Detailed Structural Analysis of the Immunoregulatory Polysaccharides from the Mycobacterium Bovis BCG

Abstract

Bacillus Calmette-Guérin polysaccharide and nucleic acid (BCG-PSN), extracted from Mycobacterium bovis, is an immunoregulatory medicine commonly used in clinic. However, the structural characteristics and potential pharmacological efficacy of the polysaccharides from BCG-PSN remain unclear. Herein, two polysaccharides (BCG-1 and BCG-2) were purified and their structures were characterized. Monosaccharide composition analysis combined with methylation analysis and NMR data indicated that BCG-1 and BCG-2 were an α-D-(1→4)-mannan with (1→2)-linked branches, and an α-D-(1→4)-glucan with (1→6)-linked branches, respectively. Herein, the mannan from BCG-PSN was first reported. Bioactivity assays showed that BCG-1 and BCG-2 dose-dependently and potently increased the production of inflammatory mediators (NO, TNF-α, IL-6, IL-1β, and IL-10), as well as their mRNA expressions in RAW264.7 cells; both have similar or stronger effects compared with BCG-PSN injection. These data suggest that BCG-1 and BCG-2 are very likely the active ingredients of BCG-PSN.

Keywords: active ingredients; glucan; immunomodulator; mannan; polysaccharide purification.

Figure 1

Purification and structural characterization of polysaccharides from BCG-PSN. (A) HPGPC profiles of BCG-PSN, BCG, BCG-1, BCG-2, and BCG-3; (B) UV absorption spectra of BCG-PSN, BCG, BCG-1, BCG-2, and BCG-3; (C) HPLC-DAD profiles of PMP derivatives of standard monosaccharides and the monosaccharides from polysaccharides; (D) FT-IR spectra of BCG-PSN, BCG, BCG-1, and BCG-2; (E) The TIC profile of PMAAs from the polysaccharide BCG-1; (F) The TIC profile of PMAAs from the polysaccharide BCG-2; noncarbohydrate signals are marked with ×.

Figure 1

Purification and structural characterization of polysaccharides from BCG-PSN. (A) HPGPC profiles of BCG-PSN, BCG, BCG-1, BCG-2, and BCG-3; (B) UV absorption spectra of BCG-PSN, BCG, BCG-1, BCG-2, and BCG-3; (C) HPLC-DAD profiles of PMP derivatives of standard monosaccharides and the monosaccharides from polysaccharides; (D) FT-IR spectra of BCG-PSN, BCG, BCG-1, and BCG-2; (E) The TIC profile of PMAAs from the polysaccharide BCG-1; (F) The TIC profile of PMAAs from the polysaccharide BCG-2; noncarbohydrate signals are marked with ×.

Figure 2

¹H (A,B), ¹³C (C), COSY (D), HSQC-TOCSY (E), ROESY (F), HMBC (G) NMR spectra of BCG-1. Chemical shifts are relative to internal trimethylsilylpropionic acid sodium at 0 ppm. The letters A (maroon) and B (blue) represented 2,6-O-α-Manp (Residue A) and t-α-Manp (Residue B), respectively.

Figure 3

¹H (A), ¹³C (B), COSY (C), TOCSY (D), HSQC-TOCSY (E), HMBC (F) NMR spectra of BCG-2. Chemical shifts are relative to external trimethylsilylpropionic acid sodium at 0 ppm. The letters A/B (maroon) represented →4)-α-Glc→1 (residue A)/→4,6)-α-Glc(1→ (residue B). The letters C (blue) represented t-α-Glc (1→ (residue C).

Figure 4

Proposed structures of BCG-1 (A) and BCG-2 (B) purified from BCG-PSN.

Figure 5

Effects of LPS, BCG-PSN, BCG-1, and BCG-2 on the production of NO (A) and mRNA expressions of iNOS (B) in RAW264.7 cells. LPS was at 100 ng/mL, BCG-PSN was at 87.5 µg/mL. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. control, one-way ANOVA, Dunnett’s multiple comparisons test.

Figure 6

Effects of LPS, BCG-PSN, BCG-1, and BCG-2 on cytokines TNF-α (A), IL-6 (B), IL-1β (C), IL-10 (D) produced by the RAW 264.7 cells. LPS was at 100 ng/mL, BCG-PSN was at 87.5 µg/mL. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. control, one-way ANOVA, Dunnett’s multiple comparisons test.

Figure 7

Effects of LPS, BCG-PSN, BCG-1, and BCG-2 on the mRNA expressions of TNF-α (A), IL-6 (B), IL-1β (C), IL-10 (D) in the RAW 264.7 cells. LPS was at 100 ng/mL, BCG-PSN was at 87.5 µg/mL. *** p < 0.001 vs. control, one-way ANOVA, Dunnett’s multiple comparisons test.