Chemical composition, biological activities and proteomic profiling of epidermal mucus in two catfish species from the genus Clarias

• Main Author: Nurul Mariam Hussin
• Format: Thesis
• Language: English; English
• Published: 2020
• Subjects: QR180-189.5 Immunology
• Online Access: https://eprints.ums.edu.my/id/eprint/40977/1/24%20PAGES.pdf; https://eprints.ums.edu.my/id/eprint/40977/2/FULLTEXT.pdf; https://eprints.ums.edu.my/id/eprint/40977/
• Institution: Universiti Malaysia Sabah

Clarias gariepinus and Clarias anfractus (genus Clarias) are potentially catfish to secrete epidermal mucus. The mucus serves as a first physical barrier of defence against pathogens, external stressors and chemical attacks. Here, we study the chemical compositions, biological activities and protein profile of epidermal mucus from both catfish species. Proximate analysis (moisture, ash, protein, lipid and carbohydrate), mineral content determined using atomic absorption spectroscopy (AAS) and inductive coupled plasma mass spectrometry (ICP-MS), amino acid determination using high-performance liquid chromatography (HPLC) and fatty acid profile analyzed using gas chromatographic with flame ionization detector (GC-FID) were the evaluation of chemical compositions for the two catfish species epidermal mucus. The moisture content was a major component in the epidermal mucus (Cgariepinus. 90.01 ± 3.33 %; Canfractus. 88.15 ± 0.32 %). The level of ash was significant (p<0.05) higher in Cgariepinus epidermal mucus (7.65 ± 1.10 %) while the level of carbohydrate was significant (p<0.05) higher in epidermal mucus of Canfractus (5.70 ± 0.37 %). Although the ash content of Canfractus epidermal mucus lowers then Cgariepinus, this study discovered its contained high levels of macro-elements minerals (K, Na, Mg, Ca and P). Both Na/K and Ca/P ratios in both catfish species epidermal mucus were less than one, agreement with the requirement of FAQ/WHO. High amounts of amino acids (8.998 ± 0.014 %) and polyunsaturated fatty acids (PUFAs; 48 %) as compared to saturated fatty acids (SFAs; 39 %) and monounsaturated fatty acids (MUFAs; 13 %) also found in Canfractus epidermal mucus. These can contribute positively to human nutritional requirements and consumptions. The antioxidant and antimicrobial activities were evaluated for the biological activities of the catfish species epidermal mucus. All the mucus were extracted using acidic, organic ( dichloromethane (DCM) and aqueous phase) and water solvents to determine the DPPH radical scavenging, reducing power, ferric reducing power (FRAP), antibacterial and antifungal activities. The antimicrobial activities were based on the disc diffusion method. Overall, the DPPH scavenging effect in epidermal mucus of both catfish extracts ranges from 35 % to 55 %. Interestingly, the acidic extract of Cgariepinus epidermal mucus showed significantly (p<0.05) higher of reducing power activity compared to BHT and ascorbic acid at a concentration of 80 μg/mL; ascorbic acid at 100 μg/ml. All epidermal mucus extracts showed low of FRAP activity compared to BHT and ascorbic acid. Although the antioxidant activities of both catfish epidermal mucus extracts showed low activities (p<0.05) compared to the BHT and ascorbic acid, it revealed these catfish epidermal mucus could be potentially rich sources of natural antioxidants. These findings can be used as a basis for more advanced research on catfish epidermal mucus antioxidant capability. Of the screened epidermal mucus extracts, the acidic epidermal mucus extract of Canfractus exhibited significant (p<0.05) high levels of inhibitory activity (12.2 ± 2.54 mm) against Gram-positive bacteria, Bacillus cereus. All epidermal mucus extracts also showed inhibition zone against Gram-negative bacteria, Escherichia coli (range 6.77 to 9.44 mm) and Pseudomonas aeruginosa (range 6.16 to 8.60 mm). None of the acidic extracts was active against Candida albicans. From the biological activities experiments, its demonstrate that Cgariepinus and Canfractus epidermal mucus were able to give average 22.52 ± 0.40 % of acidic extract yield, 9.06 ± 1.00 % of DCM phase extract yield, 13.36 ± 1.21 % of aqueous phase extract and 35.43 ± 3.15 % of water extract yield. The yields expressed as grams of dry extract per 100 g of catfish epidermal mucus. Furthermore, the study also was done to find one the most efficient extraction method for proteomic analysis of the catfish epidermal mucus. It found that precipitation with trichloroacetic acid (TCA) and acetone result was an efficient sample concentration and desalting for proteomic analysis, compared to precipitation with ammonium sulfate. Therefore, this protein extraction method was chosen for a routine procedure for the proteomic analysis of C.gariepinus and C.anfractus epidermal mucus. A distinctive protein pattern was observed on the SDS-PAGE gels between two different catfish epidermal mucus and managed to get 2D-PAGE proteome map. The image analysis of 2D-PAGE gel was performed with Progenesis SameSpots version 4.5 and two sets of comparison of the 2D-PAGE gel images were done, Set 1: C.gariepinusvs Canfractus, and Set 2: C.anfractus vs C.gariepinus to detect differentially expressed protein spots. Results of image analysis revealed 277 differentially expressed proteins for experiment set of C.gariepinus vs C.anfractus and 211 differentially expressed proteins for experiment set of C.anfractus vs C.gariepinus. Only 20 differentially expressed proteins from all two sets of experiments have successfully identified and characterized by the Shimadzu Prominence nano HPLC system [Shimadzu] coupled to a 5600 TripleTOF mass spectrometer [Sciex] and Mascot sequence matching software [Matrix Science]. A reference proteome map of Cgariepinus and C.anfractus epidermal mucus were obtained for the first time. The proteins identified in C.gariepinus and C.anfractus epidermal mucus can be categorized (protein biological functions) into three types of the general group of protein-based on the gene ontology (GO) analysis. The 55 % of structural proteins (two proteins of keratin, type II cytoskeletal 8; tropomyosin alpha-1 chain; actin, cytoplasmic 1; intermediate filament protein ON3 and glial fibrillary acidic protein), followed by 36 % of metabolic proteins (alpha-enolase, transcription factor AP-2-delta; Cyclindependent kinase 8 and MI COS complex subunit mic25a) and 9 % of protectionrelated protein (heat shock cognate 71 kDa protein) were successfully identified in C.gariepinus epidermal mucus. In C.anfractus epidermal mucus, the majority of the proteins were identified as metabolic proteins (67 %: glyceraldehyde-3-phosphate dehydrogenase; alpha-enolase; nascent polypeptide-associated complex subunit alpha; protein PATl homolog 1; kynurenine--oxoglutarate transaminase and triosephosphate isomerase B), followed by protection-related proteins (22 %: heat shock cognate 71 kDa protein and ferritin, middle subunit) and structural proteins (11 %: keratin, type I cytoskeletal 18). We can summarize from all the three main functions of protein, the epidermal mucus of catfish genus of Clarias consist of 51 % of metabolic proteins, 33 % of structural proteins and 16 % of protective-related proteins. The protein findings from epidermal mucus of C.gariepinus and C.anfractus may also be related to the biological activity screening evaluation that showed both species able to give some antioxidant, antibacterial and antifungal activities. Furthermore, the proteomic data provide systematic protein information together with the other chemical compositions data to comprehensively understand the biological function of C.gariepinus and C.anfractus epidermal mucus. In conclusion, the wild catfish, C.anfractus epidermal mucus contain various functional proteins and other beneficial chemical compounds compared to cultured catfish, C.gariepinus epidermal mucus. Therefore, mucus of this species has the potential too to be used in the nutraceutical and pharmaceutical industries. These were as indicated through the biological activities, bioactive compounds and identified functional proteins found in C.anfractus epidermal mucus.