EndoS2 is a unique and conserved enzyme of serotype M49 group A Streptococcus that hydrolyses N-linked glycans on IgG and α1-acid glycoprotein

Many bacteria have evolved ways to interact with glycosylation functions of the immune system of their hosts. Streptococcus pyogenes [GAS (group A Streptococcus)] secretes the enzyme EndoS that cleaves glycans on human IgG and impairs the effector functions of the antibody. The ndoS gene, encoding EndoS, has, until now, been thought to be conserved throughout the serotypes. However, in the present study, we identify EndoS2, an endoglycosidase in serotype M49 GAS strains. We characterized EndoS2 and the corresponding ndoS2 gene using sequencing, bioinformatics, phylogenetic analysis, recombinant expression and LC–MS analysis of glycosidic activity. This revealed that EndoS2 is present exclusively, and highly conserved, in serotype M49 of GAS and is only 37% identical with EndoS. EndoS2 showed endo-β-N-acetylglucosaminidase activity on all N-linked glycans of IgG and on biantennary and sialylated glycans of AGP (α1-acid glycoprotein). The enzyme was found to act only on native IgG and AGP and to be specific for free biantennary glycans with or without terminal sialylation. GAS M49 expression of EndoS2 was monitored in relation to carbohydrates present in the culture medium and was linked to the presence of sucrose. We conclude that EndoS2 is a unique endoglycosidase in serotype M49 and differs from EndoS of other GAS strains by targeting both IgG and AGP. EndoS2 expands the repertoire of GAS effectors that modify key glycosylated molecules of host defence

Glycomics

Paramount to our understanding of carbohydrates in biology has been the development of methods for analyzing and characterizing the essential features of glycans. This has resulted in a comprehensive appreciation for the various roles of glycans and the pathways associated with their synthesis. Indeed, the importance of glycosylation has been recognized by the biopharmaceutical industry. Therapeutics for a multitude of formerly untreatable diseases are coming on stream and this is largely due to the ability of the biopharmaceutical industry to produce complex proteins carrying appropriate posttranslational modifications. Importantly, glycosylation as a posttranslational modification has taken a prominent role in the focus of bioproduction, as subtle changes in protein glycosylation can significantly impact the safety, efficacy, and biological activity of a therapeutic. In response, the biopharmaceutical industry is dedicated to the generation of expression systems that provide humanized glycosylation to maximize the potency, safety, and pharmacokinetic behavior of glycoprotein therapies. Central to this strategy is the ability to manipulate glycosylation, which is largely dependent on the development of techniques that allow the assessment and characterization of glycans. To be of benefit to the biopharmaceutical industry, methods for glycan analysis will be heavily reliant on platforms that offer robust, rapid, quantifiable, sensitive, and cheap solutions, which can be performed at-line in a high-throughput format. This article provides an introduction to the field of glycomics and how specific aspects of glycosylation have been employed by the biopharmaceutical industry to generate therapeutics with enhanced biological attributes. Methods for the analysis of glycans and associated curation and access of glycan data through bioinformatics are presented.20 page(s

EndoE from <i>Enterococcus faecalis</i> Hydrolyzes the Glycans of the Biofilm Inhibiting Protein Lactoferrin and Mediates Growth

<div><p>Glycosidases are widespread among bacteria. The opportunistic human pathogen <i>Enterococcus faecalis</i> encodes several putative glycosidases but little is known about their functions. The identified endo-β-<i>N</i>-acetylglucosaminidase EndoE has activity on the N-linked glycans of the human immunoglobulin G (IgG). In this report we identified the human glycoprotein lactoferrin (hLF) as a new substrate for EndoE. Hydrolysis of the N-glycans from hLF was investigated using lectin blot, UHPLC and mass spectrometry, showing that EndoE releases major glycoforms from this protein. hLF was shown to inhibit biofilm formation of <i>E. faecalis in vitro</i>. Glycans of hLF influence the binding to <i>E. faecalis,</i> and EndoE-hydrolyzed hLF inhibits biofilm formation to lesser extent than intact hLF indicating that EndoE prevents the inhibition of biofilm. In addition, hLF binds to a surface-associated enolase of <i>E. faecalis</i>. Culture experiments showed that the activity of EndoE enables <i>E. faecalis</i> to use the glycans derived from lactoferrin as a carbon source indicating that they could be used as nutrients <i>in vivo</i> when no other preferred carbon source is available. This report adds important information about the enzymatic activity of EndoE from the commensal and opportunist <i>E. faecalis</i>. The activity on the human glycoprotein hLF, and the functional consequences with reduced inhibition of biofilm formation highlights both innate immunity functions of hLF and a bacterial mechanism to evade this innate immunity function. Taken together, our results underline the importance of glycans in the interplay between bacteria and the human host, with possible implications for both commensalism and opportunism.</p></div

Glycan analysis of lactoferrin.

<p>Hydrophilic interaction liquid chromatography (HILIC)-fluorescence chromatogram of 2-AB labeled glycans released from human lactoferrin by the endoglycosidase EndoE (A) and the endoglycosidase PNGaseF, respectively (B). Identified glycans are separated into peaks. The numbers correspond to the glycan structures depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091035#pone-0091035-g003" target="_blank">Figure 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091035#pone-0091035-g004" target="_blank">4</a>.</p

PNGaseF released N-glycans identified from human lactoferrin.

<p>The depicted glycan structure is based on the Oxford glycan nomenclature <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091035#pone.0091035-Harvey1" target="_blank">[56]</a>. Glycans were detected as [M-2H]²⁻ ions. GU values were generated as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091035#pone.0091035-Guile1" target="_blank">[54]</a>. In situations where chromatographic peaks containing multiple structures, the associated peak area was divided equally among the structures for simplicity.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

Influence of human lactoferrin on biofilm formation of <i>Enterococcus faecalis</i>.

<p>Biofilm formation of <i>E. faecalis</i> was measured using the crystal violet assay and is expressed as OD₅₅₀. Human lactoferrin (hLF) was added either fully glycosylated (hLF) or deglycosylated (de-hLF) due to the treatment with EndoE. Error bars indicate the standard deviation from the mean of three independent experiments with three replicates. w/o: no hLF added.</p