Population growth and the declining efficiency of traditional fisheries necessitate an increase in both the production and diversity of cultured species. This drives the application of multiomic molecular techniques to study the immune response of fish, with proteomics being one such technique. Proteomics offers a precise and comprehensive view of the proteome and the changes it may undergo due to intrinsic or extrinsic factors affecting the animal. In this context, we have examined the impact of hepcidin 1 (Hamp1) on the proteome of the brain and skin of sea bream (Sparus aurata), a significant species for marine aquaculture in temperate and warm waters [1]. Hamp1 is a host defense peptide (HDP) [2]. HDPs are small, mostly cationic peptides with potent biocidal and immunomodulatory effects. Studying them could enhance fish health in aquaculture, thereby improving fish production and quality. Consequently, in this study, we intramuscularly injected synthetic Hamp1 peptide (50 μL at 25 μM in sterile phosphate buffer). Brain and skin samples were collected for protein extraction using RIPA Buffer and trypsin digestion at 4 and 10 days post-injection. The proteome in the samples was then analyzed by HPLC-MS/MS. Data analysis with Spectrum Mill MS Proteomics software revealed that Hamp1 injection induced changes in the sea bream proteome, as observed in the PCA and PLS-DA, in the brain at 4 days post-injection and in the skin at both 4 and 10 days post-injection.
Among the proteins whose expression varied most in the brain of seabream, sampled four days after peptide injection, were helicase-like transcription factor (HLTF), myelin basic protein (LOC115578167), and α-2-HS-glycoprotein 2 (A0A671UQD9). Additionally, ten days post-injection, RYR3 and α-1,6-mannosyl-glycoprotein 6-β-N-acetylglucosaminyltransferase (MGAT5B) showed increased levels in this organ. Curiously, in the brain, Hamp1 elevated the concentration of proteins within the nucleus, such as HLTF, which plays a role in DNA repair, ATP binding, or helicase/hydrolase activity, as well as proteins in the myelin membrane that serve a structural function.
In contrast, the skin exhibited a greater increase in protein expression compared to the brain. Notably, protein disulfide isomerase (A0A671Y1J8, which is involved in the endoplasmic reticulum stress response), Galectin (LOC115574816), and Alsin Rho guanine guanine (LOC115574816) were elevated four days after injection. Furthermore, Alsin Rho guanine nucleotide exchange factor ALS2 b (ALS2, which is found in the centrosome and plays an active role in cell division) and Peptidyl-prolyl cis-trans isomerase (Ppib, which is associated with RNA polymerase II, protein folding, and isomerase activity) increased at four days, pression of proteins located in the lumen of the endoplasmic reticulum. The protein Galectin, which binds to carbohydrates, galactosides, and laminin and is present in the extracellular matrix or collagen-containing protein, also showed increased expression. Finally, in the skin, we detected an enhanced response to Hamp1 at 10 days post-injection, resulting in a significant increase in proteins while α-1,4 glucan phosphorylase (A0A671V311), Phosphoglycerate kinase (A0A671UGW5), and Rabphilin 3A homolog (mouse), b (RPH3A), among others. A0A671V311 and A0A671UGW5 are present in the cytoplasm and involved in the cell’s energy metabolism, including carbohydrate storage, glycogen catabolic process, carbohydrate degradation, glycolysis, pyruvate metabolism, and gluconeogenesis, among others. Furthermore, the expression of RPH3, a protein capable of binding metal ions, GTPase, and involved in intracellular protein transport, exocytosis, and spontaneous neurotransmitter secretion, was also greatly increased. This protein is present in the synaptic vesicle membrane and neurons.
The findings indicate that the impact of each host defense peptide (HDP) is contingent upon the specific organ under investigation. Furthermore, the results suggest the involvement of certain proteins, either directly or indirectly, in the immunomodulatory effects of HDPs, which may differ depending on the organ. To the best of our knowledge, this study presents the inaugural findings on the effects of HDPs in the brain of a commercially significant fish species. The administration of Hamp1 induces alterations in the proteome of the seabream brain, with more pronounced changes observed at 4 days compared to 10 days. Notably, many of the upregulated proteins are localized within the cell nucleus and are associated with DNA. Conversely, in the skin, the effects of Hamp1 are evident at both 4 and 10 days, with a more substantial proteomic alteration occurring at 10 days. This is characterized by the overexpression of proteins within the endoplasmic reticulum and cytoplasm, facilitating processes such as cell division and neuronal signaling.
Acknowledgements
This research has been financed by the proyecto PID2020-113637RB-C21 de investigación financiado por MCIN/ AEI /10.13039/501100011033 and it forms part of the ThinkInAzul programme supported by MCIN with funding from European Union Next Generation EU (PRTR-C17.I01) and by Comunidad Autónoma de la Región de Murcia - Fundación Séneca.
References
[1] FAO, The State of World Fisheries and Aquaculture 2024, FAO ;, 2024. https://openknowledge.fao.org/handle/20.500.14283/cd0683en (accessed March 15, 2025).
[2] A. Cuesta, J. Meseguer, M.Á. Esteban, The antimicrobial peptide hepcidin exerts an important role in the innate immunity against bacteria in the bony fish gilthead seabream, Molecular Immunology 45 (2008) 2333–2342. https://doi.org/10.1016/j.molimm.2007.11.007.