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Trametes versicolor protein YZP activates regulatory B lymphocytes - gene identification through de novo assembly and function analysis in a murine acute colitis model.

Kuan YC, Wu YJ, Hung CL, Sheu F - PLoS ONE (2013)

Bottom Line: YZP induced a greater than 60-fold increase in IL-10 secretion in mice B lymphocytes; moreover, YZP specifically triggered the differentiation of CD1d(+) B cells into IL-10-producing regulatory B cells (Bregs) and enhanced the expression of CD1d.Furthermore, YZP activated Breg function via interaction with TLR2 and TLR4 and up-regulation of the TLR-mediated signaling pathway.We purified a novel Breg-stimulating protein, YZP, from T. versicolor and developed an advanced approach combining RNA-seq and de novo assembly technologies.to clone its gene.

View Article: PubMed Central - PubMed

Affiliation: Center for Biotechnology, National Taiwan University, Taipei, Taiwan ; Department of Horticulture, National Taiwan University, Taipei, Taiwan.

ABSTRACT

Background: Trametes versicolor (Yun-Zhi) is a medicinal fungus used as a chemotherapy co-treatment to enhance anti-tumor immunity. Although the efficacies of T. versicolor extracts have been documented, the active ingredients and mechanisms underlying the actions of these extracts remain uncharacterized.

Results: We purified a new protein, YZP, from the fruiting bodies of T. versicolor and identified the gene encoding YZP using RNA-seq and de novo assembly technologies. YZP is a 12-kDa non-glycosylated protein comprising 139 amino acids, including an 18-amino acids signal peptide. YZP induced a greater than 60-fold increase in IL-10 secretion in mice B lymphocytes; moreover, YZP specifically triggered the differentiation of CD1d(+) B cells into IL-10-producing regulatory B cells (Bregs) and enhanced the expression of CD1d. YZP-induced B cells suppressed approximately 40% of the LPS-activated macrophage production of inflammatory cytokines in a mixed leukocyte reaction and significantly alleviated the disease activity and colonic inflammation in a DSS-induced acute colitis murine model. Furthermore, YZP activated Breg function via interaction with TLR2 and TLR4 and up-regulation of the TLR-mediated signaling pathway.

Conclusions: We purified a novel Breg-stimulating protein, YZP, from T. versicolor and developed an advanced approach combining RNA-seq and de novo assembly technologies.to clone its gene. We demonstrated that YZP activated CD1d(+) Breg differentiation through TLR2/4-mediated signaling pathway, and the YZP-stimulated B cells exhibited anti-inflammatory efficacies in vitro and in murine acute colitis models.

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Complete nucleotide and amino acid sequences of YZP.RNA-seq and de novo contig assembly were conducted to identify the YZP gene. The complete nucleotide sequence of the YZP cDNA was confirmed by PCR cloning using the primers shown in Supporting Information Table S1 and Table S2, and is presented in plain capital letters with the start and termination codons presented in red. The nucleotide sequences of one set of cloning primers (YZP1) are indicated with blue arrows. The translated amino acid sequence is presented in bold capital letters, whereas the N-terminal amino acids are underlined. The putative signal peptide predicted using the SignalP 4.0 Server (Supporting Information Figure S3) is indicated with a grey background.
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pone-0072422-g002: Complete nucleotide and amino acid sequences of YZP.RNA-seq and de novo contig assembly were conducted to identify the YZP gene. The complete nucleotide sequence of the YZP cDNA was confirmed by PCR cloning using the primers shown in Supporting Information Table S1 and Table S2, and is presented in plain capital letters with the start and termination codons presented in red. The nucleotide sequences of one set of cloning primers (YZP1) are indicated with blue arrows. The translated amino acid sequence is presented in bold capital letters, whereas the N-terminal amino acids are underlined. The putative signal peptide predicted using the SignalP 4.0 Server (Supporting Information Figure S3) is indicated with a grey background.

Mentions: SDS-PAGE followed by periodic acid Schiff staining was performed to confirm the lack of polysaccharide contamination in the protein samples. Purple bands corresponding to positive signals for carbohydrate content were observed for the crude protein (lane 5 in Figure 1E). However, under the same conditions, the purple bands at the 12-kDa position diminished in the sample corresponding to the purified 12-kDa protein, suggesting that after purification via 2 chromatographic steps, the 12-kDa protein was free of carbohydrate content (lane 6 in Figure 1D). This protein significantly activated IL-10 production and cell proliferation in mice splenic immune cells (Figure S2) and was subsequently referred to as the Yun-Zhi protein (YZP). Endotoxin contamination in the YZP sample was less than 0.013 EU/mg, as examined with a ToxinSensor™ Chromogenic LAL Endotoxin Assay Kit. The following protein yields were generated from each purification step: 0.21±0.11 g of crude protein was extracted from 1 kg of T. versicolor fruiting bodies; 85.3±36.1 mg of low MW proteins fraction was isolated from 1 g of crude proteins; and 0.6±0.1 mg of YZP was purified from 1 mg of low MW proteins. In summary, the yield of purified YZP was 9.8±2.4 mg per kg of T. versicolor fruiting bodies. Edman degradation permitted the identification of the first 15 amino acids (aa) of the N-terminus of YZP as [A], [L], [T], [S], [V], [S], [F], [D], [P], [V], [Y], [D], [N], [A], and [A] (underlined letters in Figure 2). The N-terminal amino acid sequence was used as the target for YZP gene identification.


Trametes versicolor protein YZP activates regulatory B lymphocytes - gene identification through de novo assembly and function analysis in a murine acute colitis model.

Kuan YC, Wu YJ, Hung CL, Sheu F - PLoS ONE (2013)

Complete nucleotide and amino acid sequences of YZP.RNA-seq and de novo contig assembly were conducted to identify the YZP gene. The complete nucleotide sequence of the YZP cDNA was confirmed by PCR cloning using the primers shown in Supporting Information Table S1 and Table S2, and is presented in plain capital letters with the start and termination codons presented in red. The nucleotide sequences of one set of cloning primers (YZP1) are indicated with blue arrows. The translated amino acid sequence is presented in bold capital letters, whereas the N-terminal amino acids are underlined. The putative signal peptide predicted using the SignalP 4.0 Server (Supporting Information Figure S3) is indicated with a grey background.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3760908&req=5

pone-0072422-g002: Complete nucleotide and amino acid sequences of YZP.RNA-seq and de novo contig assembly were conducted to identify the YZP gene. The complete nucleotide sequence of the YZP cDNA was confirmed by PCR cloning using the primers shown in Supporting Information Table S1 and Table S2, and is presented in plain capital letters with the start and termination codons presented in red. The nucleotide sequences of one set of cloning primers (YZP1) are indicated with blue arrows. The translated amino acid sequence is presented in bold capital letters, whereas the N-terminal amino acids are underlined. The putative signal peptide predicted using the SignalP 4.0 Server (Supporting Information Figure S3) is indicated with a grey background.
Mentions: SDS-PAGE followed by periodic acid Schiff staining was performed to confirm the lack of polysaccharide contamination in the protein samples. Purple bands corresponding to positive signals for carbohydrate content were observed for the crude protein (lane 5 in Figure 1E). However, under the same conditions, the purple bands at the 12-kDa position diminished in the sample corresponding to the purified 12-kDa protein, suggesting that after purification via 2 chromatographic steps, the 12-kDa protein was free of carbohydrate content (lane 6 in Figure 1D). This protein significantly activated IL-10 production and cell proliferation in mice splenic immune cells (Figure S2) and was subsequently referred to as the Yun-Zhi protein (YZP). Endotoxin contamination in the YZP sample was less than 0.013 EU/mg, as examined with a ToxinSensor™ Chromogenic LAL Endotoxin Assay Kit. The following protein yields were generated from each purification step: 0.21±0.11 g of crude protein was extracted from 1 kg of T. versicolor fruiting bodies; 85.3±36.1 mg of low MW proteins fraction was isolated from 1 g of crude proteins; and 0.6±0.1 mg of YZP was purified from 1 mg of low MW proteins. In summary, the yield of purified YZP was 9.8±2.4 mg per kg of T. versicolor fruiting bodies. Edman degradation permitted the identification of the first 15 amino acids (aa) of the N-terminus of YZP as [A], [L], [T], [S], [V], [S], [F], [D], [P], [V], [Y], [D], [N], [A], and [A] (underlined letters in Figure 2). The N-terminal amino acid sequence was used as the target for YZP gene identification.

Bottom Line: YZP induced a greater than 60-fold increase in IL-10 secretion in mice B lymphocytes; moreover, YZP specifically triggered the differentiation of CD1d(+) B cells into IL-10-producing regulatory B cells (Bregs) and enhanced the expression of CD1d.Furthermore, YZP activated Breg function via interaction with TLR2 and TLR4 and up-regulation of the TLR-mediated signaling pathway.We purified a novel Breg-stimulating protein, YZP, from T. versicolor and developed an advanced approach combining RNA-seq and de novo assembly technologies.to clone its gene.

View Article: PubMed Central - PubMed

Affiliation: Center for Biotechnology, National Taiwan University, Taipei, Taiwan ; Department of Horticulture, National Taiwan University, Taipei, Taiwan.

ABSTRACT

Background: Trametes versicolor (Yun-Zhi) is a medicinal fungus used as a chemotherapy co-treatment to enhance anti-tumor immunity. Although the efficacies of T. versicolor extracts have been documented, the active ingredients and mechanisms underlying the actions of these extracts remain uncharacterized.

Results: We purified a new protein, YZP, from the fruiting bodies of T. versicolor and identified the gene encoding YZP using RNA-seq and de novo assembly technologies. YZP is a 12-kDa non-glycosylated protein comprising 139 amino acids, including an 18-amino acids signal peptide. YZP induced a greater than 60-fold increase in IL-10 secretion in mice B lymphocytes; moreover, YZP specifically triggered the differentiation of CD1d(+) B cells into IL-10-producing regulatory B cells (Bregs) and enhanced the expression of CD1d. YZP-induced B cells suppressed approximately 40% of the LPS-activated macrophage production of inflammatory cytokines in a mixed leukocyte reaction and significantly alleviated the disease activity and colonic inflammation in a DSS-induced acute colitis murine model. Furthermore, YZP activated Breg function via interaction with TLR2 and TLR4 and up-regulation of the TLR-mediated signaling pathway.

Conclusions: We purified a novel Breg-stimulating protein, YZP, from T. versicolor and developed an advanced approach combining RNA-seq and de novo assembly technologies.to clone its gene. We demonstrated that YZP activated CD1d(+) Breg differentiation through TLR2/4-mediated signaling pathway, and the YZP-stimulated B cells exhibited anti-inflammatory efficacies in vitro and in murine acute colitis models.

Show MeSH
Related in: MedlinePlus