Molecular Cloning of a New Immunomodulatory Protein from Anoectochilus formosanus which Induces B Cell IgM Secretion through a T-Independent Mechanism

Abstract

An immunomodulatory protein (IPAF) was purified and cloned from Anoectochilus formosanus, an Orchidaceae herbal plant in Asia. The major targeting immune cells of IPAF and its modulating effects toward B lymphocytes were investigated. Rapid amplification of cDNA ends (RACE) was conducted to clone the IPAF gene, and the obtained sequence was BLAST compared on the NCBI database. MACS-purified mouse T and B lymphocytes were stimulated with IPAF and the cell proliferation, activation, and Igs production were examined. IPAF comprised a 25 amino acids signal peptide and a 138 amino acids protein which was homologous to the lectins from Orchidaceae plant. IPAF selectively induced the cell proliferation in mouse splenic B lymphocytes but not T lymphocytes. The IPAF-induced B cells exhibited increased CD69 and MHC class II expression, and a dose- and time-dependent enhancement in IgM production. These results suggested potential benefits of IPAF to strengthen the humoral immunity.

Investigating the function of a novel protein from Anoectochilus formosanus which induced macrophage differentiation through TLR4-mediated NF-κB activation

“…These data indicated that IPAF activated murine macrophages through TLR4-dependent NF-κB activation. Intriguingly, the IPAF-induced B cell activation was not qualitatively affected by TLR4 deficiency [9]. This implied that TLR4 might participate partially in the interaction between IPAF and B cells, and that other receptors such as the B cell receptor might be involved in the IPAF-induced B cell activation.…”

“…The IPAF used in this study was prepared as described in our previous work [9]. Briefly, the whole plant of A. formosanus was homogenized by sonication, and the crude protein was precipitated by applying 90% of ammonium sulfate.…”

“…In our recent report [9], we have purified a new protein molecule, the immunomodulatory protein from A. formosanus (IPAF), through anion exchange chromatography approach, and cloned the complete nucleotide sequence of IPAF (ADK74829.1) by means of the rapid amplification of cDNA ends PCR methods. The amino acid (aa) sequence of IPAF comprised a 25-aa signal peptide and a 138-aa mature protein, and shared high sequence homology to the lectins from Orchidaceae plants.…”

Trametes versicolor Protein YZP Activates Regulatory B Lymphocytes – Gene Identification through De Novo Assembly and Function Analysis in a Murine Acute Colitis Model

“…), de novo contig assembly was performed by combining the processed-reads based on sequence overlap. To identify the gene encoding YZP, the contigs were translated into protein sequences, which were subsequently matched to the N-terminal amino acid sequence of the YZP derived using previously described methods [15]. The YZP gene was PCR-cloned using primers designed based on the sequence of the contig encoding the N-terminus of YZP (Table S1 and Table S2).…”

“…Mice splenic cells were acquired as previously described [15]. Briefly, the spleens were excised from euthanized mice, and the total splenocytes and splenic CD19 + B cells or CD90 + T cells were purified using MACS system (Miltenyi Biotec, Bergisch Gladbach, Germany).…”