Mitf Involved in Innate Immunity by Activating Tyrosinase-Mediated Melanin Synthesis in Pteria penguin

Abstract

The microphthalmia-associated transcription factor (MITF) is an important transcription factor that plays a key role in melanogenesis, cell proliferation, survival and immune defense in vertebrate. However, its function and function mechanism in bivalve are still rarely known. In this research, first, a Mitf gene was characterized from Pteria penguin (P. penguin). The PpMitf contained an open reading frame of 1,350 bp, encoding a peptide of 449 deduced amino acids with a highly conserved basic helix-loop-helix-leucine zipper (bHLH-LZ) domain. The PpMITF shared 55.7% identity with amino acid sequence of Crassostrea gigas (C. gigas). Tissue distribution analysis revealed that PpMitf was highly expressed in mantle and hemocytes, which were important tissues for color formation and innate immunity. Second, the functions of PpMitf in melanin synthesis and innate immunity were identified. The PpMitf silencing significantly decreased the tyrosinase activity and melanin content, indicating PpMitf involved in melanin synthesis of P. penguin. Meanwhile, the PpMitf silencing clearly down-regulated the expression of PpBcl2 (B cell lymphoma/leukemia-2 gene) and antibacterial activity of hemolymph supernatant, indicating that PpMitf involved in innate immunity of P. penguin. Third, the function mechanism of PpMitf in immunity was analyzed. The promoter sequence analysis of tyrosinase (Tyr) revealed two highly conserved E-box elements, which were specifically recognized by HLH-LZ of MITF. The luciferase activities analysis showed that Mitf could activate the E-box in Tyr promoter through highly conserved bHLH-LZ domain, and demonstrated that PpMitf involved in melanin synthesis and innate immunity by regulating tyrosinase expression. Finally, melanin from P. penguin, the final production of Mitf-Tyr-melanin pathway, was confirmed to have direct antibacterial activity. The results collectively demonstrated that PpMitf played a key role in innate immunity through activating tyrosinase-mediated melanin synthesis in P. penguin.

Keywords: Mitf; Pteria penguin; innate immunity; melanin; tyrosinase.

Figure 1

The full-length nucleotide and deduced amino acid sequences of PpMitf. The ORF sequence was displayed in uppercase, the 5’ UTR and 3’ UTR sequences were displayed in lowercase. The initiation codon (ATG) and termination codon (TGA) were boxed. The putative bHLH-LZ domains were boxed in gray. The signal peptide (aataa) was marked in boldface. * meant no amino acid was coded.

Figure 2

Sequence alignment and phylogenetic analysis. (A) Multiple sequence comparison of Mitf amino acid sequences in various species, including Pteria penguin (PpMitf, MN296415), Crassostrea gigas (CgMitf, XP_011447560.2), Crassostrea virginica (CvMitf-like, XP_022298291.1), Mizuhopecten yessoensis (MyMitf-like, XP_021364176.1), Pecten maximus (PmMitf-like, XP_033745263.1), Mytilus coruscus (McMitf, CAC5409749.1), Hyriopsis cumingii (HcMitf, QBG58751.1), Pomacea canaliculata (PcMitf, XP_025106304.1), Octopus vulgaris (OvMitf-like, XP_029634800.1), Danio rerio (DrMitf, NP_571922.2), Xenopus laevis (XlMitf, NP_001165646.1), Gallus gallus (GgMitf, BAA25648.1), Mus musculus (MsMitf, AAF81266.2). The black background showed highly conserved amino acids, the green and pink background showed similar amino acids. HLH-LZ domain was boxed in red, and N-terminal conserved domain was boxed in blue. (B) Phylogenetic tree of Mitf genes. The phylogenetic tree was constructed using neighbor-joining method. Numbers at the nodes represented the bootstrap values determined by bootstrap analysis of 1,000 replicates. The scale bar indicated the number of amino acid substitutions per site.

Figure 3

The expression of PpMitf in different tissues was validated by qRT-PCR. The abundance of PpMitf mRNA was normalized to that of β-actin of P. penguin. The data were represented as mean ± SD (N = 5). Different letters (a, b, bc and d) meant significant difference among these columns (P <0.05).

Figure 4

The effect of Mitf silencing on Tyr expression and tyrosinase activity. (A) The position and length of siRNA1 and siRNA2 (B) The PpMitf expression and tyrosinase activity analysis. The samples were from blank group, NC group (GFP-siRNA), PpMitf-siRNA1 and PpMitf-siRNA2 group. The β-actin of P. penguin was used as an internal control in qRT-PCR. The arbutin group was used as positive control in tyrosinase activity analysis. The data were represented as mean ± SD (N = 5). *P <0.05; **P <0.01; No signal means no difference.

Figure 5

The content of melanin from samples in blank, NC, RNAi groups and arbutin group using LC-MS/MS analysis. (A) HPLC (High Performance Liquid Chromatography) chromatograms. (B) The content of PDCA and PTCA. The data were represented as the mean ± SD (N = 5). *P <0.05; **P <0.01.

Figure 6

Transcriptional levels of PpTyr, PpCdk2 and PpBcl2 after PpMitf RNAi. The qRT-PCR was performed using samples from blank, NC, siRNA1 and siRNA2 group. The abundance of mRNA was normalized to that of β-actin of P. penguin. The data were represented as the mean ± SD (N = 5). *P <0.05; **P <0.01.

Figure 7

Sequence of the Tyr promoter. The putative transcriptional start site was indicated with box, and the initiation codon was bolded. The two conserved E-box, six CRE sites, two AP-2 sites were shaded in gray. The putative CAAT box and TATA box were underlined.

Figure 8

The PpTyr promoter activity was induced by PpMitf. (A) PpTyr promoter activity analysis. The 293T cells in 24-well plates were transfected with 0.4 μg of Tyr-promoter-Luc (pGL3-Basic in control) and 0.04 μg pRL-cmv vector. 48 h post transfection, cells were collected for luciferase activity assays. (B) The construction of Mitf-pcDNA3.1, Mitf-△HLHLZ-pcDNA3.1, Tyr-△Eboxes-promoter-Luc, Tyr-△Ebox1-promoter-Luc and Tyr-△Ebox2-promoter-Luc plasmids. (C) The function analysis of HLHLZ domain in MITF and E-box in Tyr promoter by luciferase activity analysis and western blot. Column and lane 1, the 293T cells were cotransfected with pcDNA3.1 and Tyr-promoter-Luc vector; Column and lane 2, Mitf-pcDNA3.1 and Tyr-promoter-Luc; Column and lane 3, Mitf-△HLHLZ-pcDNA3.1 and Tyr-promoter-Luc; Column and lane 4, Mitf-pcDNA3.1 and △Eboxes-promoter-Luc; Column and lane 5, Mitf-pcDNA3.1 and △Ebox1-promoter-Luc; Column and lane 6, Mitf-pcDNA3.1 and △Ebox2-promoter-Luc. The data were represented as the mean ± SD (N = 5). *P <0.05; **P <0.01; No signal means no difference.

Figure 9

Antibacterial activity of haemolymph supernatant from samples after PpMitf RNAi. (A) Growth curves of E. coli exposed to haemolymph supernatants of NC, siRNA1 and siRNA2 groups. (B) OD600 value of E. coli at T50 in different groups. Each value was shown as mean ± SD (N = 5). *P <0.05; **P <0.01.

Figure 10

Antibacterial activity of melanin oxidation products from P. penguin samples. (A) Photographs showed the number of E. coli after Mitf silencing and adding melanin oxidation products. (B) Histogram showed the number of E. coli after Mitf silencing and adding melanin oxidation products. Each value was shown as mean ± SD (N = 5; *P <0.05; **P <0.01).