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. 2009 Sep 18;284(38):26161-73.
doi: 10.1074/jbc.M109.024067. Epub 2009 Jul 16.

Differential recognition and hydrolysis of host carbohydrate antigens by Streptococcus pneumoniae family 98 glycoside hydrolases

Melanie A Higgins  1 , Garrett E Whitworth, Nahida El Warry, Mialy Randriantsoa, Eric Samain, Robert D Burke, David J Vocadlo, Alisdair B Boraston
Affiliations

Differential recognition and hydrolysis of host carbohydrate antigens by Streptococcus pneumoniae family 98 glycoside hydrolases

Melanie A Higgins et al. J Biol Chem. .

Abstract

The presence of a fucose utilization operon in the Streptococcus pneumoniae genome and its established importance in virulence indicates a reliance of this bacterium on the harvesting of host fucose-containing glycans. The identities of these glycans, however, and how they are harvested is presently unknown. The biochemical and high resolution x-ray crystallographic analysis of two family 98 glycoside hydrolases (GH98s) from distinctive forms of the fucose utilization operon that originate from different S. pneumoniae strains reveal that one enzyme, the predominant type among pneumococcal isolates, has a unique endo-beta-galactosidase activity on the LewisY antigen. Altered active site topography in the other species of GH98 enzyme tune its endo-beta-galactosidase activity to the blood group A and B antigens. Despite their different specificities, these enzymes, and by extension all family 98 glycoside hydrolases, use an inverting catalytic mechanism. Many bacterial and viral pathogens exploit host carbohydrate antigens for adherence as a precursor to colonization or infection. However, this is the first evidence of bacterial endoglycosidase enzymes that are known to play a role in virulence and are specific for distinct host carbohydrate antigens. The strain-specific distribution of two distinct types of GH98 enzymes further suggests that S. pneumoniae strains may specialize to exploit host-specific antigens that vary from host to host, a factor that may feature in whether a strain is capable of colonizing a host or establishing an invasive infection.

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Figures

FIGURE 1.
FIGURE 1.
Kinetic properties of S. pneumoniae family 98 enzymes. Shown are kinetic plots of the activity of Sp3GH98 on the type 2 A pentasaccharide (A) and the type 2 B pentasaccharide (B). C, a corresponding analysis of the activity of Sp4GH98 on the LewisY tetrasaccharide.
FIGURE 2.
FIGURE 2.
GH98 enzymes remove carbohydrate antigens from a model cell line. Shown are epifluorescent images of A549 cells treated with Sp4GH98 and Sp3GH98 enzymes and then prepared with antibodies to the LewisY antigen (red) or the A/B antigens (red) and counterstained with 4′,6-diamidino-2-phenylindole (blue). A–D, cells were probed with anti-LewisY antibody after no treatment (A), Sp4GH98 treatment (B), Sp3GH98 treatment (C), or treatment with both enzymes (D). E–H, cells were probed with anti-A/B antibody after no treatment (E), Sp4GH98 treatment (F), Sp3GH98 treatment (G), or treatment with both enzymes (H). Bar, 5 μm. I, quantification of fluorescence of A549 cells treated as in A–H (treatment is indicated below the graph). Bars, mean ± S.E. fluorescence per unit area for a sampling of five randomly selected regions of cytoplasm. Bars marked with an asterisk, significantly different from cells not treated with an enzyme as judged by one-way analysis of variance.
FIGURE 3.
FIGURE 3.
The structural features of S. pneumoniae family 98 enzymes. A, divergent stereo schematic representation of the structure of Sp4GH98. The N-terminal (α/β)8 barrel housing the catalytic residues in shown in green, the C-terminal β-sandwich domain in purple, and the smaller intervening domain in yellow. Shown are solvent-accessible surface representations (gray) of the active site of Sp4GH98 containing the H disaccharide (shown in a stick representation) (B) and Sp3GH98 containing the A trisaccharide (shown in a stick representation) (C). Green, galactose; blue, fucose; magenta, N-acetylgalactosamine. The yellow mesh shows the maximum likelihood (27)/σa-weighted (29) Fo − Fc maps for the carbohydrates (contoured at 3σ; 0.24 electrons/Å3 for Sp4GH98 and 0.20 electrons/Å3 for Sp3GH98). Subsites of the active site are labeled in white. D, schematic of the interactions in the −1 and −2 subsites of Sp4GH98 and Sp3GH98. Residues in black are conserved in both enzymes and labeled in black (Sp4GH98) or gray (Sp3GH98). Red residues are present only in Sp4GH98. E, schematic of the −2′ subsite of Sp3GH98. Black residues are shown for interactions that are conserved for the galactose of the B-antigen and the N-acetylgalactosamine of the A antigen. The red residue represents an interaction that is unique to the A antigen. F, divergent stereo representation of a structural overlay of the active sites of Sp4GH98 (blue) and Sp3GH98 (orange). The A trisaccharide product in the Sp3GH98 active site is shown in sticks and colored as in C. The loop protruding from the β-sandwich domain is shown in a schematic diagram representation. Trp512 of Sp4GH98 that blocks the −2′ subsite in this protein is shown in a stick representation.
FIGURE 4.
FIGURE 4.
Aglycon recognition in GH98 enzymes. Solvent-accessible surface representation (gray) of the active site of Sp4GH98E158A containing the LewisY tetrasaccharide substrate (shown in a stick representation) (A) and Sp3GH98E558A containing the A-LewisY pentasaccharide (shown in a stick representation) (B). Green, galactose; blue, fucose; cyan, N-acetylglucosamine; magenta, N-acetylgalactosamine. The yellow mesh shows the maximum likelihood/σa-weighted Fo − Fc maps for the carbohydrates contoured at 2.5σ (0.16 electrons/Å3) for Sp4GH98E158A and 3σ (0.22 electrons/Å3) for Sp3GH98E558A. Subsites of the active site are labeled in white. C, representative overlay of the GH98 active site. All GH98 structures were overlaid. Because of the virtually identical positioning of the active site structures, the Sp3GH98 A trisaccharide complex was chosen as a reference point to display key features. The backbone of the Sp3GH98 A trisaccharide product complex is shown in a schematic diagram with relevant active site residues shown in a stick representation. The A trisaccharide sugar is shown as yellow sticks. The A-LewisY pentasaccharide from the Sp3GH98E558A substrate complex is shown as green sticks. Residues in Sp3GH98 are labeled in gray, and analogous residues in Sp4GH98, which were identically positioned, are labeled in black. Sugar residues in the +1 and −1 subsites of Sp4GH98 were also positioned virtually identically to those of the Sp3GH98 complexes. Relevant interresidue hydrogen bonds and protein-substrate distances are shown. The putative catalytic acid is colored pink and putative catalytic bases are colored blue. D, schematic of the interactions in the +1 and +1′ subsites. Interactions conserved between Sp4GH98 and Sp3GH98 are shown with green amino acids. Black amino acids are those only in Sp4GH98, and red amino acids are those only in Sp3GH98.
FIGURE 5.
FIGURE 5.
NMR analysis of GH98 catalytic mechanism. A, structure of the LewisY tetrasaccharide substrate. B, structure of the Fucα1–2Gal (H disaccharide) product. C, the hydrolysis of LewisY tetrasaccharide measured as a function of time by 1H NMR spectroscopy. Peaks corresponding to the chemical shifts of the proton on the anomeric carbon for substrate (S) and product (P) are labeled for the α- and β-anomers.
FIGURE 6.
FIGURE 6.
Schematics of the two proposed pathways for fucose utilization. A, the fucose utilization pathway involving the Sp4GH98-type enzyme; B, the fucose utilization pathway involving the Sp3GH98-type enzyme. Enzymes are represented by ovals and color-coded by general function: fucose processing (green), carbohydrate transport (purple), and glycan hydrolysis (blue). FcsA, -I, -K, and -U are enzymes putatively acting as an aldolase, isomerase, kinase, and mutarotase, respectively. The EII components are those of a PTS transporter. SBP, 1, and 2 represent the solute-binding protein (FcsSBP (14)) and two permease components, respectively, of an ABC transporter. The ATPase component of the ABC transporter is unidentified and is represented by gray ovals. GH95 and GH29, putative α-1,2-fucosidases; GH36A, a putative α-N-acetylgalactosaminidase; GH36B, a putative α-galactosidase. PG+C, peptidglycan and capsule layers; M, membrane. Details of the sugar notation and glycan structures are shown below B. Further details of the homology based prediction of component functions are given in supplemental Tables S2–S4.
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