More than two/thirds of the eukaryotic proteins are predicted to be glycosylated (Apweiler et al., 1999), including many proteins of the

immune system. It has been shown that pathogenic and commensal Gram-positive bacteria produce enzymes that hydrolyze the glycans of glycoproteins, thus liberating nutrients supporting bacterial growth (Roberts et al., 2000, 2001; Collin & Fischetti, 2004; Sanchez et al., 2010; Ruiz et al., 2011). Glycan hydrolysis may also serve the purpose of modifying the functionality of specific host Roscovitine clinical trial proteins, e.g. in the immune system (Collin and Olsen, 2001, 2003). Enterococcus faecalis is known to have extracellular endo-β-N-acetylglucosaminidase activity that enables growth on RNaseB by releasing high-mannose type glycans from a single N-glycosylation site (Roberts, et al., 2000, 2001; Collin & Fischetti, 2004). There are three main types of N-linked glycans that are all build on a common core pentasaccharide (Man3-GlcNAc2) that is linked to the asparagine through one of two consecutive GlcNAc units (Supporting Information, Fig. S1): (1) the ‘high-mannose’

Selleckchem MG-132 type found in RNaseB, containing additional α-linked mannose residues, (2) the ‘complex’ type, which has no additional mannose residues but may have as many as five antennae containing a variety of additional sugar types, and (3) the ‘hybrid’ type, comprising a combination of high-mannose and complex-type branches. Hydrolysis of the N-linked glycans by endo-β-N-acetylglucosaminidases entails hydrolysis of the glycosidic bond between the two GlcNAc residues in the (Man3-GlcNAc2) core, which implies that the deglycosylated protein retains one GlcNAc (Morelle and Michalski, 2005, 2007). The ability of E. faecalis to release high-mannose type glycans from RNaseB has been ascribed to a protein called EndoE from E. faecalis SPTLC1 HER1044, corresponding to EF0114 of E. faecalis V583 (99% sequence identity; Collin & Fischetti, 2004). This is a two-domain protein consisting of a family 18 glycoside hydrolase (GH18) with the originally detected endo-β-N-acetylglucosaminidase activity and a GH20 domain that

hydrolyzes complex-type glycans of IgG. In addition to EF0114, the genome of E. faecalis V583 encodes two other GH18 proteins, EF0361 and EF2863 (Cantarel et al., 2009). EF0361 is a chitinase (Leisner et al., 2009) and its gene is followed by ef0362, encoding a CBM33 protein that belongs to a family of enzymes known to play a critical role in chitin degradation (Vaaje-Kolstad et al., 2010). EF2863 is predicted to be a secreted endo-β-N-acetylglucosaminidase that has not been investigated so far, despite its potential importance for the ability of E. faecalis V583 to exploit host glycoproteins. In this study, we have characterized the putative endo-β-N-acetylglucosaminidase, EF2863, from E. faecalis V583. The results confirm the predicted activity and provide information concerning the ability of this novel enzyme to hydrolyze different types of glycoproteins.