S. coelicolor has a well characterised protein O-glycosylation pathway, but little is known about the constitution of the glycoproteome. To better understand the physiological processes affected by protein O-glycosylation in S. coelicolor we decided that an in-depth characterisation of the glycoproteome was required. The first step towards glycoproteome characterisation was the optimisation of methods for glycoprotein detection and enrichment. Previous work has shown that a disruption of protein O-glycosylation in S. coelicolor results in changes in growth, colony morphology, φC31cΔ25 phage sensitivity and sensitivity to antibiotics, when grown on DNA (Cowlishaw and Smith 2001; Cowlishaw and Smith 2002; Howlett et al. 2016). F134 medium was chosen for the cultivation of strains in this study as the timing for the expression of the S. coelicolor glycoprotein SCO4142 (PstS) was well known (Nieselt et al. 2010; Thomas et al. 2012) This protein could therefore serve as a positive control for the glycoprotein enrichment in this study. We also hoped to further the characterisation of SCO4142 using glycoproteomics approaches, since glycopeptides mapping this glycoprotein were not previously observed by Wehmeier et al. (2009).
The phenotypes associated with defective glycosylation in S. coelicolor were studied on F134 agar to investigate whether these effects were growth medium dependent. We hypothesised that similar phenotypes on both growth media could suggest that glycoproteins associated with these phenotypes on DNA, are also expressed on F134 agar. The overall growth rates of all strains tested were reduced on F134 agar compared to DNA. However, the slow growth phenotype of DT3017 (ppm1) previously observed on DNA was
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still visible on F134 agar, albeit with altered overall growth kinetics. The small colony phenotype of DT3017 (ppm1) compared to J1929 and DT1025 (pmt) was evident on F134 agar suggesting that this phenotype is not medium dependent. S. coelicolor J1929 was sensitive to infection by the phage φC31cΔ25 on both media, suggesting that the phage receptor glycoprotein is expressed when grown on DNA and F134 agar. Similarly, the glycosylation deficient strains were consistently resistant to infection by the phage φC31cΔ25 on both media, suggesting that the phage receptor is not glycosylated on DNA or F134 agar. The greatest phenotypic difference between the strains on F134 agar and DNA was observed in the differences in their antibiotic susceptibilities. The overall reduced sensitivity of the glycosylation deficient strains to ampicillin, imipenem, meropenem, bacitracin and vancomycin on F134 agar compared to DNA could be explained by the slow growth rate on F134 agar. β-lactam antibiotics (e.g. ampicillin, imipenem and meropenem) target proteins required for peptidoglycan crosslinking, while bacitracin inhibits steps in peptidoglycan biosynthesis (Stone and Strominger 1971; Waxman and Strominger 1983). Vancomycin acts by binding to the terminal D-alanyl-D-alanine dipeptide of peptidoglycan precursors and inhibiting their incorporation (Barna and Williams 1984). While a defective glycosylation system might alter the activity of cell-wall biosynthetic enzymes or limit cell- wall precursors leading to greater antibiotic sensitivity under rapid growth conditions, under slow growth conditions such as those observed on F134 medium these effects could be reduced. It is however unclear why J1929 and DT1025 (pmt) displayed increased sensitivity to penicillin, teicoplanin and rifampicin on F134 agar compared to DNA.
The analysis of the membrane using Con A-HRP after western blotting suggests that glycoproteins are present in S. coelicolor J1929, and are absent from the glycosylation deficient DT1025 (pmt) and DT3017 (ppm1). The concomitant loss of Con A-HRP reactivity in the presence of the competitive inhibitor of mannose binding, methyl α-D glucopyranoside suggests that Con A is binding to the glycan on glycoproteins in the membrane of J1929. This observation is further supported by the fact that glycoproteins were enriched from the S. coelicolor J1929 membrane and culture filtrate, but not from DT1025 (pmt) after Con A affinity chromatography. The peak eluted from the Con A column after the enrichment of DT1025 (pmt) membranes proteins (Figure 3.8), despite the apparent absence of Coomassie stained protein could be explained by the presence of other glycoconjugates in S. coelicolor membrane, such as teichoic acids. This is supported by the observation that it is also retained longer than the glycoprotein peak in the J1929 sample.
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The Con A agarose (Vector Laboratories) used in this study was far more effective for glycoprotein enrichment than the Con A sepharose used previously by Wehmeier et al. (2009). The use of the latter resulted in a larger amount of non-specific binding, which was not observed with Con A agarose. In this work, the lack of non-specific binding to the Con A agarose was demonstrated in the Con A enrichments of the membrane and culture filtrate proteomes of DT1025 (pmt). The use of Con A agarose for glycoprotein enrichment was highly reproducible; all of the Con A-HRP reactive bands observed during the small-scale enrichments corresponded to the molecular masses of proteins observed by Coomassie staining and Con A-HRP reactivity after the large-scale enrichment.
Five candidate glycoproteins were successfully enriched during this study. Included in these was the previously characterised S. coelicolor glycoprotein SCO4142 (PstS) (Wehmeier et al. 2009). Four of the proteins are predicted to be lipoproteins, which are well known targets of O-glycosylation in M. tuberculosis (Dobos et al. 1995; Sutcliffe and Harrington 2004; Sartain and Belisle 2009). The homologue of SCO5776 in M. tuberculosis (Rv0411c) was previously identified by LC-ESI MS/MS after the enrichment of the culture filtrate by Con A affinity chromatography (Gonzalez-Zamorano et al. 2009). SCO4856, a putative succinate dehydrogenase flavoprotein subunit is part of a membrane-located complex facing the cytoplasm. This protein is homologous to the E. coli protein SdhA which has a role in the tricarboxylic acid (TCA) cycle (Yankovskaya et al. 2003). It is probable that the enrichment of this protein was non-specific due to its high abundance in the cell. However, the possibility that this protein is glycosylated cannot be excluded.
The identification of SCO4471 is exciting because of its possible role in cell wall biosynthesis. The characterisation of the glycosylated amino acids in SCO4471 using non-reductive β- elimination and mass spectrometry was unsuccessful, but glycosylation of SCO4471 cannot be ruled out. The O-glycosylation target site prediction algorithm NetOGlyc 4.0 identified seven putative glycosylated residues near the N-terminus, as well as three putative glycosylated residues near the C-terminus of SCO4471. It is possible that the C-terminal peptide (ETPASSGAPANAA) had difficulty ionising, because unlike most tryptic peptides the C-terminal amino acid is not basic. O-glycosylation sites in M. tuberculosis glycoproteins have been shown to occur more frequently at the C-terminal end of the protein (Smith et al. 2014). These results provide further evidence of a glycoproteome in S. coelicolor. The combination of glycoprotein enrichment using Con A and Con A-HRP as a glycoprotein detection strategy
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provide promising first steps towards characterising the S. coelicolor glycoproteome. The next step was to validate the glycoproteins using glycoproteomics approaches.