Characterisation of a New Fungal Immunomodulatory Protein from Tiger Milk mushroom, Lignosus rhinocerotis

Abstract

Lignosus rhinocerotis (Tiger milk mushroom) is an important folk medicine for indigenous peoples in Southeast Asia. We previously reported its de novo assembled 34.3 Mb genome encoding a repertoire of proteins including a putative bioactive fungal immunomodulatory protein. Here we report the cDNA of this new member (FIP-Lrh) with a homology range of 54-64% to FIPs from other mushroom species, the closest is with FIP-glu (LZ-8) (64%) from Ganoderma lucidum. The FIP-Lrh of 112 amino acids (12.59 kDa) has a relatively hydrophobic N-terminal. Its predicted 3-dimensional model has identical folding patterns to FIP-fve and contains a partially conserved and more positively charged carbohydrates binding pocket. Docking predictions of FIP-Lrh on 14 glycans commonly found on cellular surfaces showed the best binding energy of -3.98 kcal/mol to N-acetylgalactosamine and N-acetylglucosamine. Overexpression of a 14.9 kDa soluble 6xHisFIP-Lrh was achieved in pET-28a(+)/BL21 and the purified recombinant protein was sequence verified by LC-MS/MS (QTOF) analysis. The ability to haemagglutinate both mouse and human blood at concentration ≥0.34 μM, further demonstrated its lectin nature. In addition, the cytotoxic effect of 6xHisFIP-Lrh on MCF-7, HeLa and A549 cancer cell lines was detected at IC50 of 0.34 μM, 0.58 μM and 0.60 μM, respectively.

Figure 1. The structure of FIP-fve dimer (1OSY.pdb).

Figure 2. RT-PCR and cloning of FIP-Lrh cDNA.

(a) Total RNA extracted from sclerotia of L. rhinocerotis as analysed on a 1% agarose gel electrophoresis. Lane M₁ contained 1.5 μg of λ HindIII DNA marker (NEB, USA) whereas Lane 1 and 2 contained ~0.5 μg and 1.5 μg of total RNA. (b) An approximately 500 bp PCR product of FIP-Lrh cDNA was obtained through RT-PCR using FIPf and FIPr primers, as shown in Lane 3. M₂ contained 1.25 μg of 100 bp DNA marker (NEB, USA). (c) Four pGEMT clones containing FIP-Lrh cDNA were subjected to PCR using FIPf and FIPr. A total of 5 μL PCR product obtained using clone pGEM_FIP_Lrh_1, pGEM_FIP_Lrh_2, pGEM_FIP_Lrh_3 and pGEM_FIP_Lrh_4 as template was loaded in Lane 4–7 respectively. Arrow indicates the presence of insert of approximately 400–500 bp in size.

Figure 3. Multiple protein sequence alignment, protein and DNA sequence comparison of the four cloned FIP-Lrh cDNAs and the putative FIP-Lrh.

The Met (M) initiation codon is as indicated (). The position of the splice sites (donor, GT and acceptor, AG) in the intron is in bold and underlined and was predicted using the SplicePort tool (http://spliceport.cbcb.umd.edu). The predicted intron (27 bp) is shown in grey.

Figure 4. Multiple alignment of protein sequence and phylogeny of the four cloned FIP-Lrh cDNA with reported FIPs.

The alignment is performed with the Clustal Omega (1.2.1) (EMBL-EBI) using the published FIP sequences of FIP-gja (G. japonicum) (GenBank: AAX98241.1), FIP-gsi (Ganoderma sinense), FIP-glu (G. lucidum) (UniProtKB/Swiss-Prot: P14945.2), FIP-gap (G. applanatum, GenBank: AEP68179.1), FIP-fve (Flammulina velutipes, GenBank: ADB24832.1), FIP-tvc (Trametes versicolor) and FIP-vvo (Volvariella volvacea) and gaps are introduced for optimal alignment and maximum similarity between all compared sequences. The identical amino acids among all the aligned sequences are indicated as ‘*’ whereas “:” conserved substitutions and empty space represents a non-conserved substitution. and the identical amino acids with FIP-gsi are shown in a gray background. The key residues in FIP-fve (W24, T28, D34, T90, I91 and W111) and the corresponding residues in FIP-Lrh (W25, D35, I92 and W112) present in the carbohydrate binding module (CBM) are shown as a solid line (). A Neighbour-joining phylogram (with real Branch length, distance corrected) was generated using the Omega Clustal program with FIPs from the representatives of genera Lignosus, Ganoderma, Flammulina, Volvariella and Trametes.

Figure 5. Hydrophobicity profile, predicted 3-D model of FIP-Lrh homodimer and Ramachandran plot.

(a) The hydropathy plot of the deduced amino acid sequence of FIP-Lrh was plotted using the Kyte-Doolittle (http://web.expasy.org/protscale/). The 3-D structure of the homodimer showed α-helix at the N-terminal and the seven β-sheets (a,b).

Figure 6. Comparison of the predicted binding energy of FIP-Lrh and FIP-fve to 14 different glycans.

The structure of two glycans with the most stable binding, N-acetylgalactosamine and N-acetylglucosamine and the least binding glycan, Glycogen, are as shown.

Figure 7

N-acetylgalactosamine docking into the active binding pocket of (a) FIP-fve and (b) FIP-Lrh and their corresponding LIGPLOT. The residues involved in binding of N-acetylgalactosamine are identified and are labelled for the 3-D structures of both proteins. Protein residues that are in equivalent 3D positions when the two structures are superposed are circled in red. H-bonds and their distances are indicated in green dashed lines. Residues closest to the ligand (indicated by stick and ball figures) whereas protein residues making hydrophobic interactions with ligand are indicated by spoked arcs.

Figure 8. Ni-NTA purification of 6xHisFIP-Lrh protein.

A total of 24 μL sample were loaded into each well, as follows: 1: cell lysate containing the soluble 6xHisFIP-Lrh protein; 2: contained the flow through collected after the Ni-NTA resin binding; W_1–2, W_3–4, W_5–6, W_7–8: contained flow through of washes in wash buffer with 20 mM, 30 mM, 40 mM and 50 mM Imidazole (pH8.0) respectively; the E_2–8: eluents containing the 14.9 kDa recombinant 6xHisFIP-Lrh (arrow) eluted at 250 mM Imidazole. (P) the purified 6xHisFIP-Lrh (arrow). M: Broad range protein marker (p7710s, NEB, UK).

Figure 9. Haemagglutination assay of 6xHisFIP-Lrh.

Different concentration of 6xHisFIP-Lrh (0–30 μg/mL, 0–2.01 μM) were incubated with 2% human or mouse RBCs and monitored under inverted microscope after 2 hr of incubation. The 6xHisFIP-Lrh was able to haemagglutinate both human and mouse whole blood at concentration of ≥0.34 μM (5 μ/mL), with greater degree of cells agglutination observed in mouse compared to human.

Figure 10. Cytotoxicity of 6xHisFIP-Lrh on human breast, cervical and lung cancer cell lines.

Cell viability curve of 6xHisFIP-Lrh (0–3.36 μM) treated MCF-7 (ATCC HTB-22), A549 (ATCC CCL-185) and HeLa (ATCC CCL-2) cancer cell lines was obtained from triplicate assays. The IC₅₀ of MCF-7, HeLa and A549 cells was 0.34 μM, 0.58 μM and 0.60 μM of recombinant protein, respectively.