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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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Purification and biochemical characterization of YZP.A. Fruiting bodies of Trametes versicolor (Yun-Zhi). B. Chromatogram of the crude protein extract of T. versicolor fractionated on a HiTrap Q anion exchange column. C. Chromatogram of the pooled YZP-containing fractions fractionated on a Resource Q anion exchange column. D. Chromatogram of the purified YZP samples analyzed by PROTEIN KW-802.5 SHODEX gel-filtration chromatography. E. SDS-PAGE analysis of the crude protein extracts from T. versicolor (lanes 1 and 5), the YZP-containing fraction (lane 2), non-YZP fraction (lane 3), and purified YZP (lanes 4 and 6). Lanes 1 to 4 were stained with CBR, whereas lanes 5 and 6 were stained with PAS reagent. Lane M was loaded with PageRuler Prestained Protein Ladder #SM0671. F. The calibration curve was established via linear regression of the molecular weights of the proteins present in the PageRuler Prestained Protein Ladder #SM0671 versus the nature logarithm of their relative mobility shifts. The molecular weights of T. versicolor proteins are indicated with red arrows.
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pone-0072422-g001: Purification and biochemical characterization of YZP.A. Fruiting bodies of Trametes versicolor (Yun-Zhi). B. Chromatogram of the crude protein extract of T. versicolor fractionated on a HiTrap Q anion exchange column. C. Chromatogram of the pooled YZP-containing fractions fractionated on a Resource Q anion exchange column. D. Chromatogram of the purified YZP samples analyzed by PROTEIN KW-802.5 SHODEX gel-filtration chromatography. E. SDS-PAGE analysis of the crude protein extracts from T. versicolor (lanes 1 and 5), the YZP-containing fraction (lane 2), non-YZP fraction (lane 3), and purified YZP (lanes 4 and 6). Lanes 1 to 4 were stained with CBR, whereas lanes 5 and 6 were stained with PAS reagent. Lane M was loaded with PageRuler Prestained Protein Ladder #SM0671. F. The calibration curve was established via linear regression of the molecular weights of the proteins present in the PageRuler Prestained Protein Ladder #SM0671 versus the nature logarithm of their relative mobility shifts. The molecular weights of T. versicolor proteins are indicated with red arrows.

Mentions: The crude proteins extracted from T. versicolor fruiting bodies (Figure 1A) were analyzed by SDS-PAGE, and 4 major bands (lane 1 in Figure 1E) with molecular weights (MW) of 87, 45, 16, and 12 kDa were detected. To separate these proteins, the crude extracts were fractionated on a HiTrap Q anion exchange column (Figure 1B). When eluted with 0.15 to 0.16 M NaCl-Tris buffer (N-TB, pH 8.2), a single peak comprising the 16 and 12 kDa proteins (lane 2 in Figure 1E) was obtained, whereas the 87 and 45 kDa proteins were eluted with 0.5 M N-TB (lane 3 in Figure 1E). The fractions containing the 16- and 12-kDa proteins were pooled and re-dialyzed into TB for further purification on a Resource Q anion exchange column (Figure 1C). The column was eluted with 0.15 to 0.16 M N-TB, and a sample containing the 12-kDa protein was obtained (lane 4 in Figure 1E). This sample was analyzed with a Shodex gel-filtration column (Figure 1D), and the purity of the sample was greater than 95%, based on the calculation of the peak area relative to the total area under the curve. The immuno-modulating activity of the crude proteins extract, the lower-MW fraction (YZP-enriched), and the higher-MW fraction (non-YZP) was analyzed using murine peritoneal macrophages (MΦ) and splenocytes. The crude proteins extract stimulated TNF-α production in MΦ and increased cellular enzyme activity in splenocytes. These effects were significantly elevated in YZP-enriched fraction purified through HiTrap Q column (Figure S1). The purity of YZP was significantly improved from ∼90% to above 96% through further purification with Resource Q column; however, only mild enhancement in immuno-modulating activity was observed.


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)

Purification and biochemical characterization of YZP.A. Fruiting bodies of Trametes versicolor (Yun-Zhi). B. Chromatogram of the crude protein extract of T. versicolor fractionated on a HiTrap Q anion exchange column. C. Chromatogram of the pooled YZP-containing fractions fractionated on a Resource Q anion exchange column. D. Chromatogram of the purified YZP samples analyzed by PROTEIN KW-802.5 SHODEX gel-filtration chromatography. E. SDS-PAGE analysis of the crude protein extracts from T. versicolor (lanes 1 and 5), the YZP-containing fraction (lane 2), non-YZP fraction (lane 3), and purified YZP (lanes 4 and 6). Lanes 1 to 4 were stained with CBR, whereas lanes 5 and 6 were stained with PAS reagent. Lane M was loaded with PageRuler Prestained Protein Ladder #SM0671. F. The calibration curve was established via linear regression of the molecular weights of the proteins present in the PageRuler Prestained Protein Ladder #SM0671 versus the nature logarithm of their relative mobility shifts. The molecular weights of T. versicolor proteins are indicated with red arrows.
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Related In: Results  -  Collection

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pone-0072422-g001: Purification and biochemical characterization of YZP.A. Fruiting bodies of Trametes versicolor (Yun-Zhi). B. Chromatogram of the crude protein extract of T. versicolor fractionated on a HiTrap Q anion exchange column. C. Chromatogram of the pooled YZP-containing fractions fractionated on a Resource Q anion exchange column. D. Chromatogram of the purified YZP samples analyzed by PROTEIN KW-802.5 SHODEX gel-filtration chromatography. E. SDS-PAGE analysis of the crude protein extracts from T. versicolor (lanes 1 and 5), the YZP-containing fraction (lane 2), non-YZP fraction (lane 3), and purified YZP (lanes 4 and 6). Lanes 1 to 4 were stained with CBR, whereas lanes 5 and 6 were stained with PAS reagent. Lane M was loaded with PageRuler Prestained Protein Ladder #SM0671. F. The calibration curve was established via linear regression of the molecular weights of the proteins present in the PageRuler Prestained Protein Ladder #SM0671 versus the nature logarithm of their relative mobility shifts. The molecular weights of T. versicolor proteins are indicated with red arrows.
Mentions: The crude proteins extracted from T. versicolor fruiting bodies (Figure 1A) were analyzed by SDS-PAGE, and 4 major bands (lane 1 in Figure 1E) with molecular weights (MW) of 87, 45, 16, and 12 kDa were detected. To separate these proteins, the crude extracts were fractionated on a HiTrap Q anion exchange column (Figure 1B). When eluted with 0.15 to 0.16 M NaCl-Tris buffer (N-TB, pH 8.2), a single peak comprising the 16 and 12 kDa proteins (lane 2 in Figure 1E) was obtained, whereas the 87 and 45 kDa proteins were eluted with 0.5 M N-TB (lane 3 in Figure 1E). The fractions containing the 16- and 12-kDa proteins were pooled and re-dialyzed into TB for further purification on a Resource Q anion exchange column (Figure 1C). The column was eluted with 0.15 to 0.16 M N-TB, and a sample containing the 12-kDa protein was obtained (lane 4 in Figure 1E). This sample was analyzed with a Shodex gel-filtration column (Figure 1D), and the purity of the sample was greater than 95%, based on the calculation of the peak area relative to the total area under the curve. The immuno-modulating activity of the crude proteins extract, the lower-MW fraction (YZP-enriched), and the higher-MW fraction (non-YZP) was analyzed using murine peritoneal macrophages (MΦ) and splenocytes. The crude proteins extract stimulated TNF-α production in MΦ and increased cellular enzyme activity in splenocytes. These effects were significantly elevated in YZP-enriched fraction purified through HiTrap Q column (Figure S1). The purity of YZP was significantly improved from ∼90% to above 96% through further purification with Resource Q column; however, only mild enhancement in immuno-modulating activity was observed.

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