Table 1.
Used primers for cloning of igdE genes.
Table 2.
Used antibodies and dilutions for Western Blot analysis.
Table 3.
Number of identified putative IgdE family protease sequences and searched genomes.
Fig 1.
Phylogenetic tree of identified putative IgdE proteases.
Phylogenic analysis of the IgdE_domain of 55 identified putative IgdE proteases. The maximum likelihood (ML) tree was constructed using a Jones-Taylor-Thornton (JTT) model with PhyML. Bootstrap values greater than or equal to 80% are shown. Putative IgdE protease sequences that corresponded to the genes cloned for expression of recombinant protein are marked in bold. Tree scale is given as average number of substitutions per site.
Table 4.
Location of igdE genes within the genomes of the respective Streptococcus species and flanking genes.
Fig 2.
IgG host species specificity of IgdE family proteases.
0.5 mg/ml human, porcine, equine, bovine and murine IgG were incubated for 16h at 37°C with (A) 20 μg/ml purified rIgdEagalactiae, (B) 20 μg/ml purified rIgdEporcinus, (C) 5% soluble fraction of E. coli cells expressing rIgdEpseudoporcinus, (D) 5% soluble fraction of E. coli cells expressing rIgdEequi. PBS (A and B) or 5% soluble fraction of E. coli cells without recombinant construct (C and D) were used as negative controls (-). Reactions were analyzed by Coomassie blue SDS-PAGE under reducing conditions. Images of different SDS-PAGE run in parallel have been assembled into one figure. The diagnostic 32 kDa IgG cleavage product (*) appeared when rIgdEagalactiae was incubated with human IgG, rIgdEporcinus with porcine IgG, rIgdEpseudoporcinus with human IgG and porcine IgG, and rIgdEequi with equine IgG.
Fig 3.
IgdE family proteases are specific for IgG compared to IgM and IgA.
2% porcine plasma was incubated with (+) or without (-) 20 μg/ml purified rIgdEporcinus (A) or 5% soluble fraction of E. coli cells expressing rIgdEpseudoporcinus (B) respectively for 16h at 37°C. 2% equine serum was incubated with 5% soluble fraction of E. coli cells expressing rIgdEequi (C) for 16h at 37°C. The reactions were analyzed by anti-porcine or anti-equine IgG, IgM and IgA Western blots under reducing conditions. Only degradation products of IgG (*) could be observed.
Fig 4.
IgdEagalactiae and IgdEpseudoporcinus are specific for human IgG compared to IgM and IgA.
0.5 mg/ml human IgG, IgM and IgA were incubated for 16h at 37°C with (+) or without (-) 20 μg/ml purified rIgdEagalactiae (A) or 5% soluble fraction of E. coli cells expressing rIgdEpseudoporcinus (B). Reactions were analyzed by SDS-PAGE under reducing conditions. SDS-PAGE was stained with Coomassie blue. Order of lanes within SDS-PAGE was adjusted.
Fig 5.
IgdEagalactiae, IgdEpseudoporcinus and IgdEequi are IgG subtype specific.
(A) 0.25 mg/ml human IgG subtypes were incubated for 16h at 37°C with (+) or without (-) 20 μg/ml purified rIgdEagalactiae. Reactions were analyzed by SDS-PAGE under reducing conditions. IgG cleavage (*) occurred only upon incubation with IgG1. SDS-PAGE was stained with Coomassie blue. Order of lanes within SDS-PAGE was adjusted. (B) 0.25 mg/ml human IgG subtypes were incubated for 16h at 37°C with (+) or without (-) 5% soluble fraction of E. coli cells expressing rIgdEpseudoporcinus. Reactions were analyzed by SDS-PAGE under reducing conditions. IgG cleavage (*) occurred only upon incubation with IgG1. SDS-PAGE was stained with Coomassie blue. (C) 0.09 mg/ml recombinant equine IgG subtypes were incubated for 16h at 37°C with (+) or without (-) 5% soluble fraction of E. coli cells expressing rIgdEequi. Reactions were analyzed by SDS-PAGE under reducing conditions. IgG cleavage (*) occurred only upon incubation with IgG7. SDS-PAGE was stained with Coomassie Fluor Orange Protein Gel Stain. Images of different SDS-PAGE run in parallel have been assembled into one figure.
Fig 6.
IgdE family proteases cleave IgG in the hinge region.
(A) The cleavage sites within IgG molecules were determined through N-terminal Edman sequencing of the 32 kDa IgG cleavage products generated by IgdE family proteases. The identified aa sequences (bold) were found in the hinge regions of the respective IgG heavy chains. Homodimer disulfide bond cysteine residues are underlined. 10 aa N- and C-terminal from the identified cleavage site (scissor symbol) of the respective IgG heavy chain are shown. Porcine IgG4a was chosen as a representative for porcine IgG. (B) Sequences of the hinge regions and adjacent parts of the CH1 and CH2 domains of human, porcine and equine IgG subtypes were aligned using T-COFFEE (Version_8.93) [38] to illustrate hinge region diversity. Alignment reliability assessed by TCS [39] is color coded (blue to red).