Cytoplasmic N-Glycosyltransferase of Actinobacillus pleuropneumoniae Is an Inverting Enzyme and Recognizes the NX(S/T) Consensus Sequence

Abstract

N-Linked glycosylation is a frequent protein modification that occurs in all three domains of life. This process involves the transfer of a preassembled oligosaccharide from a lipid donor to asparagine side chains of polypeptides and is catalyzed by the membrane-bound oligosaccharyltransferase (OST). We characterized an alternative bacterial pathway wherein a cytoplasmic N-glycosyltransferase uses nucleotide-activated monosaccharides as donors to modify asparagine residues of peptides and proteins. N-Glycosyltransferase is an inverting glycosyltransferase and recognizes the NX(S/T) consensus sequence. It therefore exhibits similar acceptor site specificity as eukaryotic OST, despite the unrelated predicted structural architecture and the apparently different catalytic mechanism. The identification of an enzyme that integrates some of the features of OST in a cytoplasmic pathway defines a novel class of N-linked protein glycosylation found in pathogenic bacteria.N-Linked glycosylation is characterized by an N-glycosidic linkage between the side chain amide of an asparagine residue of proteins and an oligosaccharide. This type of glycosylation occurs in both prokaryotes and eukaryotes and requires the assembly of an oligosaccharide on a polyisoprenoid lipid by sequential addition of monosaccharides, catalyzed by cytosolic glycosyltransferase. The resulting lipid-linked oligosaccharide is translocated to the luminal side of the endoplasmic reticulum membrane or the plasma membrane of prokaryotes, where it may be further elongated. The glycan is then transferred to the ␦-amino group of asparagine residues within the consensus sequence NX(S/T) of polypeptides. This reaction is catalyzed by oligosaccharyltransferase (OST), 4 a membrane-bound enzyme that can be composed of several different subunits (1). As oligosaccharide transfer takes place in the endoplasmic reticulum or in the periplasm, N-glycosylation affects proteins trafficking along the secretory pathway.N-Glycosylation exhibits important physiological functions. In the early secretory pathway of eukaryotes, N-glycans present on newly synthesized proteins orchestrate the folding of glycoproteins and act as a signal for directing misfolded polypeptides to degradation (2). After being processed in the Golgi organelle, N-linked glycans are relevant, among other processes, for the modulation of the immune system and for the control of immune cell homeostasis and inflammation (3, 4). The importance of this protein modification is supported by its incidence: more than half of all eukaryotic proteins are glycosylated (5).About 8 years ago, a study uncovered that the extracellular HMW1A adhesin of the Gram-negative bacterium Haemophilus influenzae is modified with hexose monosaccharides on asparagine residues (6). The pioneering work of St Geme and co-workers (7, 8) subsequently showed that the modified asparagine residues are found within the same consensus sequence recognized by OST (i.e. NX(S/T)) and that the enzyme responsible for this modification is the HMW1C p...

N-Linked Glycosylation in Archaea: a Structural, Functional, and Genetic Analysis

“…In this PglB-independent case, the N-linked glycan does not contain an N-acetylated linking sugar (33). More recently, a similar system was reported for Actinobacillus pleuropneumoniae, where an N-glycosyltransferase homologue of HMW1C transferred a glucose or galactose to asparagine (218). In Archaea, many N-linked glycan structures have been determined, and in several cases, the linking sugar is a hexose, indicating that AglB does not require a C-2 acetamido sugar at the linking position, as do Stt3 and PglB.…”

The Sweet Tooth of Bacteria: Common Themes in Bacterial Glycoconjugates

“…N-glycosylation of proteins has been described for only a few species, and its occurrence in bacteria is believed to be rare. En bloc N-glycosylation has so far mainly been studied in detail in Campylobacter species (77), while H. influenzae and Actinobacillus pleuropneumoniae harbor a sequential N-glycosylation mechanism (296,297). Recently, genomic analysis revealed the presence of an en bloc N-glycosylation mechanism in some Helicobacter species (298).…”

Beyond the Sequon: Sites of N-Glycosylation

“…In turn, the HMW1A adhesin is important for NTHi colonisation and pathogenesis (St Geme et al, 1993, St Geme, 1994. Similar to several other described bacterial protein glycosyltransferases, HMW1C glycosylates its HMW1A substrate protein in the cytoplasm, before secretion across the inner membrane (Fleckenstein et al, 2006, Charbonneau et al, 2012, Choi et al, 2010, Schwarz et al, 2011a. Most of these other reported bacterial glycosyltransferases are Oglycosyltransferases, transferring nucleotide-activated monosaccharides to the hydroxyl groups of Ser or Thr.…”

“…In contrast, the HMW-C cytoplasmic system of some bacteria is catalysed by a soluble glycosyltransferase that transfers a nucleotide-activated monosaccharide to protein in the cytoplasm. However, it is striking that the bacterial cytoplasmic HMW-C enzymes have very similar site recognition to 'traditional' OTase enzymes: they efficiently glycosylate Asn in 'sequons' with Asn-Xaa-Ser/Thr (Xaa≠Pro), but are capable of glycosylating some selected asparagines lacking S/T at the +2 position (Choi et al, 2010, Schwarz et al, 2011a, Schwarz & Aebi, 2011. HMW-C enzymes also share the substrate requirement of OTase for unfolded protein, or flexible loops in folded protein (Schwarz et al, 2011a).…”