O-Glycosylation of the N-terminal Region of the Serine-rich Adhesin Srr1 of Streptococcus agalactiae Explored by Mass Spectrometry

Abstract

Serine-rich (Srr) proteins exposed at the surface of Gram-positive bacteria are a family of adhesins that contribute to the virulence of pathogenic staphylococci and streptococci. Lectin-binding experiments have previously shown that Srr proteins are heavily glycosylated. We report here the first mass-spectrometry analysis of the glycosylation of Streptococcus agalactiae Srr1. After Srr1 enrichment and trypsin digestion, potential glycopeptides were identified in collision induced dissociation spectra using X! Tandem. The approach was then refined using higher energy collisional dissociation fragmentation which led to the simultaneous loss of sugar residues, production of diagnostic oxonium ions and backbone fragmentation for glycopeptides. This Protein glycosylation takes place in bacteria. A considerable amount of information on the biochemical pathways involved in protein glycosylation has been obtained in the genera Campylobacter and Neisseria (1-3). Hence, functional studies performed on Gram-negative bacteria have revealed important properties of protein glycosylation pathways. Like in eukaryotes, proteins can be glycosylated on asparagines (N-glycosylation) or serine/threonine residues (O-glycosylation). Two modes of glycan transfer onto proteins have been characterized in Gram-negative bacteria. In the first, the activated glycan is synthesized on a lipid carrier on the cytoplasmic side of the membrane. After membrane translocation, the glycan portion of the polyprenyl-phosphate-glycan intermediate is transferred en bloc to the protein by an oligosaccharyltransferase active in the periplasm. Such mechanism has been demonstrated for N-glycosylation in Campylobacter jejuni (4 -6) and O-glycosylation in Neisseria gonorrheae (7-9). These pathways are responsible for the glycosylation of more than 65 proteins in C. jejuni (10) and 12 different proteins in N.

Insights into bacterial protein glycosylation in human microbiota

“…Structural studies of GtfC identified a conserved loop region that is crucial for acceptor substrate binding from GtfC homologs in streptococci (Zhu et al, 2015a). Mass spectrometry analysis of purified Srr1 unambiguously demonstrates that Serine or Threonine resides on Srr1 was modified by GlcNAc and Glc, which resembles the findings of Srr2 (Chaze et al, 2014). Interestingly there is a novel modification found on Srr1, which was identified as O -acetylated- N -acetylhex-osamine (Chaze et al, 2014).…”

“…Mass spectrometry analysis of purified Srr1 unambiguously demonstrates that Serine or Threonine resides on Srr1 was modified by GlcNAc and Glc, which resembles the findings of Srr2 (Chaze et al, 2014). Interestingly there is a novel modification found on Srr1, which was identified as O -acetylated- N -acetylhex-osamine (Chaze et al, 2014). However, it still remains unknown what enzyme is responsible for the modification.…”

Three glycosylated serine‐rich repeat proteins play a pivotal role in adhesion and colonization of the pioneer commensal bacterium, Streptococcus salivarius

“…E). This finding provides further indication that the SSR variants are glycosylated: the glycan content of SRR proteins is known to alter their electrophoretic mobility, leading to a slower migration pattern and to detection of protein bands with a higher apparent molecular mass (Takamatsu et al ., ; Mistou et al ., ; Yen et al ., ; Chaze et al ., ). As SrpA was expressed at a higher level than SrpB and SrpC, the band representing SrpA 1000flag was excised and digested with trypsin, and the resulting peptides extracted and subjected to liquid chromatography coupled to mass spectrometry analysis (LC‐MS/MS).…”

“…For the 8 peptides located in the SRR2 domain, the maximum number of HexNAc and O‐ AcHexNAc residues anchored to a peptide sequence was close to the number of serine/threonine residues in the sequence. This observation suggests that these peptides contain either HexNAc or O ‐AcHexNAc modifications, as shown for Srr1 of S. agalactiae (Chaze et al ., ), and that all serine and threonine residues located in the C‐terminal SRR2 domain are glycosylated. Glycosylation with a Hex residue was found only on peptides carrying at least 3 HexNAc residues, suggesting that HexNAc modification is a prerequisite for Hex glycosylation.…”

Glycosyltransferase-mediated Sweet Modification in Oral Streptococci

“…The glycan sequence of the Fap1 peptide backbone has been determined as Rha1-3Glc1-(Glc1-3Glc NAc1-) 2, 6Glc1-6GlcNAc . The glycosyl composition of other SRRPs has been determined for GspB of Streptococcus gordonii (Takamatsu et al 2004) and Srr1 of Streptococcus agalactiae (Chaze et al 2014), but the sequential order of the glycans are unknown.…”