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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.

SUBMITTER: Kuan YC

PROVIDER: S-EPMC3116865 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Molecular cloning of a new immunomodulatory protein from Anoectochilus formosanus which induces B cell IgM secretion through a T-independent mechanism.

Kuan Yen-Chou YC Wu Tsai-Jen TJ Kuo Che-Yu CY Hsu Ju-Chun JC Chang Wen-Ying WY Sheu Fuu F

PloS one 20110616 6

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 produc ...[more]

PMID: 21698210

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Project description:IntroductionPolysaccharide and alcohol extracts of Anoectochilus formosanus Hayata have attracted great attention as they exhibit noteworthy properties such as prebiotic and anti-hyperglycemic effects. However, the antioxidant and wound-healing activities of the polysaccharide extract as well as the antibacterial and cytotoxic effects of the ethanol extracts have not been thoroughly uncovered. Therefore, our study investigated these bioactivities of the two extracts prepared from Anoectochilus formosanus to broaden understandings of medical benefits of the plant.MethodsThe monosaccharide composition was analyzed by HPAEC-PAD. The antioxidant and wound-healing activities of the polysaccharide extract were evaluated by ABTS and scratch assays, respectively. The broth dilution method was used to determine the antibacterial ability of the ethanol extract. Additionally, the cytotoxic and mechanistic effects of this extract against hepatocellular carcinoma HUH-7 cells was assessed by MTT assay, qRT-PCR and Western blotting methods.ResultsThe polysaccharide extract possessed an effective free radical scavenging ability in an ABTS assay (IC50 = 44.92 μg/ml). The extract also ameliorated wound recovery in a fibroblast scratch assay. Meanwhile, the ethanol extract was able to inhibit the growth of Staphylococcus aureus (MIC = 2500 μg/ml), Bacillus cereus (MIC = 2500 μg/ml), Escherichia coli (MIC = 2500 μg/ml), and Pseudomonas aeruginosa (MIC = 1250 μg/ml). Additionally, it repressed the viability of HUH-7 cells (IC50 = 53.44 μg/ml), possibly through upregulating the expression of caspase 3 (CASP3), CASP8, and CASP9 at both mRNA and protein levels.ConclusionThe polysaccharide extract of A. formosanus exhibited the antioxidant and wound-healing properties whereas the ethanol extract showed the antibacterial activity and cytotoxicity against HUH-7 cells. These findings specify notable biological effects of the two extracts which could be of potential use in human healthcare.

Project description:Human selenium-dependent glutathione peroxidase (GP) is implicated as a mechanism of resistance against oxygen free radicals. The 5' flanking sequence upstream from the coding region of GP contained an oxygen-responsive element termed ORE1 that is responsive to hypoxia, as well as several copies of the activator protein-1 (AP-1)- and AP-1-like-binding sites. In this study, we sought to define the molecular events that lead to GP gene transcription in response to hyperoxia in human umbilical-vein endothelial cells, and asked whether such induction is mimicked and sustained by activation of protein kinase C (PKC) by phorbol esters. Treatment of cells with 100 nM phorbol 12,13-dibutyrate (PdBu) induced a delayed (24-48 h) but significant (2-fold) increase in steady-state GP mRNA levels. Steady-state GP mRNA levels also rose after exposure to 95% O2, again after considerable delay (48-72 h). For both PdBu and oxygen, induction was transcriptionally regulated, as demonstrated by nuclear run-on experiments. The simulations by PdBu and oxygen were additive. In contrast with PdBu, hyperoxia did not stimulate translocation of PKC from the cytosol to the particulate fraction, although the specific activity of both cytosolic and particulate-associated PKC was increased 2-fold in cells exposed to 95% O2 for 5 days. In addition, gel mobility-shift assays using double-stranded tumour-promoting-agent-responsive element (TRE) and nuclear extracts derived from phorbol- and oxygen-treated cells revealed that PdBu, but not hyperoxia, increased AP-1 DNA-binding activity. On the other hand, the up-regulation of GP expression by oxygen could not be accounted for by the ORE1 core sequence, since no specific protein-DNA binding activity could be detected using nuclear extracts from hyperoxic cells and ORE1. Taken together, these results suggest that there may be different molecular mechanisms controlling GP expression. After exposure to PdBu, GP undergoes transcriptional activation via a process that can be readily explained by a classic AP-1 interaction with the TRE sites in the GP promoter. During hyperoxia, GP also undergoes transcriptional activity, but via a process that appears to involve neither TRE nor ORE1.

Dataset Information

Molecular cloning of a new immunomodulatory protein from Anoectochilus formosanus which induces B cell IgM secretion through a T-independent mechanism.

Publications

Molecular cloning of a new immunomodulatory protein from Anoectochilus formosanus which induces B cell IgM secretion through a T-independent mechanism.

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