Functional Divergence of the N-Lobe and C-Lobe of Transferrin Gene in Pungitius sinensis (Amur Stickleback)

Simple Summary As an iron-binding glycosylated protein, transferrin plays key roles in iron metabolism and immune response. Here, a transferrin transcript was identified from Amur stickleback, which encoded 679 amino acid peptides harbored obvious N-lobe and C-lobe domains. This transferrin was highly expressed in liver and increased by pathogen stimulation. Molecular evolution was performed on the N-lobe and C-lobe domains. Iron-binding capacity and antibacterial characteristics were also compared between N-lobe and C-lobe. These results will have important reference significance for the subsequent functional research. Abstract Transferrin is an important iron-binding glycosylated protein and plays key roles in iron-binding and immune response. Here, a 2037-bp open reading frame was obtained from our previous transcriptome sequencing data of Amur stickleback, which encoded a 679 amino acid putative transferrin protein harbored obvious N-lobe and C-lobe domains. The tissue-specific expression pattern showed that the transcript was detected in a variety of tissues, with the highest signal in liver. Moreover, Streptococcus iniae pathogen stimulation can increase the expression level of this transcript, implying important immune properties for organisms. Next, N-lobes and C-lobes were obtained from 45 fish species. The phylogenetic tree showed that N-lobes and C-lobes were in two different evolutionary branches, and they had different motif composition. Functional divergence indicated a higher evolutionary rate or site-specific alteration among the N-lobe and C-lobe groups. Ka/Ks value of C-lobe group was relatively higher than that of N-lobe group, indicating a faster change rate of C-lobe sequences in evolution. Moreover, some sites experiencing positive selection were also found, which may be involved in the iron- or anion-binding, pathogen resistance and diversification of transferrin protein. Differential iron-binding activity was also detected between N-lobe and C-lobe of Amur stickleback transferrin protein with Chrome Azurol S assay. Compared with the C-lobe, the N-lobe showed stronger growth inhibitory activity of Escherichia coli, implying their potential antibacterial properties. This study will give a reference for subsequent research of transferrin proteins.


Introduction
As an essential trace element of an organism, iron functions in many metabolic and physiological processes such as electron transfer, DNA synthesis, etc. [1][2][3]. The bioavailability of iron depends on the Fe 2+ and Fe 3+ forms. Among them, the free ferrous iron (Fe 2+ ) is highly toxic and can form hydroxyl radicals, causing oxidative damage of macromolecules. Therefore, the free ferric form (Fe 3+ ) is necessary for its transportation [4,5]. Fe 3+ is insoluble, so organisms usually use specialized iron-binding proteins to transport or store it [6]. In addition, excessive iron promotes the production of free radicals and the propagation of pathogens [1,2]. It is thus clear that maintaining an appropriate iron balance is essential for organisms [7]. The maintenance of iron homeostasis requires a series of with NanoDrop (Thermo Scientific, Wilmington, NC, USA). Next, reverse transcription was performed with M-MLV (TakaRa, Dalian, China) and random primers. The TruSeq Stranded mRNA LTSample prep kit (Illumina, San Diego, CA, USA) was used to construct cDNA. To explore the response of the TF gene to a pathogen, healthy fish was injected with 100 µL of 1 × 10 4 CFU/mL live Streptococcus iniae (Pier and Madin strain) purchased from MingzhouBio, a Gram-positive bacteria and a serious aquaculture pathogen that can infect a variety of aquaculture fish. Each test group was sampled at 3 h, 6 h, 12 h, 24 h, 2 d, 3 d, and 4 d after post-challenge, and liver tissue was collected to extract RNA and reverse transcribe into cDNA as previously described. Quantitative PCR with the LightCycler ® 480 II Realtime PCR Instrument (Roche, Basel, Switzerland) was performed using specific primers ( Table 1). Actin gene was used as an internal reference (Supplementary Data S1). Relative expression level of the TF was measured by the 2 −∆∆Ct method [44]. Three biological replicates were used for this analysis.

Sequence Retrieval of Fish TFs
To identify potential TF proteins in the 45 fish species, the TF protein sequence of Amur stickleback identified by our previous transcriptome sequencing data [45] was used to BLAST the Ensembl database (http://www.ensembl.org/index.html, accessed on 8 June 2021) [46] with cutoff E-value of 1 × 10 −1 . For each species, the candidate with the highest homology with the Amur stickleback TF protein was selected for subsequent analysis (Supplementary Data S2). Next, Pfam database [47] was used to confirm the candidates based on whether the conserved transferrin domain existed or not and to distinguish sequences of N-lobe and C-lobe.

Phylogeny and Motif Analysis
Multiple sequence alignment of these N-lobes and C-lobes was performed with MUS-CLE 3.52 [48]. Subsequently, MEGA v6 [49] was used for phylogenetic analysis using neighbor joining (NJ) method with pairwise-deletion, p-distance, and 1000 bootstrap replicates parameters. Conserved motifs were identified with MEME (http://meme-suite.org, accessed on 28 August 2021) [50] in each amino acid sequence with parameters: maximum 6 motif and 6-50 widths.

Functional Divergence Analyses
DIVERGE V2.0 [51] was used to estimate functional divergence among the N-lobes and the C-lobes of fish TF genes. Type I functional divergence can detect specific residue regions, which means that these residues undergo functional change [51]. Type II functional divergence can detect some conserved residues with different biochemical characteristics that function for protein specification. θ I and θ II stand for the coefficients among different groups predicted by type I and type II functional divergence, respectively. When these coefficients are greater than 0, it means that the site-specific selection changes the physicochemical properties of amino acids. In addition, significant differences in evolutionary rate and amino acid physicochemical properties existing between different group members were also indicated with posterior analysis [52].

Site-Specific Selection Assessment and Testing
The selective pressure of each residue of TF proteins was estimated with the synonymous rate (Ks) and non-synonymous rate (Ka) of nucleotide substitution. Here, Ka/Ks values were calculated with M8, M8a, M7, and M5 models. All of these models calculate the Ka/Ks value between sites with eight discrete classes and posterior distribution [53,54]. I-TASSER [55] was used to predict the tertiary structure of TF protein in Amur stickleback.

Recombinant N-Lobe and C-Lobe Plasmid Construction, Expression, and Purification
The cDNA fragments of N-lobe and C-lobe were amplified by the specific primers with Nde I and Xba I restriction sites ( Table 1). The PCR products were digested, connected to the expression vector pCZN1, and transformed into BL21 strain. Cells were cultured until the concentration of 0.6 at OD600 in LB liquid medium adding ampicillin at 37 • C and inducted with 0.2 mM IPTG (isopropyl β-D-thiogalactopyranpside) at 15 • C overnight. The recombinant peptides were purified with His Band Resin columns (Novagen, Madison, WI, USA) after sonication. Their purity was analyzed through 12% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). NanoDrop (Thermo Scientific, Wilmington, NC, USA) was used to measure the peptide concentration. It can quantify protein by detecting the absorbance value of protein at 280 nm without using protein standard.

Iron-Binding Assay of N-Lobe and C-Lobe of TF in Amur Stickleback
The iron-binding characteristics of N-lobe and C-lobe were evaluated with Chrome Azurol S (CAS) assay [56]. A 500 µL CAS assay solution (Sigma Aldrich, USA) was used to mix with expressed N-lobe, C-lobe, and maltose binding protein (MBP, Abcam; a protein tag, which can increase the solubility of protein and assist in protein folding but does not have any antibacterial and iron-binding capacity), and the mixture was incubated at room temperature in dark conditions for 1 h and measured the light absorbance value at 630 nm. Here, protein with different concentrations was used to detect the effect of sample concentration on iron-binding ability. The negative control is represented by an equivalent amount of elution buffer. Finally, the average of the relative absorbance of the three repetitions was calculated to represent their iron-binding activities.

Antibacterial Assay of N-Lobe and C-Lobe of TF in Amur Stickleback
Escherichia coli was first cultured in 37 • C LB medium at 200 rpm for about 6 h and diluted to the absorbance value of 1. Then, 100 µL diluted E. coli liquid (DH5α) and FeCl 3 with final concentration of 1µM were added to the 96 well microtiter plate, respectively. Then, equal amounts of N-lobes, C-lobes, MBP or elution buffer (control) were added into each well to 2 µM final concentration. Finally, the light absorption value of bacterial solution at 600 nm in different time periods was measured to verify the growth of the strain. The test is repeated three times.

Identification and Expression Patterns of TF Gene in Amur Stickleback
A 2037-bp open reading frame was obtained from our transcriptome sequencing data of Amur stickleback [45], which encodes a TF protein with 679 amino acid residues. This TF transcript of Amur stickleback, similar to the zebrafish transferrin-a gene, has high homology with the mammalian serum transferrin. It has N-lobe and C-lobe typical functional domains, separated by a short bridge (Supplementary Data S2). This TF protein of Amur stickleback was homologous to those of other species (Figure 1).
A 2037-bp open reading frame was obtained from our transcriptome sequencing data of Amur stickleback [45], which encodes a TF protein with 679 amino acid residues. This TF transcript of Amur stickleback, similar to the zebrafish transferrin-a gene, has high homology with the mammalian serum transferrin. It has N-lobe and C-lobe typical functional domains, separated by a short bridge (Supplementary Data S2). This TF protein of Amur stickleback was homologous to those of other species (Figure 1). Figure 1. Alignment of transferrin amino acid sequences between P. sinensis (marked with red asterisks) and other seven fish species randomly selected. The N-lobe and C-lobe areas are marked with arrows.
Next, the tissue distribution of PsTF transcripts was also detected. The results indicated that the expression of PsTF transcripts was differentially distributed in the six examined tissues. The highest expression pattern of PsTF mRNA was detected in liver, followed by intestine, kidney, and muscle. Moreover, the lowest expression level of PsTF transcripts was detected in gill and heart ( Figure 2A). Next, the tissue distribution of PsTF transcripts was also detected. The results indicated that the expression of PsTF transcripts was differentially distributed in the six examined tissues. The highest expression pattern of PsTF mRNA was detected in liver, followed by intestine, kidney, and muscle. Moreover, the lowest expression level of PsTF transcripts was detected in gill and heart ( Figure 2A).
To study the effect of pathogen stimuli to the PsTF, the dynamic expression patterns of PsTF gene in liver tissue infected with S. iniae were analyzed ( Figure 2B). The result indicated that at 24 h and 2 days after bacterial infection, the expression level of PsTF was the highest. Compared with the control, the increase fold of PsTF transcripts was about 4.7 and 3.8 at these two points of time, respectively. Therefore, the transcriptional changes of PsTF gene may be an immune mechanism against pathogenic stimuli. To study the effect of pathogen stimuli to the PsTF, the dynamic expression patterns of PsTF gene in liver tissue infected with S. iniae were analyzed ( Figure 2B). The result indicated that at 24 h and 2 days after bacterial infection, the expression level of PsTF was the highest. Compared with the control, the increase fold of PsTF transcripts was about 4.7 and 3.8 at these two points of time, respectively. Therefore, the transcriptional changes of PsTF gene may be an immune mechanism against pathogenic stimuli.

Phylogenetic Analysis of the N-Lobe and C-Lobe
To further elucidate the evolutionary relationship of the N-lobe and C-lobe of TF family genes from 45 fish species, an NJ phylogenetic tree was constructed. According to the sequence similarity and phylogeny, these members were classed into two groups, named N-lobe and C-lobe groups ( Figure 3). This structure is believed to originate from a gene duplication event about 670 million years ago [18,19].

Phylogenetic Analysis of the N-Lobe and C-Lobe
To further elucidate the evolutionary relationship of the N-lobe and C-lobe of TF family genes from 45 fish species, an NJ phylogenetic tree was constructed. According to the sequence similarity and phylogeny, these members were classed into two groups, named N-lobe and C-lobe groups ( Figure 3). This structure is believed to originate from a gene duplication event about 670 million years ago [18,19].

Motif Distribution of the N-Lobe and C-Lobe Sequences in Fish TFs
To further understand the diversity of N-lobe and C-lobe in fish, six conserved motifs were identified in these sequences (Figure 4).

Motif Distribution of the N-Lobe and C-Lobe Sequences in Fish TFs
To further understand the diversity of N-lobe and C-lobe in fish, six conserved motifs were identified in these sequences (Figure 4).

Motif Distribution of the N-Lobe and C-Lobe Sequences in Fish TFs
To further understand the diversity of N-lobe and C-lobe in fish, six conserved motifs were identified in these sequences (Figure 4).  These N-lobes and C-lobes share most of the motif distribution, while the C-lobes specifically contain motif 6, suggesting that these sequences from ancient gene duplication have changed in evolution. The specific motif 6 in C-lobe group may confer different functions of this family protein in evolution.

Functional Divergence Analysis between N-Lobe and C-Lobe Groups
Type I and Type II functional divergence were estimated to investigate the residue substitution leading to adaptive functional diversification. The results showed that the type I divergence coefficient (θ I ) of these two groups was 0.599 (Table 2), indicating the change of specific sites among them. Selective restriction on different lobe members may lead to changes in specific functions after divergence. In addition, the type II divergence coefficient (θ II ) was also estimated with high standard error (Table 3). Table 2. Type I functional divergence between the N-lobe and the C-lobe of fish transferrin gene family. The critical sites are displayed by residues on the N-lobe.

Comparison
Type  To identify key residues associated with functional changes among N-lobe and C-lobe groups of TF proteins, posterior divergence probability of each site was estimated. The larger value of posterior probability indicates a great possibility of change in site-specific amino acids [52]. As a result, some key residues were identified related to functional divergence in these groups (Tables 2 and 3).

Selective Pressure Estimation
To identify which residues experienced positive selection, the Ka/Ks value was calculated for each site of N-lobe and C-lobe sequences ( Table 4). The Ka/Ks value of the C-lobe group was relatively higher than that of the N-lobe, indicating a faster change rate in C-lobe. All values of Ka/Ks were less than 1, indicating that each group had undergone purification selection. However, seventeen sites were identified to undergo positive selection in the N-lobe group predicted by M8 model (Table 4). At the same time, thirty-nine and forty-six sites were also found in the C-lobe group predicted by M8 and M5 model, respectively (Table 4). For instance, among the 17 sites of N-lobe group, 23V, 33L, 95R, 127K, and 278V sites were located in the motifs 3, 1, and 5 ( Figure 5).  Next, N-lobes and C-lobes of TF in Amur stickleback were first expressed and purified in vitro (Figures 6A and S1).

Figure 5.
Three-dimensional structure of the N-lobe of TF protein in Amur stickleback predicted with the I-TASSER server [55]. Seventeen positive selection sites predicted by M8 model are marked with arrows. Table 4. Likelihood values and parameter estimates for N-lobe and C-lobe of TF genes. Ka/Ks: the synonymous rate (Ks) and non-synonymous rate (Ka) of nucleotide substitution; ωs: beta + w.

Iron-Binding Activity of the N-Lobe and C-Lobe of TF Protein in Amur Stickleback
Next, N-lobes and C-lobes of TF in Amur stickleback were first expressed and purified in vitro (Figures 6A and S1). Firstly, CAS assay reagent was used to detect the potential binding ability of N and C-lobe to ferric ions (Fe 3+ ). The results indicate that both N-lobe and C-lobe hav ability to bind Fe 3+ , and the level increases with the increase in concentration ( Figure  Compared with C-lobe, N-lobe has a stronger ability to bind iron ions. However, pared with the control, MBP has no reaction to iron-binding activity.

Antimicrobial Properties of the N-Lobe and C-Lobe of TF Protein in Amur Stickleback
Next, their potential antimicrobial properties were also analyzed. The results sho that both N-lobe and C-lobe had potential antibacterial ability (Figure 7). Lines: M-marker; N1 and N2-soluble fraction after sonication of N-lobe expressed cells; N3 and N4-purified N-lobe fragments after elution; C1 and C2-soluble fraction after sonication of C-lobe expressed cells; C3 and C4-purified C-lobe fragments after elution; (B) Ironbinding activity of the TF N-lobe and C-lobe in Amur stickleback. The x-axis represents the protein concentration, and the y-axis represents the light absorption value at 630 nm. Values were described as mean ± SE (n = 3). Asterisks (* p < 0.05; ** p < 0.01) indicated the statistically significant differences between treatments.
Firstly, CAS assay reagent was used to detect the potential binding ability of N-lobe and C-lobe to ferric ions (Fe 3+ ). The results indicate that both N-lobe and C-lobe have the ability to bind Fe 3+ , and the level increases with the increase in concentration ( Figure 6B). Compared with C-lobe, N-lobe has a stronger ability to bind iron ions. However, compared with the control, MBP has no reaction to iron-binding activity.

Antimicrobial Properties of the N-Lobe and C-Lobe of TF Protein in Amur Stickleback
Next, their potential antimicrobial properties were also analyzed. The results showed that both N-lobe and C-lobe had potential antibacterial ability (Figure 7).
Compared with C-lobe, N-lobe showed stronger bacterial growth inhibitory activity. The high Fe 3+ -binding efficiency of N-lobe may play key roles in inhibiting bacterial growth. Here, the results also showed that MBP did not produce any significant changes in bacterial growth.
Compared with C-lobe, N-lobe has a stronger ability to bind iron ions. However, com-pared with the control, MBP has no reaction to iron-binding activity.

Antimicrobial Properties of the N-Lobe and C-Lobe of TF Protein in Amur Stickleback
Next, their potential antimicrobial properties were also analyzed. The results showed that both N-lobe and C-lobe had potential antibacterial ability (Figure 7). Figure 7. Analysis of iron-dependent bacterial growth inhibition. The x-axis represents time, and the y-axis represents the light absorption value at 600 nm. Values were described as mean ± SE (n = Figure 7. Analysis of iron-dependent bacterial growth inhibition. The x-axis represents time, and the y-axis represents the light absorption value at 600 nm. Values were described as mean ± SE (n = 3). Asterisks (* p < 0.05; ** p < 0.01) indicated the statistically significant differences between treatments. MBP: maltose binding protein; OD: optical density.

Discussion
Here, a transcript was obtained from Amur stickleback, which encodes a TF protein with obvious N-lobe and C-lobe domains. First, the differential tissue distribution and bacterial stimulation of this transcript were detected ( Figure 2). Liver tissue is the main site for TF synthesis in vertebrates [57]. Therefore, high levels of PsTF transcripts were detected in the liver in this study. Similar results were also observed in other species of fish [10,36,37,39,40,58,59]. In some species, a large amount of TF gene expression was also detected in blood and other tissues [12]. This indicates that TF may play important roles in multiple tissues. Previous studies have also reported that bacterial stimulation can upregulate the expression of TF gene [36,38]. In fish, high levels of TF transcripts were usually detected under pathological conditions [36]. In addition, insect TF has also been shown to be up-regulated under pathological conditions such as bacterial stimulation [60,61]. These results suggest that PsTF may exhibit an immune response to pathogenic infection.
Some iron-and anion-binding residues were also described by previous study [17]. In this study, four iron-and two anion-binding sites were also found in the N-lobe and C-lobe of Amur stickleback TF protein, respectively. They are residues D73, Y103, Y196, and H255 of N-lobe and D394, Y429, Y521, and H581 of C-lobe, which are located in motifs 3, 1, 2, and 4, respectively. In addition, anion-binding residues of the T128 and K132 in N-lobe and T454 and R458 in C-lobe were also found to locate in the motif 1. Mutation of some equivalent ligands resulted in a significant sharp reduction or loss of iron-binding capacity of TFs in human, rat, pufferfish, and chicken [17,62,63]. This suggests that N-lobes and C-lobes from duplication have had functional divergence in evolution.
Functional divergence identified some critical amino acid sites (Tables 2 and 3). For instance, seventeen key functional divergent were identified between N-lobe and C-lobe groups predicted by type I functional divergence. Among them, fifteen sites were in the motifs 3 and 1. Some sites are near the Fe-binding residues and anion-binding residues described by previous study [17]. These sites may affect the Fe-binding and anion-binding function. The different evolution rates of some key residues may lead to functional differentiation of fish TF gene family.
In the process of evolution, selection pressure, evaluated by Ka/Ks value, usually causes specific peptides to mutate, thereby changing the structure and function of proteins. When the Ka/Ks value was greater than or less than 1, it indicated positive selection or purification selection, respectively [64]. Here, some positive selection sites were predicted in the N-lobe and C-lobe groups (Table 4). Among them, some sites are close to the ironand anion-binding residues and may affect the ability of binding iron or anion [17]. The positive selection of fish TFs is considered to be caused by its competition with pathogenic bacterial proteins [29]. In addition, positive selection plays key roles in the diversification of TF proteins [31,35,65]. It suggests that selection pressure promotes the rapid change of TFs, which may be related to organisms' adaptation to different environments.
Here, N-lobe was identified to have a stronger ability to bind iron ions compared with C-lobe. Previous studies have also indicated that the Fe-binding abilities are different among N-lobe and C-lobe, and N-lobe are easier to bind iron than C-lobe [16,17,66]. Moreover, the differential iron-binding activity also existed in amphioxus (Branchiostoma belcheri) [67], disk abalone (Haliotis discus discus) [68], and rock bream (Oplegnathus fasciatus) [10]. In addition, the linger regions of N-and C-lobes also affect the flexibility and Fe-binding rate of TF protein [69]. TF can perform some antibacterial activity through combining free iron and then creating a low iron level environment [70,71]. This study further confirmed that N-lobe are easier to bind iron than C-lobe [16,17,66]. Iron is a basic nutrient necessary for biology and participates in many important biological processes [1][2][3]. Therefore, maintaining a certain concentration of iron ions is necessary for the growth and metabolism of organisms. Under pathological conditions, the combination of TF and iron ions leads to the decrease in available iron concentration, which inhibits the growth of pathogens.

Conclusions
One TF gene from Amur stickleback was characterized by analyzing its expression, motif distribution, functional divergence, molecular evolution, iron-binding ability, and antibacterial properties. This TF gene was mainly expressed in liver and strongly induced by pathogenic bacteria. The N-lobe and C-lobe regions have different motif composition, selection pressure, and functional divergence in evolution. Moreover, different iron-binding activity and antibacterial characteristics also existed in the N-lobe and C-lobe of Amur stickleback TF protein. Therefore, in the process of evolution, the N-lobe and C-lobe domains of TF genes experienced different selection pressures, resulting in the changes of their motif composition and functional divergence and the differences of their iron-binding activity and antibacterial properties. This study will provide a basis for the subsequent study of TF family genes.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/ani12243458/s1, Figure S1: Quality detection and Western blot analysis of the expressed N-lobe and C-lobe segments after gel cutting; Supplementary Data S1: Transferrin and Actin sequences obtained from previous transcriptome sequencing data of Amur stickleback for primer designing in this study; Supplementary Data S2: Transferrin sequences used in this study. N-lobe and C-lobe segments are also marked.