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IgG glycan hydrolysis by a bacterial enzyme as a therapy against autoimmune conditions

Mattias Collin^*, Oonagh Shannon, and Lars Björck

Division of Infection Medicine, Department of Clinical Sciences, Lund University, SE-22184 Lund, Sweden

View larger version (41K): [in this window] [in a new window]   Fig. 1. Structure and glycosylation of IgG. (A) Schematic representation of the fully substituted IgG heavy-chain glycan. S2 indicates the fully sialylated glycoform G0, and brackets indicate the extent of the G0 glycoform. LCA indicates the binding site for the LCA used in lectin experiments and EndoS the cleavage site for the enzyme. (B) Structural model of human IgG. IgG heavy chains are colored blue, and the light chains are colored red. Brackets indicate the antigen-binding Fab' portion and the Fc effector portion of IgG. Arrow indicates the two conserved glycans (yellow) attached to Asn-297 of the heavy chains. The model was generated by using VMD 1.8.5 from a model deposited in the Protein Data Bank by M. Clark (Cambridge University, Cambridge, U.K.).

View larger version (21K): [in this window] [in a new window]   Fig. 2. EndoS hydrolyzes IgG in whole human blood. (A) SDS/PAGE analysis of purified IgG from human blood incubated with increasing concentrations of EndoS. (B) LCA lectin blot analysis of purified IgG from whole human blood incubated with increasing concentrations of EndoS. (C) Densitometric analysis of the lectin blot on IgG purified from human blood incubated with increasing concentrations of EndoS. Values are presented as the percentage of signal from control sample incubated with buffer alone. Means and standard errors were calculated from three independent experiments using blood from three different donors.

View larger version (38K): [in this window] [in a new window]   Fig. 3. EndoS hydrolyzes IgG in vivo in rabbits. (A) SDS/PAGE (stain) and lectin blot analysis (LCA blot) of purified IgG from serum samples withdrawn from a representative rabbit at indicated time points after the first i.v. injection of 500 µg of EndoS (0 days). (B) SDS/PAGE (stain) and lectin blot analysis (LCA blot) of purified IgG from serum samples withdrawn from the rabbit at indicated time points after a second administration (35 days) of EndoS. (C) SDS/PAGE (stain) and lectin blot analysis (LCA blot) of purified IgG from serum samples withdrawn from the rabbit at indicated time points after a third administration (135 days) of EndoS. (D) Lectin blot analysis of total serum taken before (0) and 12 h (12) after the first injection of EndoS. ConA, Concavalin A; SNA, Sambucus nigra lectin. Arrow to the right indicates the position of the -chain of IgG.

View larger version (25K): [in this window] [in a new window]   Fig. 4. Rabbit antibody response to EndoS. Serum samples were withdrawn from the rabbit at indicated time points after the first (0 days), second (35 days), and third (135 days) injections of EndoS. Serum samples after the first (0 days), second (35 days), and third (135 days) injections were used as primary antisera in an ELISA experiment with immobilized EndoS. Increase in concentration (ng/ml) of anti-EndoS IgG compared with concentration before first injection is presented. One representative experiment is shown. (Inset) The serum samples after the first injection were used as primary antisera in a Western blot on separate membrane strips with SDS/PAGE-separated purified EndoS.

View larger version (8K): [in this window] [in a new window]   Fig. 5. EndoS pretreatment of pathogenic IgG antibodies inhibits antibody-mediated thrombocytopenia in mice. (A) Female BALB/c mice (n = 3) received i.p. injections of rabbit anti-mouse platelet IgG (PLT-IgG). Blood samples were taken at regular intervals, and platelet counts were determined by using flow cytometry. (B) Survival plots of BALB/c mice injected with PLT-IgG that were pretreated with GST-EndoS (n = 4) or GST (n = 4). (C) Platelet counts over time as determined by flow cytometry on blood samples from mice that had received PLT-IgG pretreated with GST-EndoS.

View larger version (25K): [in this window] [in a new window]   Fig. 6. EndoS rescues mice from lethal IgG-mediated thrombocytopenia. (A) Survival plots of BALB/c mice injected with PLT-IgG, followed by GST-EndoS (n = 8) or GST (n = 8) treatment 3 h after PLT-IgG administration. (B) SDS/PAGE analysis (stain) and LCA lectin blot analysis (LCA blot) of IgG purified from GST-EndoS or GST-treated mice 24, 48, or 72 (only GST-EndoS) h after injection of PLT-Ig. (C) Blood samples were taken at regular intervals, and platelet counts were determined by using flow cytometry in mice that received GST-EndoS treatment. (D) Survival plots of BALB/c mice injected with PLT-Ig, followed by GST-EndoS (n = 7) or GST (n = 7) treatment at the onset of clear signs of intraabdominal bleeding (5–7 h after PLT-Ig administration).

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