Functional Evaluation of Stand-Alone CBM (carbohydrate-binding module) and Sialidase domain Constructs.

(A) SDS-PAGE gel showing the purification of the C-terminal sialidase domain construct (PG0352CT). The band pertaining to PG0352CT is indicated by the red arrow. FT: flow-through, Insol.: insoluble fraction. (B) Representative image from the sialidase filter spot assay of recombinant PG0352CT. (C) SDS-PAGE gel showing the purified recombinant CBM. (D) Co-immunoprecipitation (Co-IP) using rCBM and human serum. Recombinant PG1589 was included as a negative control. Red color numbers are three protein bands found in the CBM pulldown sample that are absent in the control samples. (E) Co-IP using rCBM and C1q untreated and treated with PG0352. For the treated sample, prior to Co-IP, C1q was incubated with PG0352 for 2 hours at 37°C. (F) Cartoon representation of fructose bound to the CBM domain of PG0352. FO-FC omit electron density, contoured at 3σ, is shown as green mesh. Residues making interactions with fructose are labeled and shown as sticks, with carbon, nitrogen, and oxygen atoms colored yellow, blue, and red, respectively. Ordered water molecules are depicted as red spheres. The chemical structure for fructose is shown in S6 Fig.</p

Data collection and refinement statistics for six PG0352 crystal structures.

Data collection and refinement statistics for six PG0352 crystal structures.</p

PG0352 is monomeric.

The oligomeric state of PG0352 was determined using size-exclusion chromatography (SEC). The estimated molecular weight of proteins under each peak were determined from the standard curve as described in the methods. (TIFF)</p

PG0352 Ligand Interactions.

Cartoon representation of the sialidase active site bound with (A) Neu5Ac, (B) DANA, (C) 3’SL, and (D) 6’SL. FO-FC omit electron density maps, contoured at 3s, are shown as green mesh in each panel. Residues making interactions with ligands are labeled and shown as sticks, with carbon, nitrogen, and oxygen atoms colored yellow, blue, and red, respectively. Ordered water molecules are depicted as red spheres. The D219A mutation is labeled in red in panels C and D. Coordination of the carboxylate head of sialic acid is mediated by the Tri-arg motif (Arg-194, Arg-398, Arg-460). The glycerol moiety is stabilized by interactions with Asp-381, while the N-acetyl moiety is stabilized by polar interactions with Asp-256 and insertion into a hydrophobic pocket formed by Leu-220, Val-272, Leu-277, Trp-278, and Phe-327. Chemical structures for each ligand are shown in S6 Fig.</p

SDS-PAGE analysis of complement factors (cFactors) treated with PG0352.

For this experiment, individual complement factors were treated with wild-type PG0352 or inactive PG0352 (PG0352AAAA) for 60 minutes and then subjected to 10% SDS-PAGE followed by Coomassie blue staining. FH: factor H; FI: factor I. (TIFF)</p

PG0352 Interactions with Citrate.

Cartoon representation of the sialidase active site bound with citrate. Residues belonging to the active site are labeled and shown as sticks, with carbon, nitrogen, and oxygen atoms colored salmon, blue, and red, respectively. Ordered water molecules are depicted as red spheres. Note Gln-400, Asp-219, and Asp-256 display alternate rotameric conformations. Interactions between one carboxyethyl head and the tri-Arg (Arg-194, Arg-398, Arg-460) motif are observed, proposed to mimic the interaction of the carboxylate head of sialic acid. Additional stabilizing interactions are observed between a second carboxyethyl group and Arg-213, mimicking the interaction between this side chain and the O-4 of sialic acid. The third carboxyethyl group is oriented away from the active site and does not directly interact with the enzyme. (TIFF)</p

PG0352 Inhibits the Bactericidal Activity of Serum.

Serum killing assays were conducted on E. coli NEB5α using normal human serum pre-treated with wild-type PG0352 or its inactive form PG0352AAAA, as described in the Materials and Methods section. Heat-inactivated serum was used as a control. (A) 2% serum; (B) 5% serum. Statistical analysis was determined using one-way ANOVA followed by Tukey’s multiple comparison at P .05. (C) Complement deposition assays. A total of 107 cells of E. coli cells were co-incubated with 5% normal human serum (-) or PG0352 treated (+) serum for 20 minutes at 37°C. The resultant serum-treated cells were harvested, washed, and resuspended in 50 μl PBS. Approximately 10 μl samples were subjected to SDS-PAGE, followed by immunoblotting analysis with four different antibodies as labelled: αC3 (a polyclonal antibody against C3), αC9 (a monoclonal antibody against C9), αC5-9 (a monoclonal antibody against C5-9 complex), and αGroEL (a polyclonal antibody against E. coli GroEL). These antibodies were purchased from Abcam.</p

PG0352 desialylates human Factor H (FH).

(A) SDS-PAGE of FH untreated (band A) and treated (band B) with recombinant PG0352 or PG0352AAAA for 3 hours at 37°C. Bands A and B were excised for nano-LC-ESI/MS/MS to examine N-linked glycans. (B) & (C) A representative LC-ESI-MS/MS spectrum of FH glycopeptide untreated (B) and treated (C) with PG0352.</p

Functional Evaluation of PG0352.

(A) PG0352 was treated with the fluorescence substrate 4-methylumbelliferyl-α-D-N-acetylneuraminic acid (4-MUNANA) under different pH conditions to evaluate the optimal pH for its sialidase activity as described in the Methods section. PG0352 displays a broad activity range (pH 3–8) with maximal activity at pH 5. Top: a representative image of the level of fluorescence obtained under each pH condition based on the liberation of 4-MU. (B) PG0352 sialidase activity was evaluated as described in the Methods section utilizing 4-MUNANA and 4-methylumbelliferyl-α-D-glycolylneuraminic acid (4-MUNAGc) as substrates. Inactive PG0352 (PG0352AAAA) was utilized as a negative control.</p

MS/MS spectra for nano-LC/MS/MS for human Factor H (FH) before and after the treatment of PG0352.

MS/MS spectra for nano-LC/MS/MS for human Factor H (FH) before and after the treatment of PG0352.</p