkaline phosphatase, cathepsin B and proteases in comparison to epidermal mucus. Protease characterization using specific inhibitors showed that the extruded slime had higher levels of serine trypsin-like proteases compared to metalloproteases whereas epidermal mucus showed equal proportion of both serine and metalloproteases. SDS-PAGE analysis showed high levels of three proteins with molecular masses in the range of 13–16 kDa in the extruded slime. The LC/MS/MS analysis of protein bands 1, 2 and 3 showed closest matches to hemoglobulin-3, histone H3 and H2B proteins, respectively. The observation of elevated levels of innate immune parameters in the extruded slime suggested that the extruded slime has a significant role in innate immunity of hagfish against infectious pathogens. Crown Copyright Ó 2008 Published by Elsevier Ltd. All rights reserved.
Keywords: Myxine glutinosa Extruded slime Hydrolytic enzymes Proteases Histone H3 Histone H2B Hemoglobin
1. Introduction Hagfish, also referred to as slime eel or slime hag, are members of the myxinidae family, which along with the lampreys (family petromyzonidae), form the only living class of jawless vertebrates called the agnatha [1,2]. Fossil studies have shown that hagfish have been in existence for about 330 million years [3]. Morphological characteristics of hagfish such as degenerated eyes, soft skeleton, pouched gills and lack of true jaws, paired fins, scales and lateral lines suggests they are the ancestors of modern vertebrates or craniates (teleost and cartilaginous fish) and that hagfish are evolutionarily the most primitive form of living vertebrates [1,4,5]. Atlantic hagfish, Myxine glutinosa (Linnaeus), are found in deep cold waters of the Atlantic and Arctic oceans at depths of 100– 300 m and up to 1100 m [4]. They inhabit burrows in the soft clay or muddy sediments of the sea bottom [4,6]. As scavengers they are often found inside dead fish [4,6] but also prey on worms, small
* Corresponding author. Present address: National Research Council, Institute for Marine Biosciences, 1411 Oxford Street, Halifax, Nova Scotia B3H 3Z1, Canada. Tel.: þ1 902 426 6351; fax: þ1 902 426 9413. E-mail address: [email protected] (S.L. MacKinnon).
shrimps and crabs and feed on restrained or moribund fish caught on longlines and fixed gill nets [7]. The two types of exudates that hagfish secrete are normal epidermal mucus and extruded slime [8]. The epidermal mucus is produced continuously under normal conditions and thought to be the collective product of various epidermal or epithelial mucus cells [8,9]. The epidermal mucus layer has been shown to serve as a physical and biological protective barrier between fish and its aquatic environment [9]. In contrast, the extruded slime of hagfish is produced during feeding and when hagfish are stressed or provoked [10]. The slime is extruded upon stimulation from the numerous slime glands that line either side of the ventrolateral body walls [8]. The extruded slime is thought to provide protection from predators or to repulse other scavengers [10]. The epidermal mucus layer provides a critical defense since fish are continually confronted with microbes [11]. The mucus consists of components such as lysozyme, proteolytic enzymes, flavoenzymes and antimicrobial peptides, which have bactericidal activities [12,13]. These innate immune substances are constitutively expressed in mucus to provide immediate protection to fish from potential pathogens [12,14]. The epithelial mucus layers are therefore considered a key component of fish innate defense mechanisms [11,13,15].
1050-4648/$ – see front matter Crown Copyright Ó 2008 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2008.08.012
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S. Subramanian et al. / Fish & Shellfish Immunology 25 (2008) 625–632
Lysozyme is a ubiquitous enzyme identified in a wide range of organisms including the epidermal mucus of various fish species [16]. The bacteriolytic activity of lysozyme in fish epidermal mucus and other tissues is believed to contribute to its host defense activity against bacterial infection [17–19]. Enhanced levels of lysozyme have been found in fish epidermal mucus during pathogenic stress [20]. The level of alkaline phosphatase (AP), a lysosomal enzyme, has been shown to be higher in the epidermal mucus of parasite infected (Lepeophtheirus salmonis) Atlantic salmon (Salmo salar) versus uninfected fish [21]. Elevated levels of AP activity have also been noticed in the epidermal mucus of experimentally wounded carp (Cyprinus carpio) [22,23]. These suggest that AP may play a role in the innate immunity of fish against pathogenic infection. Fish epidermal mucus also contains a variety of proteases, which have a significant role in innate immune mechanisms. Proteases are classified into serine, cysteine, aspartic and metalloproteases based on the chemical nature of the groups responsible for catalysis [24]. Fish mucus has been found to have serine proteases such as trypsin [19,25], cysteine proteases such as cathepsins B and L [26], aspartic proteases such as cathepsin D [27] and metalloproteases [18,28]. Proteases found in the epidermal mucus may arise from either host innate immunity or pathogenic infection. Increased levels of cathepsin activities have been observed following environmental stressors in eel (Anguilla anguilla, Anguilla japonica) and after bacterial infection in Japanese flounder (Paralichthys olivaceus) [29,30]. Extruded slime secretions have also been observed in other teleost fish such as parrotfish (Scarus dubius and Scarus perspicillatus) and cleaner-wrasse (Labroides dimidiatus) which produce copious amounts of slime from large goblet cells in the opercular region at night which results in the formation of cocoons around their bodies [31]. It has been suggested that the slime envelope protects the fish from predation by nocturnal predators [31]. Antimicrobial properties have also been observed in the slime cocoon of queen parrotfish (Scarus vetula) [32]. Similarly, the African catfish (Arius thalassinus) secretes a thick gel-like layer of proteinaceous material onto its skin surface from epidermal proteinaceous cells, or club cells, when thermally shocked, threatened or injured [33,34]. The threat-induced slime secretions of catfish differed greatly from its normal epidermal mucus and venom secretions in terms of composition [34], having significant biochemical and pharmacological properties in innate defense responses and in wound healing [33]. The extruded slime of hagfish is excreted by specialised glands that produce fine treads that strengthen the integrity of the slime [8]. The compositional differences and biological functions of hagfish extruded slime versus epidermal mucus have not been investigated. In our previous study, we demonstrated that hagfish epidermal mucus had higher levels of innate immune enzymes in comparison to various other teleosts [18]. The extruded slime of hagfish appears to be physically very different than normal epidermal mucus. To date, the role of extruded slime in hagfish protection against infectious pathogens is poorly defined. The present study therefore compares the levels of innate immune parameters including lysozyme, AP, cathepsin B and protease of extruded slime to that of normal epidermal mucus of hagfish (M. glutinosa). The protein profile of hagfish extruded slime and that of epidermal mucus was examined. Upregulated proteins of extruded slime were identified using mass spectrometry. The focus of this study was to investigate the role of extruded slime in the physiological functions of hagfish. 2. Materials and methods 2.1. Hagfish and their maintenance Hagfish (M. glutinosa) weighing 150 35 g were obtained from fishermen of Cape Sable Island, Nova Scotia, Canada. The fish were
maintained at National Research Council – Institute for Marine Biosciences (NRC – IMB), Marine Research Station, Ketch Harbour, NS. They were placed in a 1500 L capacity flow-through tank at a stocking density of 6–7 kg m3 (w 45 fish per tank) and a water temperature of 8 2 C. Canadian Council on Animal Care (CCAC) guidelines were strictly adhered to while maintaining and handling the fish. Only healthy fish were used for collection. Dead fish or fish with lesions were removed from the tanks. The fish were fed twice a month with oven-dried herring. 2.2. Epidermal mucus collection Epidermal mucus was collected from 10 fish as previously described in Ref. [18]. The epidermal mucus from individual fish was frozen immediately and freeze dried. 2.3. Extruded slime collection Hagfish (n ¼ 10) were induced to produce slime by a mild physical challenge. Individual fish were transferred carefully into a bucket containing 3 L of seawater and the water was agitated by hand. The slime extruded rapidly as a white fluid into the water, which upon hydration swelled and was scooped out of the bucket. To remove the excess water that was trapped between the slime threads, the extruded slime from individual fish was squeezed by hand then frozen and freeze dried. 2.4. Epidermal mucus and slime preparation The freeze dried epidermal mucus and extruded slime samples of individual fish were reconstituted in 3 ml of Milli Q water, homogenised using a polytron homogenizer (KinematicaÒ, Brinkmann Instruments Inc., NY, USA) and centrifuged at 1500 g for 10 min at 4 C. The homogenisation and the centrifugation steps were repeated three times for extruded slime samples to maximise the extraction of soluble components. The supernatants of both epidermal mucus and extruded slime samples were desalted by dialysis against Milli Q water using a 1000 Da molecular weight cut off (MWCO) membrane (Spectrum laboratories Inc., CA, USA), freeze dried and stored at 80 C until further analysis. 2.5. Enzymatic assays Enzymatic activities were assayed in parallel for both epidermal mucus and extruded slime (n ¼ 10 each). Prior to the assay, the freeze dried samples were reconstituted in 2 ml of the respective enzyme assay buffers and centrifuged at 9300 g for 2 min at 4 C. The protein concentration was determined using the protein-dye binding kit (Biorad, Hercules, CA, USA) [35] using bovine gamma globulin as the standard. Individual mucus and slime samples were diluted to obtain an equal protein concentration for the assays (0.4 0.05 mg protein ml1). The absorbance and fluorescence for the various assays were read using a Spectramax Plus384 Microplate Reader and Spectramax GeminiXS fluorescence plate reader (Molecular Devices, CA, USA), respectively. Lysozyme (EC 3.2.1.17) activity was determined using a turbidometric assay as previously described in Ref. [18]. Fifty microlitres of the epidermal mucus/extruded slime in 40 mM NaH2PO4 buffer (pH 6.5) was transferred to a 96-well plate and incubated at 30 C for 15 min. Freeze dried Micrococcus lysodeikticus cells (50 ml) (0.3 mg ml1 in 40 mM NaH2PO4 buffer (pH 6.5), Sigma) were then added and the change in absorbance was monitored at 30 C for 30 min. One unit of activity was defined as the amount of enzyme that catalyzed a decrease in absorbance at 450 nm of 0.001 min1. Alkaline phosphatase (AP) activity was determined by incubating 50 ml of epidermal mucus/extruded slime reconstituted in
S. Subramanian et al. / Fish & Shellfish Immunology 25 (2008) 625–632
100 mM NH4HCO3 containing 1 mM MgCl2 (pH 7.8) at 30 C for 15 min. Fifty microlitres of 4 mM p-nitrophenol phosphate substrate was added and the absorbance was measured at 405 nm over a 30 min period at 30 C. The initial rate of reaction was used to calculate activity. One unit of activity was defined as the amount of enzyme required to release 1 mmol of p-nitrophenol in 1 min. Cathepsin B activity was assayed using a modified method of Barrett and Kirschke [36]. Briefly, 50 ml of epidermal mucus/ extruded slime reconstituted in 0.1 M sodium phosphate buffer (pH 6.0) was incubated with 20 ml of assay buffer (0.1 M sodium phosphate, 0.08% (w/v) Brij, 1 mM EDTA, pH 6.0), 20 ml of 1 M dithiothreitol (DTT) and 60 ml of Milli Q water in a 96-well microtitre plate for 5 min at 30 C. Then 50 ml of 25 mM carbobenzoxy-Lphenylalanyl-L-arginyl-4-methylcoumaryl-7-amide substrate was added, and fluorescence of 7-amino-4-methylcoumarin (AMC) was measured for 30 min at an excitation wavelength of 380 nm and an emission wavelength of 405 nm. The initial rate of reaction was used to calculate enzyme activity and 1 U of activity was defined as the amount of enzyme required to release 1 mmol of substrate in 1 min. Protease activities were determined using the azocasein hydrolysis assay and zymography. Azocasein hydrolysis was determined by incubating 50 ml of the epidermal mucus/extruded slime re-suspended in 100 mM NH4HCO3 buffer (pH 7.8) with 50 ml azocasein substrate [0.25% (w/v), Sigma–Aldrich, Inc., MO, USA] in the same buffer for 19 h at 30 C. The reaction was stopped by adding 50 ml of 20% (w/v) trichloroacetic acid followed by a 5 min centrifugation at 15,400 g. Equal volumes (100 ml) of the resultant supernatant and 0.5 M NaOH were added to a 96-well plate and the absorbance measured at 405 nm. For zymography analysis the epidermal mucus (n ¼ 3)/extruded slime (n ¼ 5) samples dissolved in Milli Q water were combined with 1:1 with sample buffer (4% (w/v) sodium dodecyl sulphate (SDS), 125 mM Tris–HCl, 20% (v/v) glycerol and 0.5% (w/v) bromophenol blue adjusted with HCl to pH 6.8). Five micrograms of the sample were loaded onto a 12% SDS-polyacrylamide gel containing 0.1% (w/v) gelatin and electrophoresed at 4 C. Sodium dodecyl sulphate-PAGE standard markers (Broad range, Bio-Rad laboratories Inc., CA, USA) were included to estimate the molecular mass of proteases. The resulting gel was washed thrice at 4 C with 50 mM Tris–HCl (pH 7.5) containing 2.5% (w/v) Triton X-100 and incubated at 30 C for 19–20 h in 50 mM Tris–HCl (pH 7.5) containing 2.5% (w/v) Triton X-100, 20 mM MgCl2 and 6.25 mM CaCl2. After incubation the gel was stained with 0.25% Coomassie blue R-250 in MeOH/ H2O/AcOH (40:50:10) for 1 h and destained with MeOH/H2O/ AcOH (40:50:10) until the desired contrast was achieved. The effect of various inhibitors on protease activity was determined by adding specific inhibitors (i.e. 5 mM p-aminobenzamidine, 20 mg ml1 aprotinin, 5 mM EDTA, 5 mM o-phenanthroline and 5 mM iodoacetamide) during the incubation step in the azocasein hydrolysis assay and zymography analysis. When EDTA was used as the inhibitor, CaCl2 was omitted for the buffer. 2.6. Gel electrophoresis and trypsin digestion The protein profile of epidermal mucus and extruded slime were examined using tricine sodium dodecyl sulphate-polyacrylamide gel electrophoresis (Tricine SDS-PAGE) as described by Scha¨gger and Jagow [37]. Protein samples (6 mg total protein) were diluted 1:1 with sample buffer [4% (w/v) SDS, 50 mM Tris–HCl, 2% mercaptoethanol (v/v), 12% (v/v) glycerol and 0.5% (w/v) bromophenol blue adjusted with HCl to pH 6.8] and loaded onto a separating gel of 15% acrylamide with a 10% acrylamide spacer gel and 4% stacking gel. The gel was run in a Bio-Rad electrophoresis apparatus for 3.5– 4 h at 90 V. Sodium dodecyl sulphate-PAGE standard markers
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(Broad range, Bio-Rad laboratories Inc., CA, USA) were included to estimate the molecular mass of proteins. The proteins were visualized using silver staining [38] and the bands of interest were excised for identification by mass spectrometry. The in-gel trypsin digestion of the proteins was carried out following a modified method of Ebanks et al. [39]. Briefly, protein bands were carefully excised from the gel, placed separately into 1.5 ml Eppendorf tubes, washed 3 times each for 10 min with 100 ml of 50% acetonitrile (ACN) in 100 mM NH4HCO3 and then dehydrated in 100 ml of 100% ACN for 10 min. Proteins in the gel were reduced with 100 ml of 10 mM DTT in 100 mM NH4HCO3 at 56 C for 30 min, followed by alkylation in 100 ml of 100 mM NH4HCO3 containing 100 mM iodoacetamide in the dark at room temperature for 1 h. The reduced and alkylated proteins were then washed and dehydrated 2 times with 100 ml of 100 mM NH4HCO3 and 50% ACN in 100 mM NH4HCO3 for 10 min. The protein gel pieces were dehydrated with 100 ml of 100% ACN for 20 min and placed in a speedvac (Savant, Savant Instruments, Inc., NY, USA) for 10 min to remove residual ACN. The dehydrated protein gel pieces were reswollen by adding 25 ml of 12.5 ng ml1 trypsin (Promega, Madison, WI, USA) in 100 mM NH4HCO3 and incubated at 37 C overnight. The trypsin-digested peptides were extracted from the gel with 20 ml of 100 mM NH4HCO3 followed by two sequential elutions using 0.2% formic acid in 50% ACN in H2O. The eluted peptides were then concentrated using a speedvac (Savant). 2.7. Mass spectrometry (MS) protein identification The trypsin-digested peptide samples were submitted for LC/ MS/MS analysis (Atlantic Research Centre, Dalhousie University, Halifax, Canada). The on-line LC/MS/MS experiments were carried out using a Q TRAPÒLC/MS/MS (Applied Biosystems MDS SCIEX, CA, USA). The separation was carried out on a 150 0.1 mm chromolith CAP ROD C18 column (Merck, Darmstadt, Germany) using a linear gradient from 5% to 35% solvent B over 35 min (solvent A: 0.1% (v/v) formic acid in Milli Q water; solvent B: 0.1% (v/v) formic acid in ACN) at a flow rate of 1.0 ml min1. The HPLC eluates were introduced to MS via a nanoelectrospray ionization source. Data was acquired such that the mass to charge ratio (m/z) values of the tryptic peptides were measured by scan mode and this scan was used to generate a peak list of peptides for MS/MS analysis. The tandem MS spectra were submitted to the database search program MASCOT (Matrix Science, London, UK) to search against NCBInr and SwissProt databases. 3. Results Hagfish epidermal mucus was obtained by placing hagfish directly into anaesthetic containing seawater and then collected mucus from the epidermis as for other teleosts [18,21]. The volume of the epidermal mucus collected in this way was less than that of extruded slime, which was obtained following physical disturbances to the fish. Previous reports have induced hagfish to produce extruded slime using electrical stimulators or by pinching their tails with forceps [40]. We however noticed that a mild physical disturbance such as the agitation of water in the fishholding bucket was sufficient to stimulate slime extrusion. The extruded slime contains mucins, which upon hydration becomes a highly viscous gel-like mass (Fig. 1). This mode of induction was non-lethal to the fish and reduced potential contamination with body fluids. Biochemical analyses were carried out on the soluble components of the extruded slime, which presumably included the viscous gel and water soluble thread constituents [40]. A pronounced variation in specific activities of lysozyme, alkaline phosphatase (AP), cathepsin B and proteases were observed between the epidermal mucus and extruded slime of hagfish
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S. Subramanian et al. / Fish & Shellfish Immunology 25 (2008) 625–632 Table 2 Effect of specific protease inhibitors on protease specific activity (mean S.E.M.) in epidermal mucus and extruded slime of hagfish using the azocasein assay (n ¼ 10) Protease inhibitors p-Aminobenzamidine (5 mM) Aprotinin (20 mg ml1) EDTA (5 mM) o-Phenanthroline (5 mM) Iodoacetamide (5 mM)
Epidermal mucus 0 30.0 3.7 34.6 1.8 0 0
Extruded slime 52.7 2.0 47.8 1.1 36.1 2.9 5.0 3.5 0
Values are expressed as a percent inhibition of the total (100%) protease activity.
Fig. 1. Viscous gel exudate from the slime glands of stressed hagfish (Myxine glutinosa L.).
(Table 1). Lysozyme activity in the extruded slime was 198.4 6.2 U mg1 protein, which is approximately two-fold higher than that of the epidermal mucus lysozyme levels. The AP activity in extruded slime was about 3.5 times higher than that of epidermal mucus (1.4 0.1 U mg1 protein). Similarly, cathepsin B levels of extruded slime were approximately 3.4 times higher than the epidermal mucus (70.1 8.2 U mg1 protein). Protease levels, as determined by the azocasein hydrolysis assay, were about 5.0 times higher in extruded slime compared to the epidermal mucus (836.0 24.5 U mg1 protein). The effect of various protease inhibitors on the specific activity of hagfish extruded slime and epidermal mucus proteases were examined in the azocasein hydrolysis assay (Table 2). The specific trypsin-like protease inhibitor, p-aminobenzamidine, inhibited 50– 54% of the protease activity in extruded slime but showed no inhibition of protease activity in the epidermal mucus. Aprotinin caused 46–48% inhibition of protease activity in the extruded slime and 30–33% in the epidermal mucus. EDTA, showed similar levels of inhibition (34–38%) of activity in the extruded slime and epidermal mucus proteases. The zinc chelator, o-phenanthroline, was found to reduce protease activity by 2–8% in extruded slime but had no observable inhibition of the protease activity in epidermal mucus. The extruded slime and epidermal mucus protease activity were unaffected by the cysteine protease inhibitor, iodoacetamide. A variation in protease profiles was observed in the zymographic analysis of hagfish epidermal mucus and extruded slime (Fig. 2A,B). The extruded slime had low molecular mass proteases in the range Table 1 Comparison of enzyme activities (mean S.E.M.) of hagfish epidermal mucus and extruded slime (n ¼ 10) Enzyme activity
Epidermal mucus
Extruded slime
Lysozyme Alkaline phosphatase Cathepsin B Protease
105.0 5.2 1.4 0.1 70.1 8.2 836.0 24.5
198.4 6.2 4.9 0.3 239.8 10.1 4517.3 145.2
Specific activity is expressed as U mg1 protein.
of 18–22 kDa and several medium molecular mass proteases of approximately 29–32 kDa and 40 kDa, which were not observed in the epidermal mucus. The dense protease bands of high molecular mass at 90–150 kDa in the extruded slime were not observed in the epidermal mucus. Medium to high molecular mass proteases in the range of 50–90 kDa were found in both the epidermal mucus and the extruded slime. Incubation of zymography gels with the specific protease inhibitor, aprotinin, showed effective inhibition of the majority of the proteases in both the epidermal mucus and extruded slime, except for the high molecular mass proteases at 90–150 kDa (Fig. 3 section 2 lanes A and B). p-Aminobenzamidine showed similar inhibition patterns as aprotinin (Fig. 3 section 3 lanes A and B). EDTA and o-phenanthroline each showed nearly complete inhibition of protease activity in epidermal mucus (Fig. 3 section 4,5 lane A). In contrast, the EDTA showed poor inhibition of low (w18– 22 kDa) and high molecular mass proteases (w90–150 kDa) in extruded slime (Fig. 3 section 4, lane B). Similarly, the o-phenanthroline did not inhibit medium (w40 kDa) and high molecular mass proteases (w90–150 kDa) (Fig. 3 section 5 lane B) in extruded slime. Iodoacetamide exhibited minimal inhibition of epidermal mucus proteases (Fig. 3 section 6 lane A), but the low (w18–22 kDa) and most of the medium molecular mass proteases (w29–32 kDa and w40 kDa) were inhibited in the extruded slime (Fig. 3 section 6 lane B). Electrophoretic comparison of epidermal mucus and extruded slime (Fig. 4 lanes A and B) showed that the distribution of the majority of the medium and high molecular mass (>40 kDa) protein bands were similar, except for two protein bands at approximately 30 kDa and 40 kDa, which were more intense in the epidermal mucus. A marked difference in the protein composition and a strong upregulation of three proteins of molecular masses range of 13–16 kDa, was observed in the extruded slime (Fig. 4 lanes A and C band numbers 1, 2 and 3). The three distinct proteins of extruded slime (Fig. 4 lane C) were excised, in-gel trypsindigested and analysed by electrospray ionization LC/MS/MS. Search of NCBInr and SwissProt databases with the Mascot search engine found high matches for each band (Table 3). Protein 1 (16.7 kDa) matched the hemoglobin-3 of hagfish (M. glutinosa) (MASCOT score of 287), with homology to a number of distinct peptides of hemoglobin-3. Protein 2 (15.3 kDa) and protein 3 (13.6 kDa) showed poor or no similarity to any of the hagfish proteins in the database. MASCOT analysis of protein 2 showed good matches with the conserved regions of histone H3 identified in a wide range of organisms such as mouse, fruit fly, roundworm, innkeeper worm, sea star, marine copepod, staghorn coral, African clawed frog and humans with a score of 82. Protein 3 matched well with the conserved domain of histone H2B characterized from several organisms including marine copepod, mosquito, African clawed frog, yeast and different species of fruit with a score of 156. 4. Discussion The current study examines the biochemical differences between hagfish epidermal mucus and extruded slime. During the
S. Subramanian et al. / Fish & Shellfish Immunology 25 (2008) 625–632
1
2
3
4
5
6
7
629
8
194.6 95-150
116.5 97.2
50-90 50.2 40 37.6 29.3
29-32
20.0
18-22
A
B
Fig. 2. Protease zymography of A: epidermal mucus and B: extruded slime sample of hagfish without inhibitor. Numbers 1–8 on the top of gel indicates the individual fish. Molecular mass (kDa) markers are along the left side of the gels. The approximate mass range for high, medium and low molecular mass proteases in kDa is indicated in brackets along the right side of the gels. Each well contains 5 mg of mucus/extruded slime protein.
collection of epidermal mucus, the fish may have experienced some stress during the process of anaesthetization. However, studies on the effect of tricaine methanesulphonate on fish physiological stress responses have shown no significant variations in the level of stress indicators, such as plasma cortisol, of anaesthetised fish in comparison to pre-anaesthetized fish [41]. Tricaine methanesulphonate functions by interfering with the ion movements in nerves and muscles [42] thus reducing fish activity and minimising physical stress. The epidermal mucus in this study was collected very gently to minimise the induction of physical stress to fish. The consistency of hagfish epidermal mucus was similar to that of other fish species sampled using this method [18,21]. In contrast, the stress induced extruded slime secretion of hagfish was a highly viscous gel-like mass containing protein rich threads and mucins. The epidermal mucus of fish contains biologically active substances that exhibit defensive functions against infectious pathogens in the aquatic environment [13,18,19,25]. Significant variations in lysozyme, AP and cathepsin B activity have been previously reported in the epidermal mucus of hagfish in comparison to various teleosts [18]. The present study shows that the extruded slime of hagfish has elevated levels of these enzymes and antimicrobial protein/peptide precursors including
194.6 116.5 97.2
A
B
A
B
A
B
hemoglobin, histone H3 and H2B compared to its normal epidermal mucus. Lysozyme is a bacteriolytic enzyme that has been shown to lyse bacterial cells. The epidermal mucus lysozyme of yellowtail (Seriola quinqueradiata), common carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss) exhibited bacteriolytic activity against various pathogens [43]. As scavengers, hagfish encounter various pathogenic microorganisms in dead and decaying organisms. The increased level of lysozyme in extruded slime suggests that the slime might be secreted to provide increased protection against potential microbial pathogens. The precise function of AP in the mucosal innate immunity of fish is unclear. However the variation in AP levels in Atlantic salmon tissues and blood during smoltification have been correlated to stress response [44]. The observation of 3.5 times higher AP activity in the hagfish extruded slime than the epidermal mucus was thought to be due to the physical stress induced to the hagfish during collection of the extruded slime secretion. Elevation in the specific AP activity had been observed in fish epidermal mucus following the physical, chemical and microbial stress [21–23]. The cysteine protease, cathepsin B, has been observed at higher levels in the epidermal mucus of bottom dwelling fish species such as
A
B
A
B
A
B
50.2 37.6 29.3 20.0
1
2
3
4
5
6
Fig. 3. Protease zymography of representative A: epidermal mucus and B: extruded slime of hagfish. 1. no inhibitor and 2–5 incubated with specific protease inhibitors, 2: aprotinin (20 mg ml1), 3: p-aminobenzamidine (5 mM), 4: EDTA (5 mM), 5: o-phenanthroline (5 mM) and 6: iodoacetamide (5 mM). Molecular mass (kDa) markers are along the left side of the gels. Each well contains 5 mg of mucus/extruded slime protein.
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194.6 116.5 97.2
50.2
37.6 29.3 20.0
1
2
3
7.1
A
B
Fig. 4. Tricine SDS-PAGE of hagfish epidermal mucus and extruded slime on a 15% gel. Each well was loaded with 6 mg of total protein. The gel after electrophoresis was silver stained to visualize the protein bands. A: epidermal mucus, B: extruded slime. The numbers on right side indicate the bands that were selected for proteomic analysis 1: band 1, 2: band 2 and 3: band 3. Molecular mass (kDa) markers are along the left side of the gels.
koi carp, flounder and eel species [18,29,30]. Cathepsins, B and L, levels have also been found to increase in epidermal mucus of flounder and eel species following environmental and microbial stress, presumably to provide protection against microbial pathogens [30,39]. The present observation of an approximately three-fold increase in cathepsin B levels in extruded slime could be the genetic adaptation of hagfish to thrive in the microbial rich environments. A marked variation in protease activities between epidermal mucus and extruded slime was observed. In the azocasein assay, extruded slime showed a higher ratio of serine to metalloproteases, while the epidermal mucus had almost equal proportions of serine and metalloproteases. In the extruded slime, the trypsin specific
protease inhibitor, p-aminobenzamidine, reduced approximately 50% of proteases. This suggested that trypsin-like proteases might be a major constituent of hagfish extruded slime. The metalloprotease inhibitors, o-phenanthroline and EDTA, showed discrepancies in protease inhibition. The o-phenanthroline exhibited poor to no inhibition of protease activity and the EDTA reduced approximately 36% of the protease activity in the epidermal mucus and extruded slime. Such variations were not observed in the zymography, where both inhibitors had similar effects on both the epidermal mucus and extruded slime. Differences in the substrate used and assay conditions could be responsible for the discrepancies observed in the two assays. For example, the presence of detergents in zymography causes potential denaturation, which may activate latent proteases [20,45] that would not be observable using the azocasein assay. Alternatively, it is possible that some proteases may be irreversibly denatured during zymography. Both the metallo- and serine protease inhibitors showed similar inhibition of majority of proteases in the extruded slime. This indicates the presence of serine-activated metalloproteases or cation-specific serine proteases, which may be reduced by both metallo- and serine protease inhibitors. Even though epidermal mucus and extruded slime have cathepsin B activities, iodoacetamide showed no inhibition in zymography or the azocasein assay. Cysteine proteases are known to be activated under reduced conditions [36], the absence of dithiothreitol (DTT) or b-mercaptoethanol therefore may be the reason for the poor inhibition observed by iodoacetamide in these assays. The serine and metalloproteases from fish epidermal mucus has been shown to play roles in the innate defense mechanisms. Trypsin, a low molecular weight serine protease, isolated from the epidermal mucus of rainbow trout has been shown to have antibacterial activity against Vibrio anguillarum [36]. The trypsin and other serine proteases have been shown to activate immunoglobulin (IgE) [46] and antimicrobial peptides including defensin and cathelicidin in mammalian epithelial cells [47]. A metalloprotease isolated from the epidermal mucus of plaice (Pleuronectes platessa) exhibited antimicrobial activity against both Gram positive and Gram negative bacteria [48]. Metalloproteases have also been found to trigger other proteases [49], antimicrobial peptides [27] and various immune factors such as cytokines and chemokines [49]. Similar roles can be postulated for trypsin-like and metalloproteases present in hagfish extruded slime however further study needs to be carried out.
Table 3 Summary of proteins identified from Fig. 4 Protein homolog (Swissprot accession no.)
Parent ion (m/z)
Mr (Da)
Error (ppm)
Score
Peptide sequence of matched entry
Band 1 –hemoglobin-3 of Atlantic hagfish (P02209)
607.4464 525.7872 626.4332 790.5000
1818.9112 1049.5182 1250.6619 1578.7137
0.4062 0.0418 0.1899 0.2718
67 48 70 96
PITDHGQPPTLSEGDKK ESWPQIYK KSHLEQDPAVK HSTEFQVNPDMFK
Band 2 – histone H3 (Q9LR02)
516.8659 416.3018
1031.5876 830.4861
0.1297 0.1029
38 45
YRPGTVALR STELLIRa
Band 3 – H2B (P35068)
575.3752 887.4800 477.3598
1148.5753 1772.8226 952.5957
0.1604 0.1229 0.1095
45 68 43
ESYAIYIYK AMSIMNSFVNDIFERb LLLPGELAKb
The proteins of extruded slime (bands 1, 2 and 3) were identified using liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS). m/z: specific mass to charge ratio, Mr: relative molecular weight (Da), error: difference between measured mass and the predicted mass, score: score from MASCOT search; scores above 35 are highly significant, and peptide sequence: tryptic peptide sequence predicted. a LC/MS/MS sequences matched to the conserved region of histone H3 protein of a wide range of organisms (score of 82) such as Mouse-ear cress (SwissProt accession no. Q9LR02), fruit fly (SwissProt accession no. P84236), roundworm (SwissProt accession no. P08898), innkeeper worm (SwissProt accession no. P84239), Sea star (SwissProt accession no. P69071), marine copepod (SwissProt accession no. P84237), staghorn coral (SwissProt accession no. P22843), African clawed frog (SwissProt accession no. P02302) and human (SwissProt accession no. Q6NXT2). b LC/MS/MS sequences matched to the conserved domain of histone H2B protein with a score of 156 to several organisms, for example, marine copepod (SwissProt accession no. P35068), different species of fruit fly (SwissProt accession nos. P59781, Q8I1N0, Q76FF3, Q76FD7, Q76FE5, Q76FE9), African clawed frog (SwissProt accession no. NP_001086753), yeast (SwissProt accession no A5DWF0), and yellow fever mosquito (SwissProt accession no. XP_001656991).
S. Subramanian et al. / Fish & Shellfish Immunology 25 (2008) 625–632
Tricine SDS-PAGE analysis demonstrated the high levels of three major proteins of molecular mass ranging from 13 kDa to 16 kDa in hagfish extruded slime. Mass spectroscopic analysis of the extruded slime tryptic fragments suggested that protein 1 is closely related to hemoglobin-3 of Atlantic hagfish. The hagfish hemoglobins, unlike the tetrameric hemoglobins of higher vertebrates, are monomeric and had evolved before the separation of myoglobin and hemoglobin [4]. The observed molecular mass of hemoglobin in the hagfish extruded slime matched with the average molecular mass of vertebrate myoglobins and other cyclostome hemoglobins [50]. The precise function of hemoglobin in the hagfish extruded slime is unclear but, previous studies have detected hemoglobin in the epidermal mucus of fish stressed with physical and environmental stressors [51–53]. The presence of hemoglobin in epidermal mucus was often considered an indicator of stress and thought to be derived from red blood cells into the epidermal mucus [53]. This might explain the presence of hemoglobin in the stress induced hagfish extruded slime. Serum hemoglobin and hemoglobinderived peptides have been shown previously to exhibit antimicrobial activity in higher vertebrates [54,55]. Such functions are yet to de determined in extruded slime hemoglobin. Bands 2 and 3 (Fig. 4 lane C) showed close matches to the histone proteins, H3 and H2B, respectively. The matched peptides were from conserved regions of the histone proteins from microorganisms, flies, amphibians and mammals (Table 3). Histones are known for their roles as a structural component of the nucleosome, but also exhibit antimicrobial properties [56]. Antimicrobial activity of histone proteins (H2B and H3) or their cleaved products had been reported in the epidermis and epidermal mucus of catfish (Ictalurus punctatus), rainbow trout (O. mykiss) and Atlantic cod (Gadus morhua) [13,57]. The H2B proteins in murine macrophages and hemocytes of Pacific white shrimp (Litopenaeus vannamei) have also been shown to exhibit antimicrobial activity [58,59]. The observed high levels in extruded slime suggest that histone proteins might be involved in hagfish innate defense against potential pathogens. In conclusion, this study demonstrated that the stress induced extruded slime secretion of hagfish contains various innate immune parameters in comparison to its normal epidermal mucus. The hagfish lacks a defined adaptive immune system and they rely heavily on innate immunity for disease resistance [60]. The high levels of the innate immune substances suggest that the extruded slime is produced more likely to provide protection against microbial, environmental and other physiological stress. This is in addition to the already postulated function of extruded slime as a defense against predators. Further investigations into the isolation and mechanisms of these immune components from the extruded slime could provide a better understanding of their roles in the innate defenses of evolutionarily primitive fish. Acknowledgments The authors would like to thank Mr. Eric MacKinnon (Cape Sable Island, NS) for providing hagfishes and Ron Melanson for help with sampling. Elden Rowland and Roger Ebanks for assistance with the mass spectrometric analysis. This research was funded by National Research Council of Canada and the Ford Foundation International Fellowship Program. References [1] Forey P, Janvier P. Agnathans and the origin of jawed vertebrates. Nature 1993;361:129–34. [2] Bardack D. Relationships of living and fossil hagfishes. In: Jorgensen JM, Lomholt JP, Weber RE, Malte H, editors. The biology of hagfishes. London: Chapman and Hall; 1998. p. 1–31.
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Contents lists available at ScienceDirect
Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi
Comparison of the biochemical composition of normal epidermal mucus and extruded slime of hagfish (Myxine glutinosa L.) S. Subramanian a, N.W. Ross b, S.L. MacKinnon b, * a b
Department of Biology, Dalhousie University, Halifax, Nova Scotia B3H 4J1, Canada National Research Council, Institute for Marine Biosciences, Halifax, Nova Scotia B3H 3Z1, Canada
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 April 2008 Received in revised form 21 August 2008 Accepted 26 August 2008 Available online 6 September 2008
Hagfish (Myxine glutinosa) secrete normal epidermal mucus and extruded slime. The epidermal mucus is produced continuously to prevent pathogen adherence while the extruded slime is observed predominantly during feeding, provocation or stress. To date little is known about the involvement of extruded slime in the physiological functions of hagfish. In this preliminary study, innate immune enzymes and the protein composition of hagfish normal epidermal mucus and extruded slime were analysed and compared. The lysozyme specific activity of hagfish was observed approximately two-fold higher in extruded slime than that of epidermal mucus. The extruded slime had approximately 3.5–5.0 fold increased levels of alkaline phosphatase, cathepsin B and proteases in comparison to epidermal mucus. Protease characterization using specific inhibitors showed that the extruded slime had higher levels of serine trypsin-like proteases compared to metalloproteases whereas epidermal mucus showed equal proportion of both serine and metalloproteases. SDS-PAGE analysis showed high levels of three proteins with molecular masses in the range of 13–16 kDa in the extruded slime. The LC/MS/MS analysis of protein bands 1, 2 and 3 showed closest matches to hemoglobulin-3, histone H3 and H2B proteins, respectively. The observation of elevated levels of innate immune parameters in the extruded slime suggested that the extruded slime has a significant role in innate immunity of hagfish against infectious pathogens. Crown Copyright Ó 2008 Published by Elsevier Ltd. All rights reserved.
Keywords: Myxine glutinosa Extruded slime Hydrolytic enzymes Proteases Histone H3 Histone H2B Hemoglobin
1. Introduction Hagfish, also referred to as slime eel or slime hag, are members of the myxinidae family, which along with the lampreys (family petromyzonidae), form the only living class of jawless vertebrates called the agnatha [1,2]. Fossil studies have shown that hagfish have been in existence for about 330 million years [3]. Morphological characteristics of hagfish such as degenerated eyes, soft skeleton, pouched gills and lack of true jaws, paired fins, scales and lateral lines suggests they are the ancestors of modern vertebrates or craniates (teleost and cartilaginous fish) and that hagfish are evolutionarily the most primitive form of living vertebrates [1,4,5]. Atlantic hagfish, Myxine glutinosa (Linnaeus), are found in deep cold waters of the Atlantic and Arctic oceans at depths of 100– 300 m and up to 1100 m [4]. They inhabit burrows in the soft clay or muddy sediments of the sea bottom [4,6]. As scavengers they are often found inside dead fish [4,6] but also prey on worms, small
* Corresponding author. Present address: National Research Council, Institute for Marine Biosciences, 1411 Oxford Street, Halifax, Nova Scotia B3H 3Z1, Canada. Tel.: þ1 902 426 6351; fax: þ1 902 426 9413. E-mail address: [email protected] (S.L. MacKinnon).
shrimps and crabs and feed on restrained or moribund fish caught on longlines and fixed gill nets [7]. The two types of exudates that hagfish secrete are normal epidermal mucus and extruded slime [8]. The epidermal mucus is produced continuously under normal conditions and thought to be the collective product of various epidermal or epithelial mucus cells [8,9]. The epidermal mucus layer has been shown to serve as a physical and biological protective barrier between fish and its aquatic environment [9]. In contrast, the extruded slime of hagfish is produced during feeding and when hagfish are stressed or provoked [10]. The slime is extruded upon stimulation from the numerous slime glands that line either side of the ventrolateral body walls [8]. The extruded slime is thought to provide protection from predators or to repulse other scavengers [10]. The epidermal mucus layer provides a critical defense since fish are continually confronted with microbes [11]. The mucus consists of components such as lysozyme, proteolytic enzymes, flavoenzymes and antimicrobial peptides, which have bactericidal activities [12,13]. These innate immune substances are constitutively expressed in mucus to provide immediate protection to fish from potential pathogens [12,14]. The epithelial mucus layers are therefore considered a key component of fish innate defense mechanisms [11,13,15].
1050-4648/$ – see front matter Crown Copyright Ó 2008 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2008.08.012
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Lysozyme is a ubiquitous enzyme identified in a wide range of organisms including the epidermal mucus of various fish species [16]. The bacteriolytic activity of lysozyme in fish epidermal mucus and other tissues is believed to contribute to its host defense activity against bacterial infection [17–19]. Enhanced levels of lysozyme have been found in fish epidermal mucus during pathogenic stress [20]. The level of alkaline phosphatase (AP), a lysosomal enzyme, has been shown to be higher in the epidermal mucus of parasite infected (Lepeophtheirus salmonis) Atlantic salmon (Salmo salar) versus uninfected fish [21]. Elevated levels of AP activity have also been noticed in the epidermal mucus of experimentally wounded carp (Cyprinus carpio) [22,23]. These suggest that AP may play a role in the innate immunity of fish against pathogenic infection. Fish epidermal mucus also contains a variety of proteases, which have a significant role in innate immune mechanisms. Proteases are classified into serine, cysteine, aspartic and metalloproteases based on the chemical nature of the groups responsible for catalysis [24]. Fish mucus has been found to have serine proteases such as trypsin [19,25], cysteine proteases such as cathepsins B and L [26], aspartic proteases such as cathepsin D [27] and metalloproteases [18,28]. Proteases found in the epidermal mucus may arise from either host innate immunity or pathogenic infection. Increased levels of cathepsin activities have been observed following environmental stressors in eel (Anguilla anguilla, Anguilla japonica) and after bacterial infection in Japanese flounder (Paralichthys olivaceus) [29,30]. Extruded slime secretions have also been observed in other teleost fish such as parrotfish (Scarus dubius and Scarus perspicillatus) and cleaner-wrasse (Labroides dimidiatus) which produce copious amounts of slime from large goblet cells in the opercular region at night which results in the formation of cocoons around their bodies [31]. It has been suggested that the slime envelope protects the fish from predation by nocturnal predators [31]. Antimicrobial properties have also been observed in the slime cocoon of queen parrotfish (Scarus vetula) [32]. Similarly, the African catfish (Arius thalassinus) secretes a thick gel-like layer of proteinaceous material onto its skin surface from epidermal proteinaceous cells, or club cells, when thermally shocked, threatened or injured [33,34]. The threat-induced slime secretions of catfish differed greatly from its normal epidermal mucus and venom secretions in terms of composition [34], having significant biochemical and pharmacological properties in innate defense responses and in wound healing [33]. The extruded slime of hagfish is excreted by specialised glands that produce fine treads that strengthen the integrity of the slime [8]. The compositional differences and biological functions of hagfish extruded slime versus epidermal mucus have not been investigated. In our previous study, we demonstrated that hagfish epidermal mucus had higher levels of innate immune enzymes in comparison to various other teleosts [18]. The extruded slime of hagfish appears to be physically very different than normal epidermal mucus. To date, the role of extruded slime in hagfish protection against infectious pathogens is poorly defined. The present study therefore compares the levels of innate immune parameters including lysozyme, AP, cathepsin B and protease of extruded slime to that of normal epidermal mucus of hagfish (M. glutinosa). The protein profile of hagfish extruded slime and that of epidermal mucus was examined. Upregulated proteins of extruded slime were identified using mass spectrometry. The focus of this study was to investigate the role of extruded slime in the physiological functions of hagfish. 2. Materials and methods 2.1. Hagfish and their maintenance Hagfish (M. glutinosa) weighing 150 35 g were obtained from fishermen of Cape Sable Island, Nova Scotia, Canada. The fish were
maintained at National Research Council – Institute for Marine Biosciences (NRC – IMB), Marine Research Station, Ketch Harbour, NS. They were placed in a 1500 L capacity flow-through tank at a stocking density of 6–7 kg m3 (w 45 fish per tank) and a water temperature of 8 2 C. Canadian Council on Animal Care (CCAC) guidelines were strictly adhered to while maintaining and handling the fish. Only healthy fish were used for collection. Dead fish or fish with lesions were removed from the tanks. The fish were fed twice a month with oven-dried herring. 2.2. Epidermal mucus collection Epidermal mucus was collected from 10 fish as previously described in Ref. [18]. The epidermal mucus from individual fish was frozen immediately and freeze dried. 2.3. Extruded slime collection Hagfish (n ¼ 10) were induced to produce slime by a mild physical challenge. Individual fish were transferred carefully into a bucket containing 3 L of seawater and the water was agitated by hand. The slime extruded rapidly as a white fluid into the water, which upon hydration swelled and was scooped out of the bucket. To remove the excess water that was trapped between the slime threads, the extruded slime from individual fish was squeezed by hand then frozen and freeze dried. 2.4. Epidermal mucus and slime preparation The freeze dried epidermal mucus and extruded slime samples of individual fish were reconstituted in 3 ml of Milli Q water, homogenised using a polytron homogenizer (KinematicaÒ, Brinkmann Instruments Inc., NY, USA) and centrifuged at 1500 g for 10 min at 4 C. The homogenisation and the centrifugation steps were repeated three times for extruded slime samples to maximise the extraction of soluble components. The supernatants of both epidermal mucus and extruded slime samples were desalted by dialysis against Milli Q water using a 1000 Da molecular weight cut off (MWCO) membrane (Spectrum laboratories Inc., CA, USA), freeze dried and stored at 80 C until further analysis. 2.5. Enzymatic assays Enzymatic activities were assayed in parallel for both epidermal mucus and extruded slime (n ¼ 10 each). Prior to the assay, the freeze dried samples were reconstituted in 2 ml of the respective enzyme assay buffers and centrifuged at 9300 g for 2 min at 4 C. The protein concentration was determined using the protein-dye binding kit (Biorad, Hercules, CA, USA) [35] using bovine gamma globulin as the standard. Individual mucus and slime samples were diluted to obtain an equal protein concentration for the assays (0.4 0.05 mg protein ml1). The absorbance and fluorescence for the various assays were read using a Spectramax Plus384 Microplate Reader and Spectramax GeminiXS fluorescence plate reader (Molecular Devices, CA, USA), respectively. Lysozyme (EC 3.2.1.17) activity was determined using a turbidometric assay as previously described in Ref. [18]. Fifty microlitres of the epidermal mucus/extruded slime in 40 mM NaH2PO4 buffer (pH 6.5) was transferred to a 96-well plate and incubated at 30 C for 15 min. Freeze dried Micrococcus lysodeikticus cells (50 ml) (0.3 mg ml1 in 40 mM NaH2PO4 buffer (pH 6.5), Sigma) were then added and the change in absorbance was monitored at 30 C for 30 min. One unit of activity was defined as the amount of enzyme that catalyzed a decrease in absorbance at 450 nm of 0.001 min1. Alkaline phosphatase (AP) activity was determined by incubating 50 ml of epidermal mucus/extruded slime reconstituted in
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100 mM NH4HCO3 containing 1 mM MgCl2 (pH 7.8) at 30 C for 15 min. Fifty microlitres of 4 mM p-nitrophenol phosphate substrate was added and the absorbance was measured at 405 nm over a 30 min period at 30 C. The initial rate of reaction was used to calculate activity. One unit of activity was defined as the amount of enzyme required to release 1 mmol of p-nitrophenol in 1 min. Cathepsin B activity was assayed using a modified method of Barrett and Kirschke [36]. Briefly, 50 ml of epidermal mucus/ extruded slime reconstituted in 0.1 M sodium phosphate buffer (pH 6.0) was incubated with 20 ml of assay buffer (0.1 M sodium phosphate, 0.08% (w/v) Brij, 1 mM EDTA, pH 6.0), 20 ml of 1 M dithiothreitol (DTT) and 60 ml of Milli Q water in a 96-well microtitre plate for 5 min at 30 C. Then 50 ml of 25 mM carbobenzoxy-Lphenylalanyl-L-arginyl-4-methylcoumaryl-7-amide substrate was added, and fluorescence of 7-amino-4-methylcoumarin (AMC) was measured for 30 min at an excitation wavelength of 380 nm and an emission wavelength of 405 nm. The initial rate of reaction was used to calculate enzyme activity and 1 U of activity was defined as the amount of enzyme required to release 1 mmol of substrate in 1 min. Protease activities were determined using the azocasein hydrolysis assay and zymography. Azocasein hydrolysis was determined by incubating 50 ml of the epidermal mucus/extruded slime re-suspended in 100 mM NH4HCO3 buffer (pH 7.8) with 50 ml azocasein substrate [0.25% (w/v), Sigma–Aldrich, Inc., MO, USA] in the same buffer for 19 h at 30 C. The reaction was stopped by adding 50 ml of 20% (w/v) trichloroacetic acid followed by a 5 min centrifugation at 15,400 g. Equal volumes (100 ml) of the resultant supernatant and 0.5 M NaOH were added to a 96-well plate and the absorbance measured at 405 nm. For zymography analysis the epidermal mucus (n ¼ 3)/extruded slime (n ¼ 5) samples dissolved in Milli Q water were combined with 1:1 with sample buffer (4% (w/v) sodium dodecyl sulphate (SDS), 125 mM Tris–HCl, 20% (v/v) glycerol and 0.5% (w/v) bromophenol blue adjusted with HCl to pH 6.8). Five micrograms of the sample were loaded onto a 12% SDS-polyacrylamide gel containing 0.1% (w/v) gelatin and electrophoresed at 4 C. Sodium dodecyl sulphate-PAGE standard markers (Broad range, Bio-Rad laboratories Inc., CA, USA) were included to estimate the molecular mass of proteases. The resulting gel was washed thrice at 4 C with 50 mM Tris–HCl (pH 7.5) containing 2.5% (w/v) Triton X-100 and incubated at 30 C for 19–20 h in 50 mM Tris–HCl (pH 7.5) containing 2.5% (w/v) Triton X-100, 20 mM MgCl2 and 6.25 mM CaCl2. After incubation the gel was stained with 0.25% Coomassie blue R-250 in MeOH/ H2O/AcOH (40:50:10) for 1 h and destained with MeOH/H2O/ AcOH (40:50:10) until the desired contrast was achieved. The effect of various inhibitors on protease activity was determined by adding specific inhibitors (i.e. 5 mM p-aminobenzamidine, 20 mg ml1 aprotinin, 5 mM EDTA, 5 mM o-phenanthroline and 5 mM iodoacetamide) during the incubation step in the azocasein hydrolysis assay and zymography analysis. When EDTA was used as the inhibitor, CaCl2 was omitted for the buffer. 2.6. Gel electrophoresis and trypsin digestion The protein profile of epidermal mucus and extruded slime were examined using tricine sodium dodecyl sulphate-polyacrylamide gel electrophoresis (Tricine SDS-PAGE) as described by Scha¨gger and Jagow [37]. Protein samples (6 mg total protein) were diluted 1:1 with sample buffer [4% (w/v) SDS, 50 mM Tris–HCl, 2% mercaptoethanol (v/v), 12% (v/v) glycerol and 0.5% (w/v) bromophenol blue adjusted with HCl to pH 6.8] and loaded onto a separating gel of 15% acrylamide with a 10% acrylamide spacer gel and 4% stacking gel. The gel was run in a Bio-Rad electrophoresis apparatus for 3.5– 4 h at 90 V. Sodium dodecyl sulphate-PAGE standard markers
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(Broad range, Bio-Rad laboratories Inc., CA, USA) were included to estimate the molecular mass of proteins. The proteins were visualized using silver staining [38] and the bands of interest were excised for identification by mass spectrometry. The in-gel trypsin digestion of the proteins was carried out following a modified method of Ebanks et al. [39]. Briefly, protein bands were carefully excised from the gel, placed separately into 1.5 ml Eppendorf tubes, washed 3 times each for 10 min with 100 ml of 50% acetonitrile (ACN) in 100 mM NH4HCO3 and then dehydrated in 100 ml of 100% ACN for 10 min. Proteins in the gel were reduced with 100 ml of 10 mM DTT in 100 mM NH4HCO3 at 56 C for 30 min, followed by alkylation in 100 ml of 100 mM NH4HCO3 containing 100 mM iodoacetamide in the dark at room temperature for 1 h. The reduced and alkylated proteins were then washed and dehydrated 2 times with 100 ml of 100 mM NH4HCO3 and 50% ACN in 100 mM NH4HCO3 for 10 min. The protein gel pieces were dehydrated with 100 ml of 100% ACN for 20 min and placed in a speedvac (Savant, Savant Instruments, Inc., NY, USA) for 10 min to remove residual ACN. The dehydrated protein gel pieces were reswollen by adding 25 ml of 12.5 ng ml1 trypsin (Promega, Madison, WI, USA) in 100 mM NH4HCO3 and incubated at 37 C overnight. The trypsin-digested peptides were extracted from the gel with 20 ml of 100 mM NH4HCO3 followed by two sequential elutions using 0.2% formic acid in 50% ACN in H2O. The eluted peptides were then concentrated using a speedvac (Savant). 2.7. Mass spectrometry (MS) protein identification The trypsin-digested peptide samples were submitted for LC/ MS/MS analysis (Atlantic Research Centre, Dalhousie University, Halifax, Canada). The on-line LC/MS/MS experiments were carried out using a Q TRAPÒLC/MS/MS (Applied Biosystems MDS SCIEX, CA, USA). The separation was carried out on a 150 0.1 mm chromolith CAP ROD C18 column (Merck, Darmstadt, Germany) using a linear gradient from 5% to 35% solvent B over 35 min (solvent A: 0.1% (v/v) formic acid in Milli Q water; solvent B: 0.1% (v/v) formic acid in ACN) at a flow rate of 1.0 ml min1. The HPLC eluates were introduced to MS via a nanoelectrospray ionization source. Data was acquired such that the mass to charge ratio (m/z) values of the tryptic peptides were measured by scan mode and this scan was used to generate a peak list of peptides for MS/MS analysis. The tandem MS spectra were submitted to the database search program MASCOT (Matrix Science, London, UK) to search against NCBInr and SwissProt databases. 3. Results Hagfish epidermal mucus was obtained by placing hagfish directly into anaesthetic containing seawater and then collected mucus from the epidermis as for other teleosts [18,21]. The volume of the epidermal mucus collected in this way was less than that of extruded slime, which was obtained following physical disturbances to the fish. Previous reports have induced hagfish to produce extruded slime using electrical stimulators or by pinching their tails with forceps [40]. We however noticed that a mild physical disturbance such as the agitation of water in the fishholding bucket was sufficient to stimulate slime extrusion. The extruded slime contains mucins, which upon hydration becomes a highly viscous gel-like mass (Fig. 1). This mode of induction was non-lethal to the fish and reduced potential contamination with body fluids. Biochemical analyses were carried out on the soluble components of the extruded slime, which presumably included the viscous gel and water soluble thread constituents [40]. A pronounced variation in specific activities of lysozyme, alkaline phosphatase (AP), cathepsin B and proteases were observed between the epidermal mucus and extruded slime of hagfish
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S. Subramanian et al. / Fish & Shellfish Immunology 25 (2008) 625–632 Table 2 Effect of specific protease inhibitors on protease specific activity (mean S.E.M.) in epidermal mucus and extruded slime of hagfish using the azocasein assay (n ¼ 10) Protease inhibitors p-Aminobenzamidine (5 mM) Aprotinin (20 mg ml1) EDTA (5 mM) o-Phenanthroline (5 mM) Iodoacetamide (5 mM)
Epidermal mucus 0 30.0 3.7 34.6 1.8 0 0
Extruded slime 52.7 2.0 47.8 1.1 36.1 2.9 5.0 3.5 0
Values are expressed as a percent inhibition of the total (100%) protease activity.
Fig. 1. Viscous gel exudate from the slime glands of stressed hagfish (Myxine glutinosa L.).
(Table 1). Lysozyme activity in the extruded slime was 198.4 6.2 U mg1 protein, which is approximately two-fold higher than that of the epidermal mucus lysozyme levels. The AP activity in extruded slime was about 3.5 times higher than that of epidermal mucus (1.4 0.1 U mg1 protein). Similarly, cathepsin B levels of extruded slime were approximately 3.4 times higher than the epidermal mucus (70.1 8.2 U mg1 protein). Protease levels, as determined by the azocasein hydrolysis assay, were about 5.0 times higher in extruded slime compared to the epidermal mucus (836.0 24.5 U mg1 protein). The effect of various protease inhibitors on the specific activity of hagfish extruded slime and epidermal mucus proteases were examined in the azocasein hydrolysis assay (Table 2). The specific trypsin-like protease inhibitor, p-aminobenzamidine, inhibited 50– 54% of the protease activity in extruded slime but showed no inhibition of protease activity in the epidermal mucus. Aprotinin caused 46–48% inhibition of protease activity in the extruded slime and 30–33% in the epidermal mucus. EDTA, showed similar levels of inhibition (34–38%) of activity in the extruded slime and epidermal mucus proteases. The zinc chelator, o-phenanthroline, was found to reduce protease activity by 2–8% in extruded slime but had no observable inhibition of the protease activity in epidermal mucus. The extruded slime and epidermal mucus protease activity were unaffected by the cysteine protease inhibitor, iodoacetamide. A variation in protease profiles was observed in the zymographic analysis of hagfish epidermal mucus and extruded slime (Fig. 2A,B). The extruded slime had low molecular mass proteases in the range Table 1 Comparison of enzyme activities (mean S.E.M.) of hagfish epidermal mucus and extruded slime (n ¼ 10) Enzyme activity
Epidermal mucus
Extruded slime
Lysozyme Alkaline phosphatase Cathepsin B Protease
105.0 5.2 1.4 0.1 70.1 8.2 836.0 24.5
198.4 6.2 4.9 0.3 239.8 10.1 4517.3 145.2
Specific activity is expressed as U mg1 protein.
of 18–22 kDa and several medium molecular mass proteases of approximately 29–32 kDa and 40 kDa, which were not observed in the epidermal mucus. The dense protease bands of high molecular mass at 90–150 kDa in the extruded slime were not observed in the epidermal mucus. Medium to high molecular mass proteases in the range of 50–90 kDa were found in both the epidermal mucus and the extruded slime. Incubation of zymography gels with the specific protease inhibitor, aprotinin, showed effective inhibition of the majority of the proteases in both the epidermal mucus and extruded slime, except for the high molecular mass proteases at 90–150 kDa (Fig. 3 section 2 lanes A and B). p-Aminobenzamidine showed similar inhibition patterns as aprotinin (Fig. 3 section 3 lanes A and B). EDTA and o-phenanthroline each showed nearly complete inhibition of protease activity in epidermal mucus (Fig. 3 section 4,5 lane A). In contrast, the EDTA showed poor inhibition of low (w18– 22 kDa) and high molecular mass proteases (w90–150 kDa) in extruded slime (Fig. 3 section 4, lane B). Similarly, the o-phenanthroline did not inhibit medium (w40 kDa) and high molecular mass proteases (w90–150 kDa) (Fig. 3 section 5 lane B) in extruded slime. Iodoacetamide exhibited minimal inhibition of epidermal mucus proteases (Fig. 3 section 6 lane A), but the low (w18–22 kDa) and most of the medium molecular mass proteases (w29–32 kDa and w40 kDa) were inhibited in the extruded slime (Fig. 3 section 6 lane B). Electrophoretic comparison of epidermal mucus and extruded slime (Fig. 4 lanes A and B) showed that the distribution of the majority of the medium and high molecular mass (>40 kDa) protein bands were similar, except for two protein bands at approximately 30 kDa and 40 kDa, which were more intense in the epidermal mucus. A marked difference in the protein composition and a strong upregulation of three proteins of molecular masses range of 13–16 kDa, was observed in the extruded slime (Fig. 4 lanes A and C band numbers 1, 2 and 3). The three distinct proteins of extruded slime (Fig. 4 lane C) were excised, in-gel trypsindigested and analysed by electrospray ionization LC/MS/MS. Search of NCBInr and SwissProt databases with the Mascot search engine found high matches for each band (Table 3). Protein 1 (16.7 kDa) matched the hemoglobin-3 of hagfish (M. glutinosa) (MASCOT score of 287), with homology to a number of distinct peptides of hemoglobin-3. Protein 2 (15.3 kDa) and protein 3 (13.6 kDa) showed poor or no similarity to any of the hagfish proteins in the database. MASCOT analysis of protein 2 showed good matches with the conserved regions of histone H3 identified in a wide range of organisms such as mouse, fruit fly, roundworm, innkeeper worm, sea star, marine copepod, staghorn coral, African clawed frog and humans with a score of 82. Protein 3 matched well with the conserved domain of histone H2B characterized from several organisms including marine copepod, mosquito, African clawed frog, yeast and different species of fruit with a score of 156. 4. Discussion The current study examines the biochemical differences between hagfish epidermal mucus and extruded slime. During the
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1
2
3
4
5
6
7
629
8
194.6 95-150
116.5 97.2
50-90 50.2 40 37.6 29.3
29-32
20.0
18-22
A
B
Fig. 2. Protease zymography of A: epidermal mucus and B: extruded slime sample of hagfish without inhibitor. Numbers 1–8 on the top of gel indicates the individual fish. Molecular mass (kDa) markers are along the left side of the gels. The approximate mass range for high, medium and low molecular mass proteases in kDa is indicated in brackets along the right side of the gels. Each well contains 5 mg of mucus/extruded slime protein.
collection of epidermal mucus, the fish may have experienced some stress during the process of anaesthetization. However, studies on the effect of tricaine methanesulphonate on fish physiological stress responses have shown no significant variations in the level of stress indicators, such as plasma cortisol, of anaesthetised fish in comparison to pre-anaesthetized fish [41]. Tricaine methanesulphonate functions by interfering with the ion movements in nerves and muscles [42] thus reducing fish activity and minimising physical stress. The epidermal mucus in this study was collected very gently to minimise the induction of physical stress to fish. The consistency of hagfish epidermal mucus was similar to that of other fish species sampled using this method [18,21]. In contrast, the stress induced extruded slime secretion of hagfish was a highly viscous gel-like mass containing protein rich threads and mucins. The epidermal mucus of fish contains biologically active substances that exhibit defensive functions against infectious pathogens in the aquatic environment [13,18,19,25]. Significant variations in lysozyme, AP and cathepsin B activity have been previously reported in the epidermal mucus of hagfish in comparison to various teleosts [18]. The present study shows that the extruded slime of hagfish has elevated levels of these enzymes and antimicrobial protein/peptide precursors including
194.6 116.5 97.2
A
B
A
B
A
B
hemoglobin, histone H3 and H2B compared to its normal epidermal mucus. Lysozyme is a bacteriolytic enzyme that has been shown to lyse bacterial cells. The epidermal mucus lysozyme of yellowtail (Seriola quinqueradiata), common carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss) exhibited bacteriolytic activity against various pathogens [43]. As scavengers, hagfish encounter various pathogenic microorganisms in dead and decaying organisms. The increased level of lysozyme in extruded slime suggests that the slime might be secreted to provide increased protection against potential microbial pathogens. The precise function of AP in the mucosal innate immunity of fish is unclear. However the variation in AP levels in Atlantic salmon tissues and blood during smoltification have been correlated to stress response [44]. The observation of 3.5 times higher AP activity in the hagfish extruded slime than the epidermal mucus was thought to be due to the physical stress induced to the hagfish during collection of the extruded slime secretion. Elevation in the specific AP activity had been observed in fish epidermal mucus following the physical, chemical and microbial stress [21–23]. The cysteine protease, cathepsin B, has been observed at higher levels in the epidermal mucus of bottom dwelling fish species such as
A
B
A
B
A
B
50.2 37.6 29.3 20.0
1
2
3
4
5
6
Fig. 3. Protease zymography of representative A: epidermal mucus and B: extruded slime of hagfish. 1. no inhibitor and 2–5 incubated with specific protease inhibitors, 2: aprotinin (20 mg ml1), 3: p-aminobenzamidine (5 mM), 4: EDTA (5 mM), 5: o-phenanthroline (5 mM) and 6: iodoacetamide (5 mM). Molecular mass (kDa) markers are along the left side of the gels. Each well contains 5 mg of mucus/extruded slime protein.
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S. Subramanian et al. / Fish & Shellfish Immunology 25 (2008) 625–632
194.6 116.5 97.2
50.2
37.6 29.3 20.0
1
2
3
7.1
A
B
Fig. 4. Tricine SDS-PAGE of hagfish epidermal mucus and extruded slime on a 15% gel. Each well was loaded with 6 mg of total protein. The gel after electrophoresis was silver stained to visualize the protein bands. A: epidermal mucus, B: extruded slime. The numbers on right side indicate the bands that were selected for proteomic analysis 1: band 1, 2: band 2 and 3: band 3. Molecular mass (kDa) markers are along the left side of the gels.
koi carp, flounder and eel species [18,29,30]. Cathepsins, B and L, levels have also been found to increase in epidermal mucus of flounder and eel species following environmental and microbial stress, presumably to provide protection against microbial pathogens [30,39]. The present observation of an approximately three-fold increase in cathepsin B levels in extruded slime could be the genetic adaptation of hagfish to thrive in the microbial rich environments. A marked variation in protease activities between epidermal mucus and extruded slime was observed. In the azocasein assay, extruded slime showed a higher ratio of serine to metalloproteases, while the epidermal mucus had almost equal proportions of serine and metalloproteases. In the extruded slime, the trypsin specific
protease inhibitor, p-aminobenzamidine, reduced approximately 50% of proteases. This suggested that trypsin-like proteases might be a major constituent of hagfish extruded slime. The metalloprotease inhibitors, o-phenanthroline and EDTA, showed discrepancies in protease inhibition. The o-phenanthroline exhibited poor to no inhibition of protease activity and the EDTA reduced approximately 36% of the protease activity in the epidermal mucus and extruded slime. Such variations were not observed in the zymography, where both inhibitors had similar effects on both the epidermal mucus and extruded slime. Differences in the substrate used and assay conditions could be responsible for the discrepancies observed in the two assays. For example, the presence of detergents in zymography causes potential denaturation, which may activate latent proteases [20,45] that would not be observable using the azocasein assay. Alternatively, it is possible that some proteases may be irreversibly denatured during zymography. Both the metallo- and serine protease inhibitors showed similar inhibition of majority of proteases in the extruded slime. This indicates the presence of serine-activated metalloproteases or cation-specific serine proteases, which may be reduced by both metallo- and serine protease inhibitors. Even though epidermal mucus and extruded slime have cathepsin B activities, iodoacetamide showed no inhibition in zymography or the azocasein assay. Cysteine proteases are known to be activated under reduced conditions [36], the absence of dithiothreitol (DTT) or b-mercaptoethanol therefore may be the reason for the poor inhibition observed by iodoacetamide in these assays. The serine and metalloproteases from fish epidermal mucus has been shown to play roles in the innate defense mechanisms. Trypsin, a low molecular weight serine protease, isolated from the epidermal mucus of rainbow trout has been shown to have antibacterial activity against Vibrio anguillarum [36]. The trypsin and other serine proteases have been shown to activate immunoglobulin (IgE) [46] and antimicrobial peptides including defensin and cathelicidin in mammalian epithelial cells [47]. A metalloprotease isolated from the epidermal mucus of plaice (Pleuronectes platessa) exhibited antimicrobial activity against both Gram positive and Gram negative bacteria [48]. Metalloproteases have also been found to trigger other proteases [49], antimicrobial peptides [27] and various immune factors such as cytokines and chemokines [49]. Similar roles can be postulated for trypsin-like and metalloproteases present in hagfish extruded slime however further study needs to be carried out.
Table 3 Summary of proteins identified from Fig. 4 Protein homolog (Swissprot accession no.)
Parent ion (m/z)
Mr (Da)
Error (ppm)
Score
Peptide sequence of matched entry
Band 1 –hemoglobin-3 of Atlantic hagfish (P02209)
607.4464 525.7872 626.4332 790.5000
1818.9112 1049.5182 1250.6619 1578.7137
0.4062 0.0418 0.1899 0.2718
67 48 70 96
PITDHGQPPTLSEGDKK ESWPQIYK KSHLEQDPAVK HSTEFQVNPDMFK
Band 2 – histone H3 (Q9LR02)
516.8659 416.3018
1031.5876 830.4861
0.1297 0.1029
38 45
YRPGTVALR STELLIRa
Band 3 – H2B (P35068)
575.3752 887.4800 477.3598
1148.5753 1772.8226 952.5957
0.1604 0.1229 0.1095
45 68 43
ESYAIYIYK AMSIMNSFVNDIFERb LLLPGELAKb
The proteins of extruded slime (bands 1, 2 and 3) were identified using liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS). m/z: specific mass to charge ratio, Mr: relative molecular weight (Da), error: difference between measured mass and the predicted mass, score: score from MASCOT search; scores above 35 are highly significant, and peptide sequence: tryptic peptide sequence predicted. a LC/MS/MS sequences matched to the conserved region of histone H3 protein of a wide range of organisms (score of 82) such as Mouse-ear cress (SwissProt accession no. Q9LR02), fruit fly (SwissProt accession no. P84236), roundworm (SwissProt accession no. P08898), innkeeper worm (SwissProt accession no. P84239), Sea star (SwissProt accession no. P69071), marine copepod (SwissProt accession no. P84237), staghorn coral (SwissProt accession no. P22843), African clawed frog (SwissProt accession no. P02302) and human (SwissProt accession no. Q6NXT2). b LC/MS/MS sequences matched to the conserved domain of histone H2B protein with a score of 156 to several organisms, for example, marine copepod (SwissProt accession no. P35068), different species of fruit fly (SwissProt accession nos. P59781, Q8I1N0, Q76FF3, Q76FD7, Q76FE5, Q76FE9), African clawed frog (SwissProt accession no. NP_001086753), yeast (SwissProt accession no A5DWF0), and yellow fever mosquito (SwissProt accession no. XP_001656991).
S. Subramanian et al. / Fish & Shellfish Immunology 25 (2008) 625–632
Tricine SDS-PAGE analysis demonstrated the high levels of three major proteins of molecular mass ranging from 13 kDa to 16 kDa in hagfish extruded slime. Mass spectroscopic analysis of the extruded slime tryptic fragments suggested that protein 1 is closely related to hemoglobin-3 of Atlantic hagfish. The hagfish hemoglobins, unlike the tetrameric hemoglobins of higher vertebrates, are monomeric and had evolved before the separation of myoglobin and hemoglobin [4]. The observed molecular mass of hemoglobin in the hagfish extruded slime matched with the average molecular mass of vertebrate myoglobins and other cyclostome hemoglobins [50]. The precise function of hemoglobin in the hagfish extruded slime is unclear but, previous studies have detected hemoglobin in the epidermal mucus of fish stressed with physical and environmental stressors [51–53]. The presence of hemoglobin in epidermal mucus was often considered an indicator of stress and thought to be derived from red blood cells into the epidermal mucus [53]. This might explain the presence of hemoglobin in the stress induced hagfish extruded slime. Serum hemoglobin and hemoglobinderived peptides have been shown previously to exhibit antimicrobial activity in higher vertebrates [54,55]. Such functions are yet to de determined in extruded slime hemoglobin. Bands 2 and 3 (Fig. 4 lane C) showed close matches to the histone proteins, H3 and H2B, respectively. The matched peptides were from conserved regions of the histone proteins from microorganisms, flies, amphibians and mammals (Table 3). Histones are known for their roles as a structural component of the nucleosome, but also exhibit antimicrobial properties [56]. Antimicrobial activity of histone proteins (H2B and H3) or their cleaved products had been reported in the epidermis and epidermal mucus of catfish (Ictalurus punctatus), rainbow trout (O. mykiss) and Atlantic cod (Gadus morhua) [13,57]. The H2B proteins in murine macrophages and hemocytes of Pacific white shrimp (Litopenaeus vannamei) have also been shown to exhibit antimicrobial activity [58,59]. The observed high levels in extruded slime suggest that histone proteins might be involved in hagfish innate defense against potential pathogens. In conclusion, this study demonstrated that the stress induced extruded slime secretion of hagfish contains various innate immune parameters in comparison to its normal epidermal mucus. The hagfish lacks a defined adaptive immune system and they rely heavily on innate immunity for disease resistance [60]. The high levels of the innate immune substances suggest that the extruded slime is produced more likely to provide protection against microbial, environmental and other physiological stress. This is in addition to the already postulated function of extruded slime as a defense against predators. Further investigations into the isolation and mechanisms of these immune components from the extruded slime could provide a better understanding of their roles in the innate defenses of evolutionarily primitive fish. Acknowledgments The authors would like to thank Mr. Eric MacKinnon (Cape Sable Island, NS) for providing hagfishes and Ron Melanson for help with sampling. Elden Rowland and Roger Ebanks for assistance with the mass spectrometric analysis. This research was funded by National Research Council of Canada and the Ford Foundation International Fellowship Program. References [1] Forey P, Janvier P. Agnathans and the origin of jawed vertebrates. Nature 1993;361:129–34. [2] Bardack D. Relationships of living and fossil hagfishes. In: Jorgensen JM, Lomholt JP, Weber RE, Malte H, editors. The biology of hagfishes. London: Chapman and Hall; 1998. p. 1–31.
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