Identification and Functional Analysis of Tartrate-Resistant Acid Phosphatase Type 5b (TRAP5b) in Oreochromis niloticus

Abstract

Tartrate-resistant acid phosphatase type 5 (TRAP5) is an enzyme that is highly expressed in activated macrophages and osteoclasts and plays important biological functions in mammalian immune defense systems. In the study, we investigated the functions of tartrate-resistant acid phosphatase type 5b from Oreochromis niloticus (OnTRAP5b). The OnTRAP5b gene has an open reading frame of 975 bp, which encodes a mature peptide consisting of 302 amino acids with a molecular weight of 33.448 kDa. The OnTRAP5b protein contains a metallophosphatase domain with metal binding and active sites. Phylogenetic analysis revealed that OnTRAP5b is clustered with TRAP5b of teleost fish and shares a high amino acid sequence similarity with other TRAP5b in teleost fish (61.73-98.15%). Tissues expression analysis showed that OnTRAP5b was most abundant in the liver and was also widely expressed in other tissues. Upon challenge with Streptococcus agalactiae and Aeromonas hydrophila in vivo and in vitro, the expression of OnTRAP5b was significantly up-regulated. Additionally, the purified recombinant OnTRAP5b ((r)OnTRAP5) protein exhibited optimal phosphatase activity at pH 5.0 and an ideal temperature of 50 °C. The V_max, K_m, and k_cat of purified (r)OnTRAP5b were found to be 0.484 μmol × min^-1 × mg^-1, 2.112 mM, and 0.27 s^-1 with respect to pNPP as a substrate, respectively. Its phosphatase activity was differentially affected by metal ions (K⁺, Na⁺, Mg²⁺, Ca²⁺, Mn²⁺, Cu²⁺, Zn²⁺, and Fe³⁺) and inhibitors (sodium tartrate, sodium fluoride, and EDTA). Furthermore, (r)OnTRAP5b was found to promote the expression of inflammatory-related genes in head kidney macrophages and induce reactive oxygen expression and phagocytosis. Moreover, OnTRAP5b overexpression and knockdown had a significant effect on bacterial proliferation in vivo. When taken together, our findings suggest that OnTRAP5b plays a significant role in the immune response against bacterial infection in Nile tilapia.

Keywords: Oreochromis niloticus; phagocytosis; phosphatase activity; reactive oxygen species; tartrate-resistant acid phosphatase type 5b (TRAP5b).

Figure 1

Structure and sequence analysis of TRAP5b from Nile tilapia. (A) The nucleotide and deduced amino acid sequences of OnTRAP5b. The signal peptide is marked in a gray shadow. The metallophosphatase domain is marked with a single underline. The translation start (ATG) and stop (TGA) codons are boxed. (B,C) Alignment of the predicted amino acid sequences of OnTRAP5b homologs and predicted conserved domains of OnTRAP5b. Symbol (●) represents metal binding sites, and active sites are in red boxed. Symbol (▲) represents the N-glycosylation site. The numbers indicate the sequence identities between OnTRAP5b and the compared sequences. The conserved and identical residues are represented by black shading, and the residues that are ≥75% identical among the aligned are in gray. (D) Phylogenetic tree analysis of OnTRAP5b among other vertebrates. A neighbor-joining phylogenetic tree of TRAP5 proteins based on protein sequence analyzed with mega 6.0. The degree of confidence in each branch point was determined by bootstrap analysis (1000 times). (E) The predicted three-dimensional structure of protein TRAP5b from Oreochromis niloticus (left), Homo sapiens (middle), and overlapped three-dimensional structure of TRAP5 from them (right).

Figure 2

Expression analysis of OnTRAP5b in different tissues under normal physiological conditions. qRT-PCR was performed with cDNA samples prepared from the liver, stomach, intestine, terminal kidney, head kidney, spleen, heart, peripheral blood, gills, brain, skin, and muscle. The expression level in the muscle with the lowest expression was set as 1, and target gene expression was normalized against an endogenous control (β-actin). The data are presented as mean ± SD (n = 3). n, the number of times the experiments were performed.

Figure 3

Expression pattern of OnTRAP5b in vivo. The mRNA expression of OnTRAP5b after injection with S. agalactiae (A1–A3) and A. hydrophila (B1–B3) in the liver, spleen, and head kidney at various time points. Expression values are normalized against β-actin, and the folding unit was calculated, deciding the values of the vaccinated tissues by PBS. The values are shown as means ± SD (n = 3), n, the number of times the experiments were performed. ** p < 0.01, * p < 0.05.

Figure 4

The expression pattern of OnTRAP5b in vitro. The mRNA expression of OnTRAP5b in head kidney macrophages (A1,A2) and the hepatocytes (B1,B2) after treatment with S. agalactiae (1 × 10⁸ CFU/mL), A. hydrophila (1 × 10⁸ CFU/mL) and a control group treated with PBS. The mRNA level of the OnTRAP5b gene was normalized to β-actin, and the fold units were calculated, deciding the value of the PBS-treated cells. The values are shown as means ± SD (n = 3), n, the number of times the experiments were performed. ** p < 0.01, * p < 0.05.

Figure 5

Phosphatase activity of (r)OnTRAP5b. Analysis dependence on protein concentration (A); optimum temperature (B); optimum pH (C); temperature stability (D); and pH stability (E) of (r)OnTRAP5b. Phosphatase activity of (r)OnTRAP5b were expressed as percentages of the relative phosphatase activity. Data are presented as means ± SD (n = 3). n, the number of times the experiments were performed.

Figure 6

Effects of different metal ions and different inhibitors on (r)OnTRAP5b. The enzyme activities of (r)OnTRAP5b under different metal ions (A1–A8) and different inhibitors (B1–B3) were expressed as percentages of the relative phosphatase activity. Data are presented as means ± SD (n = 3). n, the number of times the experiments were performed.

Figure 7

Analysis of the expression of immune-related genes in head kidney macrophages. Nile tilapia head kidney macrophages were treated with (r)OnTRAP5b (100 μg/mL), (r)MBP (100 μg/mL), and PBS. After 3, 6, 12, and 24 h of stimulation, the expression of IL-1β (A); IL-6 (B); IL-10 (C); TNF-α (D); IL-8 (E); and IL-12 (F) were examined. The expression of the six genes was normalized against β-actin, and the expression of these six immune genes in PBS-treated cells was set to 1. The values are shown as means ± SD (n = 3), n, the number of times the experiments were performed. ** p < 0.01, * p < 0.05.

Figure 8

The analysis of ROS production and phagocytic activities in head kidney macrophages upon (r)OnTRAP5b. Head kidney macrophages were stimulated with 100 or 500 μg/mL (r)OnTRAP5b or (r)MBP, and oxidation of DCFH-DA was measured for 30 min using excitation and emission filters of 488 and 525 nm, respectively (A). The mean of DCFH-DA fluorescence with treatment for (r)OnTRAP5b or (r)MBP at doses of 100 and 500 μg/mL in head kidney macrophages (B). Histogram distribution of uptake S. agalactiae and A. hydrophila in head kidney macrophages with treatment for (r)OnTRAP5b or (r)MBP at doses of 100 and 500 μg/mL (C). The phagocytic rate of uptake S. agalactiae and A. hydrophila in head kidney macrophages with treatment for (r)OnTRAP5b or (r)MBP at doses of 100 and 500 μg/mL (D). The values are shown as means ± SD (n = 3), n, the number of times the experiments were performed. ** p < 0.01, * p < 0.05.

Figure 9

Analysis of the effects of overexpression and knockdown expression of OnTRAP5b on S. agalactiae infection. Nile tilapia administered with pcOnTRAP5b, pcDNA3.1, dsOnTRAP5b, dsGFP, or PBS (control) were infected with S. agalactiae. Bacterial loads in the liver (A,D), spleen (B,E), and head kidney (C,F) at 6, 12, and 24 h after S. agalactiae infection were determined. The values are shown as means ± SD (n = 3), n, the number of times the experiments were performed. ** p < 0.01, * p < 0.05.