Journal: PLoS Pathogens

Article Title: Methyl-CpG-binding (SmMBD2/3) and chromobox (SmCBX) proteins are required for neoblast proliferation and oviposition in the parasitic blood fluke Schistosoma mansoni

doi: 10.1371/journal.ppat.1007107

Figure Lengend Snippet: (A) Diagrammatic representation of the 314 amino acid SmMBD2/3 (encoded by Smp_138180) illustrating the methyl-CpG (mCpG) binding domain (PF01429), two putative predicted bipartite nuclear localisation signals (NLS), a coiled-coil domain (CC) and a C-terminal domain of methyl CpG binding protein 2 and 3 (PF140489). Amino acid positions are indicated in bold numbering. (B) A multiple sequence alignment of the methyl-CpG (mCpG) binding domains (PF01429) collected from SmMBD2/3 (italics and contained in a blue box) and MBD homologs was generated. MBDs unable to bind 5mC are indicated in red. Highly conserved residues are highlighted in turquoise and moderately conserved residues are shaded grey. A ‘*’ indicates amino acid residues that contribute to 5mC binding as assessed by mutational studies (summarised in ). A ‘#’ signifies additional amino acid residues that directly interact with 5mC and a ‘:’ indicates amino acid residues that interact with the DNA phosphate backbone . Amino acid insertions in AmMBD1 and DmMBD2/3 are indicated in black boxes above and below the alignment, respectively. AmMBD1: XP_003250634.1, HsMBD1: NP_002375.1, MmMBD1: NP_038622.2, HsMeCP2: NP_001104262.1, MmMeCP2: NP_001075448.1, XlMeCP2: NP_001081854.1, HsMBD4: NP_001263201.1, MmMBD4: NP_034904.2, HsMBD2: NP_003918.1, MmMBD2 NP_034903.2, GgMBD2: NP_001012403.1, HsMBD3: NP_001268382.1, MmMBD3: NP_038623.1, XlMBD3: AAD55389.1, BmMBD2/3: XP_004929675.1, ApMBD2/3: ACF05483.1, HpMBD2/3: ACF05485.1, AdMBD2/3: , SmMBD2/3: CCD59176.1, DmMBD2/3: NP_731370.1. (C) Ribbon representation of SmMBD2/3 homology model (green) depicts 5mC-interacting residues (stick representation: amino acids in red, 5mC in orange) found within PF01429. The SmMD2/3 model was generated using the solved crystal structure of G . gallus MBD2 co-complexed with methylated DNA as detailed in the Materials and Methods . The tetra-amino acid archetypal binding pocket is indicated (red).

Article Snippet: The MBD template selected for SmMBD2/3 modelling was the three-dimensional structure of G . gallus MBD2 (Protein Databank [ ] identification code: 2KY8) [ ].

Techniques: Binding Assay, Sequencing, Generated, Methylation

Journal: PLoS Pathogens

Article Title: Methyl-CpG-binding (SmMBD2/3) and chromobox (SmCBX) proteins are required for neoblast proliferation and oviposition in the parasitic blood fluke Schistosoma mansoni

doi: 10.1371/journal.ppat.1007107

Figure Lengend Snippet: (A) The 5mC binding capacity of nuclear protein extracts derived from adult male and female worms were quantified using the Epigentek MBD2 binding activity/inhibition assay. NIH-3T3 nuclear protein extracts and BSA were included as positive and negative controls, respectively. A significant difference in 5mC binding (in the CpG context) amongst protein samples was found. (B) IPTG-induced rSmMBD2/3-His 6 protein (arrowhead; 36.5 kDa) was produced in E . coli , purified by Ni 2+ -NTA column chromatography and subjected to MALDI-TOF MS ( Materials and Methods ; 22 peptides covering 67% of full length SmMBD2/3 identified). An un-induced sample was also produced and similarly processed. (C) The 5mC binding activity (within a CpG context) of purified rSmMBD2/3-His 6 was measured using the Epigentek MBD2 binding activity/inhibition assay and compared to un-induced bacterial and BSA protein samples. Significant differences in 5mC binding were observed between rSmMBD2/3-His 6 and both the BSA and un-induced samples.

Article Snippet: The MBD template selected for SmMBD2/3 modelling was the three-dimensional structure of G . gallus MBD2 (Protein Databank [ ] identification code: 2KY8) [ ].

Techniques: Binding Assay, Derivative Assay, Activity Assay, Inhibition, Produced, Purification, Column Chromatography