The accessory Sec protein Asp2 modulates GlcNAc deposition onto the serine-rich repeat glycoprotein GspB

Abstract

The accessory Sec system is a specialized transport system that exports serine-rich repeat (SRR) glycoproteins of Gram-positive bacteria. This system contains two homologues of the general secretory (Sec) pathway (SecA2 and SecY2) and several other essential proteins (Asp1 to Asp5) that share no homology to proteins of known function. In Streptococcus gordonii, Asp2 is required for the transport of the SRR adhesin GspB, but its role in export is unknown. Tertiary structure predictions suggest that the carboxyl terminus of Asp2 resembles the catalytic region of numerous enzymes that function through a Ser-Asp-His catalytic triad. Sequence alignment of all Asp2 homologues identified a highly conserved pentapeptide motif (Gly-X-Ser(362)-X-Gly) typical of most Ser-Asp-His catalytic triads, where Ser forms the reactive residue. Site-directed mutagenesis of residues comprising the predicted catalytic triad of Asp2 of S. gordonii had no effect upon GspB transport but did result in a marked change in the electrophoretic mobility of the protein. Lectin-binding studies and monosaccharide content analysis of this altered glycoform revealed an increase in glucosamine deposition. Random mutagenesis of the Asp2 region containing this catalytic domain also disrupted GspB transport. Collectively, our findings suggest that Asp2 is a bifunctional protein that is essential for both GspB transport and correct glycosylation. The catalytic domain may be responsible for controlling the glycosylation of GspB, while other surrounding regions are functionally required for glycoprotein transport.

Fig 1

Effects of Asp2 domain disruption upon the export of GspB736flag. (A) Schematic illustrating the locations of EZ-Tn5 insertions within asp2. (B) Western blot analysis of GspB736flag export by PS1243 derivative strains carrying in-frame insertions within asp2. Culture media (M) and protoplasts (P) were collected from exponentially growing strains and prepared as described in the Methods and Materials. (C) Densitometry analysis of GspB736flag levels within media or protoplasts. The y axis represents GspB736flag levels, based on band intensity analysis via LI-COR imaging. (D) Western blot detection of Asp2 and the in-frame insertion mutants. Proteins were separated by SDS-PAGE and subjected to Western blot analysis using anti-Flag and anti-His antibodies for GspB736flag and Asp2 detection, respectively.

Fig 2

Predicted structural homology analysis of Asp2. The predicted structural similarity of Asp2 to proteins with a solved crystal structure was assessed using the homology detection and structure prediction server HHpred (http://toolkit.tuebingen.mpg.de/hhpred). Proteins exhibiting structural similarity are shown aligned to the relevant region of Asp2 and are listed with their Protein Data Bank functional designation together with their species of origin.

Fig 3

Predicted structure of the conserved catalytic region of Asp2. (A) Proposed structure of the carboxyl-terminal region (aa 227 to 494) of Asp2 based on homology modeling using the crystal structure of the known thioesterase of Vibrio harveyi. Spatial arrangement of the catalytic residues Ser362, Glu452, and His482. The diagram was generated using PyMOL software. (B) Amino acid alignment of the catalytic region among Asp2 homologues. The conserved GXSXG motif and Glu/Asp and His catalytic residues are highlighted in pink.

Fig 4

Effects of mutating the catalytic triad upon the export of GspB736flag and native GspB. Western blot analysis of GspB736flag (A) and native full-length GspB (B) export by S. gordonii strains carrying the designated serine-alanine substitutions within Asp2. Residues constituting the catalytic triad of Asp2 are highlighted in red. Culture media (M) and protoplasts (P) were collected from exponentially growing strains, while cell wall (CW) proteins were released by mutanolysin digestion, as described in the Materials and Methods. Proteins were separated by SDS-PAGE (3 to 8%) and analyzed by Western blotting, using anti-Flag antibody to detect GspB736flag and anti-GspB serum to detect native GspB. GspB*, the excessively glycosylated form of GspB due to Asp2 mutation; Asp2◆, an overexposure of native GspB detected with anti-GspB serum.

Fig 5

Asp2-dependent export of GspB truncates from S. gordonii strains. Western blot analysis of secreted GspB variants in culture media (M) or in protoplasts (P). S. gordonii Δasp2 strains were complemented with either native asp2 or asp2^S362A. Proteins were separated by SDS-PAGE through 3 to 8% polyacrylamide gradient gels and subjected to Western blot analysis using anti-GspB antiserum.

Fig 6

Impact of mutating the Asp2 catalytic triad on the glycosylation of GspB. (A) Binding of a panel of biotinylated lectins to immobilized S. gordonii strain PS614 expressing full-length GspB in the designated asp2 background. Levels of lectin binding were detected with a horseradish peroxidase-conjugated antibiotin antibody and expressed as means ± SDs of triplicate measurements. *, values that are significantly different (P < 0.05) from the value for PS614 (with asp2). (B) Lectin blot analysis of GspB2061 secreted by PS615 complemented with asp2 variants. Proteins in the culture media were separated by SDS-PAGE (3 to 8%) and probed with sWGA.

Fig 7

Positional shift of secreted Gsp2061 due to asp2 mutation. M99 Δasp2 complemented with asp2^S362A (A) or wild-type asp2 (B) was grown to mid-log phase, and the culture media were analyzed by 2D electrophoresis. Different glycoforms of secreted GspB2061 were detected using anti-GspB antiserum. Arrows, positional shift of native GspB2061 (boxed in green) to a larger variant glycoform (boxed in red) due to asp2 mutation. IEF, isoelectric focusing.

Fig 8

Esterase activity of Asp2 and its influence upon other glycosylation components. (A) Relative activities of Asp2 and its catalytic mutant (Ser362) toward pNP esters with different carbon chain lengths. Release of p-nitrophenyl is shown relative to the activity of a known carboxyl esterase (Bacillus Est). Data shown are means ± SDs of triplicate measurements. (B) GST-GspB1061 glycosylation in E. coli. The presence (+) or absence (−) of individual components of the glycosylation machinery is indicated. E. coli lysates were separated by SDS-PAGE (3 to 8%), transferred to membranes, and probed with either ant-GST or sWGA.

Fig 9

Binding of S. gordonii strains to sialyl-T antigen. S. gordonii strain PS614 complemented with asp2, empty vector (Δasp2), or asp2^S362A was assessed for binding to immobilized sialyl-T antigen. Binding is expressed as the percentage (mean ± SD) of input bacteria that remained bound to sialyl-T antigen after repeated washing of the wells. *, P < 0.05 compared with WT asp2.