Comparative Proteomic Analysis of Erythropoiesis Tissue Head Kidney Among three Antarctic Fish Species

Antarctic icesh is the only known vertebrate species that lacks oxygen-carrying hemoglobin and functional erythrocytes. To reveal the unique hematopoietic process of icesh, we used an integrated approach including tandem mass tag (TMT) labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify the dynamic changes in the head kidney whole proteome of a white-blooded icesh, Chionodraco hamatus, compared to those in two other red-blooded Antarctic sh, Trematomus bernacchii and Notothenia coriiceps. Of the 4,672 identied proteins, in the Antarctic ice sh head kidney, 123 proteins were signicantly up-regulated and 95 proteins were down-regulated. The functional grouping of differentially expressed proteins based on KEGG pathway analysis shows that white blood sh and red blood sh have signicant differences in erythropoiesis, heme biogenesis, leucocyte and platelet cell development. The proteins involved in the hematopoietic process in icesh showed a clear trend of downregulation of erythroid lineage marker proteins and upregulation of lymphoid and megakaryocytic lineage marker proteins, including CD9, ITGB2, and MTOR, which suggests a shift in hematopoiesis in the icesh head kidney due to the loss of erythrocytes. The results of the present study not only provide basic datasets for the head kidney proteins of Antarctic shes, but also provide important references for studies on immunity and hematopoiesis in various species.


Introduction
The marine environment of the Antarctica is characterized by a long-term icy snow cover on the ocean, which results in the temperature of sea water being constantly maintained at -1. considerable diversi cation with signi cant ecological differences. This suborder is divided to six families that include Notothenioidae, Artedidraconidae, Bovichtidae, Harpagiferidae, Bathydraconidae, and Channichthyidae (Buonocore et al. 2006). To survive in such isolated and harsh cold conditions, these shes have evolved numerous unique specializations to adapt, particularly antifreeze glycoproteins, which prevent freezing of the body uids of the sh (Chen et al. 1997). The ice sh of the Channichthyidae family, also known as the "white-blooded" sh, are the only species that have developed in this region due to the to lack of hemoglobin and functional active erythrocytes (Ito et al. 1995).
The Antarctic ice sh Chionodraco hamatus (Perciformes: Channichthyidae) is a benthic sh that lives in the subzero waters of the Antarctic Ocean (Chen et al. 2019;Gerdol et al. 2019). As the only known vertebrate group that lacks hemoglobin (Evans et al. 2021), the blood circulation system of ice sh has undergone large compensatory physiological changes. Trematomus bernacchii (Perciformes: Nototheniidae) (Della Pelle et al. 2020) and Notothenia coriiceps (Perciformes: Nototheniidae) (Cao et al. 2016), two red-blooded notothenioid shes, share a similar living environment with Chionodraco hamatus(CH). The hematopoietic system, especially the immune system of the Antarctic shes (the ice sh in particular), has undergone a rigorous selection process due to the harsh long-term coldness (Romano et al. 2000).
Head kidney is the main hemopoietic tissue in sh and is also an important immune organ with a cellular composition of erythrocytes and leucocytes, such as macrophages, granulocytes, and B lymphocytes (Romano 1998). In our previous study, we found more than 100 kinds of microRNAs, including miR-152, with high expression in the head kidney of CH, which might ultimately result in the suppression of erythropoiesis in ice sh (Chan et al.). The Antarctic shes also serve as a good candidate to study the evolution of the immune system under extreme cold environments.
Previous studies have shown differences in the histological structure of lymphomyeloid organs of the Antarctic sh CH and Trematomus bernacchii (Romano et al. 2000). The transcriptome and microRNA analysis of the primary hematopoietic tissue (head kidney) of CH and two red-blooded revealed the evodevo mechanisms involving erythropoietic suppression through the upregulation of TGF-β signaling (Xu et al. 2015). Unique evolution of hepcidin genes was also noted in ice sh as compared to that in other Antarctic shes. However, little is known about the proteomic pro le of the primary hematopoietic and immune-related tissue (head kidney) of ice sh and other Antarctic shes. In the current study, we used an integrated method including TMT labeling and LC-MS/MS to quantify the dynamic variations in the entire proteome (Chionodraco hamatus) of head kidney of ice sh (CH) and two red-blooded notothenioids Trematomus bernacchii (TB)and Notothenia coriiceps (NC)with an aim to analyze the proteomic differences between the three Antarctic shes. The results of the present study not only provide important references for further exploration of the unique hematopoietic process of the white-blooded ice sh, but also shed a light on the innate and adaptive immune mechanisms of the Antarctic teleost.

Sample preparation
Chionodraco hamatus and Trematomus bernacchii individuals were gathered locally from Prydz Bay (69°22 S, 76°22 E).NC individuals were collected from the water near the Great Wall Station, a Chinese Antarctic research base (62°12 S, 58°57 W) located on the Fieldes Peninsula on King George Island.
Head kidney tissues were dissected from adult male sh samples and used for total protein extraction.
For each species, head kidney tissues from three different adult male sh individuals were dissected and used for total protein extraction. The average body length was approximately 22 cm for CH and NC and approximately 15 cm for TB individuals.

Protein extracts
The head kidney tissue was ground in liquid nitrogen for 20 minutes and sonicated three times on ice with a high intensity ultrasound processor. Then the samples in triplicate for each group were solubilized and centrifuged at a low-speed instantaneously, and the supernatant and intermediate layer are taken to add cocktail (ThermoFisher Scienti c, USA) and 4% SDS(Shanghai Chinese Medicine, China).The suspensions were disrupted by sonication on ice for 3 min, lysates were incubated on ice for 30min.
Subsequently, lysates were separated by centrifuge at 4°C for 20 minute and collected supernatants. Protein concentration of each sample was quanti ed by 2-D Quant kit.
Digested trypsin for digestion, take 100µg protein and replenish the volume to 100µl with lysate, add a nal concentration of 10 mM DTT (ThermoFisher Scienti c, USA) at 37 ° C for 60 min, then adding iodoacetamide with an ultimate density of 20 mM (Sigma) at 20 ºC for 45 min in the dark. Afterwards 200 mM TEAB was added to each sample for achieve urea density of less than 2 M (Wang et al., 2019).
then centrifuged 10,000g for 20 min and collected the precipitation. Solubilized precipitation digested overnight at 37°C using trypsin with the mass ratio of Trypsin to protein 1:50.

TMT labeling
Finally, the peptides were dried by vacuum pump, labeling TMT reagents was added in advance (Thermo Fisher Scienti c, Torrance, CA, USA) per 100 µg peptides, incubated for 2 hours at room temperature.
Sample labeling was performed as follows: group CH: 129, group TB: 130, group NC: 131. Add 50ul of ultrapure water and leave it at room temperature for 30min. Each group labeled samples of the peptide were mixtured and dried down in a vacuum, the mixtures sample containing high-performance liquid chromatography (HPLC) were separated on loaded onto UltraPerformance LC (Waters, USA) containing a C18 column in front of a 2.1x 150 mm X Bridge BEH300 (Waters, USA), The HPLC gradient was buffer B (100% ACN) in buffer A (ammonia water, formic acid adjusted pH to 10) started with 0% B and increased to 100% B then followed by 0% B 6 min at a ow rate of 400µl/min total over 80 min. Collect 80 fractions based on peak shape and time and combine them into 18 fractions using rotation vacuum concentrators, the samples then were dissolved in mass spectrometry loading buffer the following analysis.

LC-MS/MS analysis and database search
The peptides were dissolved in 0.1% FA and separated from the high-performance liquid chromatography (HPLC) analysis were combined with liquid chromatography and tandem mass spectrometry( LC-MS/MS) containing EASY-nLC 1200, Q-Exactive(Thermo, USA)and C18 column (75µm x 25cm, Thermo ,USA

Quantitative overview
Reproducibility analysis of three biological replicate experiments was conducted by Pearson's correlation coe cient. For each replicate, relative expression of the proteins of CH, TB and NC was determined with Pearson's correlation coe cient (Fig. 1A). 4822 proteins were identi ed, among which 4672 proteins were quanti ed. A comparison of the proteome pro le of CH with those of TB and NC showed that 123 proteins were upregulated (quantitative ratio of > 1.2 was considered as upregulation, CH vs. combined) and 95 proteins were downregulated (quantitative ratio of < 1/1.2 was considered as downregulation, CH vs. combined) in CH. The amount of the DEPs is summarized in Fig. 1B.

Functional classi cation of DEPs
The 218 DEPs were classi ed into three categories, namely biological processes, cellular components, and molecular functions, according to GO analysis (p < 0.05) (Fig. 2). These proteins were mainly clustered into 20 GO functional categories, which accounted for nine biological processes, nine cellular components, and two molecular functions ( Fig. 2A-C). The most prevalent biological processes were response to inorganic substances, response to metal ions, erythrocyte homeostasis, and myeloid cell homeostasis, which accounted for 55% ( Fig. 2A). According to cellular component annotation, the majority of the dysregulated proteins originated from the membrane (29%) and membrane part (15%).
Other signi cant components included the endomembrane system (13%) and the endoplasmic reticulum part (9%) (Fig. 2B). The common molecular functions included gated channel activity and ion-gated channel activity (Fig. 2C). Figure 3 shows the results of the GO enrichment analysis. The differentially quanti ed proteins of the upregulated pathways for the three Antarctic sh species are shown in Fig. 3A. The most signi cantly enriched cellular components were the endoplasmic reticulum membrane, the endoplasmic reticulum membrane network, and the endomembrane system. The main molecular functions were gated channel activity, ion-gated channel activity, and ion channel activity. The biological processes were mainly enriched in the glycoprotein metabolic process, gated channel activity, and divalent metal ion transport. The differentially quanti ed proteins of the downregulated pathways for the three Antarctic sh species are shown in Fig. 3B. The most signi cantly enriched cellular components were the main axon, muscle thin lament tropomyosin, and mitotic spindle. The main molecular functions were peptide binding and amide binding. The biological processes were mainly enriched in embryonic hemopoiesis, cardiac muscle contraction, hemoglobin biosynthetic process, and hemoglobin metabolic process. The GO enrichment analysis showed that the DEPs mainly participated in membrane, binding, and metabolic processes.

Pathway enrichment analysis of DEPs
KEGG analysis was performed to investigate the enriched pathways for the upregulated and downregulated proteins. The upregulated proteins were mainly mapped to 10 signaling pathways, which included hematopoietic cell lineage, malaria, cell adhesion molecules, protein processing in endoplasmic reticulum, HTLV-I infection, viral myocarditis, and others (Fig. 4). There are signi cant differences in erythropoiesis, heme biogenesis, and leucocyte and platelet cell development between the white-and redblooded shes (Souza et al. 2018). The downregulated proteins were mainly mapped to ve signaling pathways: pentose phosphate pathway, porphyrin and chlorophyll metabolism, prion diseases, focal adhesion, and amoebiasis (Fig. 4).
On the basis of the differential protein function determined from the KEGG pathway, white-blooded sh and red-blooded sh showed signi cant differences in erythropoiesis and platelet cell development. The protein expression pattern of the hematopoietic process of teleosts shows that in ice sh head kidney, erythropoiesis-related proteins are downregulated or almost inhibited and the production of megakaryocytic-and lymphopoiesis-related proteins is upregulated. The expression of B lymphocyte production-related proteins CD9 and ITGB2 was signi cantly upregulated (p < 0.05); the platelet production-related protein CD9 was signi cantly upregulated, and the expression of the myeloblast production-related proteins ITGB2, CD9, and MTOR was also signi cantly upregulated. This was in contrast to erythropoiesis-related proteins β-Spectrin, tfr1a, and hemoglobin, which were signi cantly downregulated and almost showed no expression (Fig. 5).

Discussion
In the present study, we conducted interspecies comparisons of head kidney proteomes between one ice sh (CH) and two red-blooded notothenioids (TB and NC) by using TMT labeling and LC-MS/MS. We compared the expression pro les and described the differences of 123 upregulated proteins and 95 downregulated proteins. The comparison of the expression patterns of the teleost head kidney proteomes showed that the expression of erythrocyte-related proteins such as β-Spectrin, tfr1a, and hemoglobin was signi cantly downregulated in ice sh; however, lymphoid and megakaryocytic lineage marker proteins, including CD9 and ITGB2, were signi cantly upregulated. An interesting point to note is that the lymphoid and megakaryocytic lineage marker proteins were not affected by the absence of erythrocytes and they were even upregulated in the ice sh head kidney (Table 1) . The proteomics analysis of the total protein in the head kidney of three Antarctic sh further con rmed that this general expression trend may cause a severe reduction in red blood cell differentiation of CH, while B cells and platelet cells are relatively up-regulated.
The major function of erythrocytes is not only to deliver oxygen to the organs, but they are also involved in innate immune responses, as they can capture speci c immune complexes such as certain pathogens and bacteria, partly through membrane electrostatic attraction, and then kill them in the liver and spleen by presenting the pathogens to Kupffer cells and antigen-presenting cells (APCs) (Ukidve et al. 2020). In our study, the number of erythrocytes in the ice sh was found to be severe reduced probably due to changes in the immune response; however, at present, we are unsure whether this change is more harmful or more bene cial. A comparison of the mucus microbial communities between white-blooded CH and red-blooded TB individuals sampled from the same locations showed much higher levels of bacterial species diversity in the CH mucus samples (data not published). To our knowledge, the sh skin mucus is a viscoelastic, adhesive gel that covers the exposed skin. ,including phagocytosis and acquired immune responses. Therefore, it remains unclear whether the increase in the number of mucus microorganisms indicates the increased susceptibility of the CH immune system to attack by microorganisms or whether it represents the ways through which the sh improves its acquired immunity; further studies are needed to con rm this aspect.
The present study mainly discusses the DEPs from total proteins of head kidney of three Antarctic sh species by comparing the signi cant differentially expressed proteins of red-blooded TB and NC and white-blooded CH. This study found 218 DEPs that could be used for further analysis of their related genes to provide a basis for further studies on the occurrence of red blood cells in ice sh. Because there are almost no red blood cells in the Antarctic ice sh, we found that among the three sh species, the expression of proteins is important role in B-cell production and platelet cell development in the hematopoietic cell lineage was signi cantly upregulated CH. The results of this study can serve as good references for further research and exploration of the immunity and hematopoiesis of the Antarctic ice sh.
Declarations QHX was responsible for the overall guidance of this article. RNJ and SJH were mainly responsible for the overall experimental process and experimental data analysis involved in this paper; WYZ and SWJ participated in sample collection and processing; WHL and FXW participated in the analysis of experimental data and image processing.

Ethics approval
Experimental protocols involving live animals were approved by the Ethics Committee for the Use of Animal Subjects of Shanghai Ocean University.

Consent to participate
Not applicable.

Consent for publication
Not applicable.     The red arrows indicate the upregulated proteins, in the ice sh compared with the red-blooded sh (see Table 1, Supporting information). HSC: haematopoietic stem cell; CMP: common myeloid progenitor; CLP: common lymphoid progenitor; MEP: megakaryocyte/erythroid progenitor; GMP: granulocyte/macrophage progenitor.

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