In our study, the NG and WG samples were well differentiated by label-free and TMT-labeling proteomics techniques. Differential expression analysis showed that the label-free and TMT groups had 643 and 107 DEPs, respectively. The types of DEPs detected by different proteomics methods showed significant differences, which also led to differences in the results of their corresponding functional and metabolic pathway analyses. In the label-free cluster, the detected DEPs were analyzed by GO functional enrichment, mainly in the cell part, intracellular, peptide metabolic process, cellular amide metabolic process, and structural molecule activity. These DEPs were mainly enriched in KEGG pathways such as Protein digestion and absorption (map 04974), Ribosome (map 03010), Spliceosome (map 03040), Focal adhesion (map 04510), and EGFR tyrosine kinase inhibitor resistance (map 01521). In the TMT cluster, these DEPs were mainly assembled in GO functions, including cellular component organization or biogenesis, molecular function regulator, and extracellular matrix. DEPs were mainly enriched in KEGG pathways, including EBV infection (map 05169), Pathways in cancer (map 05200), HIF-1 signaling pathway (map 04066), Small cell lung cancer (map 05222) and Tight junction (map 04530). In addition, our study found some co-DEPs in the label-free and TMT groups by PPI network interactions analysis, such as evm.model.Contig293_pilon.5, was myosin heavy chain 11 (myosin-11). The DEP is mainly involved in GO enrichment analysis in motor activity and ATP binding (MF) and myosin complex (CC). Although myosin-11 showed up-regulation in both the label-free and TMT groups, analyzed singularly in terms of p-value and CV. It was more significant in the label-free group, which could not perform well in PPI network analysis due to the excessive number of DEPs detected in the label-free group. On the contrary, myosin-11 in the TMT group showed more visualization in the PPI interaction network analysis. Therefore, we conclude that the label-free method is superior to the TMT method in terms of the number of DEPs. TMT was better in terms of reproducibility and accuracy. This result is consistent with the conclusion of the study by Megger et al. (2014).
The myosin-11 is an ATP-dependent molecular motor in eukaryotic cells that plays an important role in cell motility and intracellular substance transport. Contraction of striated muscle is caused by an increase in cytoplasmic Ca2+ concentration, and the binding of Ca2+ to troponin C shifts the "on-off" equilibrium of the filamentous state to the "on" state, facilitating actinoglobulin interactions. When the temperature rises into the body temperature range, the equilibrium shifts to the "on" state even in the absence of Ca2+. At low temperatures, filaments slide faster on skeletal myosin than on β-cardiac myosin. The mammalian striated muscle can contract efficiently in response to the physiological demands of different environments and within the body temperature range through the complementary regulation of myosin and protomyosin-troponin (Ishii et al. 2023). Mammals maintain a constant body temperature in cold environments through physiological adjustments. Under cold conditions, myosin undergoes significant physiological modifications, with increased levels of phosphorylation of myosin light chairr2 (MLC2) in favour of increased myosin ATPase activity (Katoch and Soni 1999). Fish myosin heavy chain exon sequences show a high degree of homology with mammalian myosin sequences, and they have been highly conserved throughout evolution. Myocardial metabolic enzyme activity and α-myosin heavy chain (MHC) are affected in high altitude environments. Cardiomyocytes are favourably adapted to hypoxic high-altitude environments by increasing enzyme activity and the percentage of α-MHC (Cai et al. 2010). Under hypoxic conditions, MLC kinase (MLCK) protein is highly expressed and phosphorylated by MLC, which disrupts the endothelial barrier function, and also induces HIF-1 activation by increasing HIF-1 α expression, nuclear accumulation, DNA binding activity, and HIF-1 target gene expression in endothelial cells (Qi et al. 2011). G. maculatum lives at high altitudes with low temperatures and low oxygen, and the up-regulation of myosin11 may be a response to the living environment. The activity of myosin11 inhibits the initiation of endothelial cell pseudopodial branch and regulates angiogenesis, which may promote the emergence of protrusions in the outside of the middle part of the NG, the formation of indentations, and ultimately the formation of the WG. Nitric oxide (NO) has been identified as a potent molecule that attenuates the effects of ionic stress responses in fish. The activated inducible nitric-oxide synthase (iNOS)/NO in hypoxic fish is favoured for stress adaptation processes to exhibit a cycle of adaptive responses. Fish can rely on the iNOS/NO system to restore basal homeostasis through recovery activities (Peter et al. 2022). Hypoxic conditions can lead to fibrosis of the liver, and HIF-1 signaling worsens the metabolic profile and hastens nonalcoholic fatty liver disease progression (Mesarwi et al. 2021). The suggested 'morphological remodelling' of the epithelium under hypoxia in gut-breathing fish (GBF) has many standard features with carcinogenesis (Satora et al. 2020). hypoxia upregulates marine medaka (Oryzias melastigma) telomerase reverse transcriptase expression via Oryzias melastigma HIF-1 in non-neoplastic fish liver and testis in vivo (Yu et al. 2006). Tight junction between hepatocytes, observed in zebrafish liver, plays an essential role in the remodelling process during organ morphogenesis (Cheung et al. 2012). The myosin-11 is involved in tightly connecting this pathway. evm.model.chr15.573 is essential for functions such as cytoskeletal organization, growth, differentiation, and neuronal development (Moon and Zheng 2003). These proteins may be associated with WG generation. Our study found significant differences in the KEGG metabolic pathway, cancer pathway, EB virus infection, HIF-1 signaling pathway, and small-cell lung cancer in the TMT group. HIF-1 is a key mediator of hypoxia, consisting of two subunits, HIF-1α and HIF-1β, which can activate the transcription of a variety of target genes in hypoxia. HIF-1 overexpression and enhanced transcriptional activity have been associated with tumourigenesis, and it has an important role in cellular and tissue growth, development, and physiological stress as well as in cancer and inflammation (Galanis et al. 2008; Wang et al. 2011). The 26S proteasome non-ATPase regulatory subunit is differentially expressed in cancer tissues of hepatocellular carcinoma. The p90 ribosomal S6 kinase phosphorylates neural cell adhesion molecules, leading to alterations in neural cell adhesion molecule conformation and function that contribute to neurite outgrowth (Wong et al. 1996). The development of WG involves cell adhesion (zhang et al. 2021), so we postulated that the p90 ribosomal S6 kinase is involved in the production of WG. Eph receptor tyrosine kinases and their ligands (ephrins) are key players in the development of the embryonic vascular system, and hypoxia not only upregulates the expression of HIF-1α and vascular endothelial growth factor, but also Ephs and hepatic collaterals of subclasses A and B in the skin, which have a role in neovascularisation in mice (Vihanto et al. 2005). The Villin protein are present in some lamellar ionocytes of freshwater fish and have the function of severing actin filaments. Villin genes and proteins are expressed at higher levels in freshwater fish compared to marine fish (Kang et al. 2017). Villin proteins induce cell surface microvillus growth and F-actin redistribution, favouring microvillus morphogenesis. The ferrochelatase is upregulated during hypoxia and regulates mitochondrial respiration. The small subunit of calpain plays an important role in cell migration (Friederich et al. 1990). Calpain small subunit also interacts with actin and is involved in embryonic development, angiogenesis, immune response, cancer metastasis, and other processes through cell migration.
In the label-free group, Ubiquitin-like protein 4A (UBL4A) was significantly down-regulated, and Heat shock 70 kDa protein (Hsp70) was significantly up-regulated. UBL is capable of modifying proteins and functions to regulate various biological pathways such as hypoxia response, proteostasis, and DNA damage response. UBL4A also functions as a tumor suppressor and DNA damage-mediated apoptosis; UBL4A causes autophagosome accumulation by interfering with lysosome function and leads to impaired autophagic degradation in vitro, suppressing tumor proliferation and metastasis by inhibiting autophagy (Chen et al. 2019). Mitigation of acute kidney injury (AKI) in septic mice by down-regulation of UBL4A provides a potential therapeutic strategy for AKI (He et al. 2020). Hsp70 plays a vital role in protecting organisms from environmental stresses (Fang et al. 2021). Hsp70 is involved in oxidative stress and has a protective and stabilising effect on stress-induced cellular damage. The mRNA expression level of CaHsp70 was highly significantly up-regulated in the kidney, liver, spleen and brain tissues after heat shock treatment, which had a more significant effect on the mRNA expression level of CaHsp70 (Aiguo et al. 2020). Differences in the timing of expression and regulatory mechanisms of different members of the Hsp70 family may be related to the specific response of rat liver to elevated body temperature (Schiaffonati et al. 2010). Low-temperature conditions induce significant up-regulation of Hsp70 and Hsp90, which can protect organisms from physiological and cellular damage induced by low-temperature stress (Ning et al. 2022). Conservation of the Hsp70 gene in the fish species family may be caused by climate change. Catfish (Ictalurus punctatus) respond to low-temperature environments by regulating the expression of Hsp70 and Hsp90 chaperone genes (Ju et al. 2002). Chronic exposure to cold conditions inhibits the ubiquitin-proteasome system, and oxidised proteins accumulate in the liver, causing damage to liver proteins and affecting liver function in fish (Sergio et al. 2019).
G.maculatum lives in a low-temperature, low-oxygen environment. Firstly, In the TMT group, Our study concluded that higher expression of evm.model.chr5.73, evm.model.chr18.610, evm.model.chr10.1201, and HIF-1 signaling pathway was associated with a hypoxic environment. evm.model.chr15.573, evm.model .chr5.320, evm.model.chr6.360, and evm.model.chr18.610 are associated with WG production. The myosin-11 not only responds to low temperature and low oxygen, but may also lead to WG production through Tight junction. Secondly, the significant enrichment of NG in cancer pathways, EBV infection, small cell lung cancer, and other pathways compared to WG may be due to the low temperature and low oxygen environment. We hypothesized that NG might be more sensitive than WG in preventing cancer and viral infections. Finally, the significant down-regulation of UBL4A and up-regulation of Hsp70 in the label-free group also further illustrated the adaptation of G. maculatum to low temperature and hypoxia environment, and the ability of UBL4A and Hsp70 to stress the low-temperature and low-oxygen environments and to protect the liver cells from injury. However, the current study has some limitations, such as the lack of validation analyses. Therefore, a validation study is needed to confirm all the speculations in this study.
In summary, both proteomics techniques, label-free and TMT, revealed the differences between NG and WG of G. maculatum well, and the label-free method was superior to the TMT method in terms of the number of DEPs. TMT was better in terms of reproducibility and accuracy. Moreover, the upregulation of the myosin11 is an adaptation to low-temperature, low-oxygen environments and may lead to WG production via the Tight junction. In future studies we should investigate WG in other related catfish species and compare catfish species containing both NG and WG with those containing only NG. The functional mechanism of WG should also be investigated by stress treatments such as low temperature and low oxygen. Our study provides a theoretical basis for the adaptation of G. maculatum in highland areas.