The HMW1C-Like Glycosyltransferases—An Enzyme Family with a Sweet Tooth for Simple Sugars

St. Geme JW III (2014) The HMW1C-Like Glycosyltransferases-An Enzyme Family with a Sweet Tooth for Simple Sugars. PLoS Pathog 10(4): e1003977. doi:10.1371/journal.ppat.1003977 The HMW1C-Like Glycosyltransferases-An Enzyme Family with a Sweet Tooth for Simple Sugars Jessica R. McCann 0 1 Joseph W. St. Geme 0 1 III 0 1 The HMW 0 1 /HMW 0 1 Two-Partner Secretion Systems Have a Third Partner 0 1 William E. Goldman, The University of North Carolina at Chapel Hill, United States of America 0 Funding: Much of the work described in this article was supported by National Institutes of Health grant RO1- DC02873 to JWSG. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript 1 1 The Department of Pediatrics, Duke University Medical Center , Durham , North Carolina, United States of America, 2 The Department of Pediatrics, The Children's Hospital of Philadelphia and the Perelman School of Medicine at the University of Pennsylvania , Philadelphia, Pennsylvania , United States of America - The HMW1 and HMW2 adhesins of nontypeable Haemophilus influenzae are high-molecular weight proteins that are secreted by the two-partner secretion (TPS) pathway, also known as the Type Vb secretion pathway [1,2]. TPS systems typically consist of a large extracellular protein called a TpsA protein (encoded by a tpsA gene) and a cognate outer membrane pore-forming translocator protein called a TpsB protein (encoded by a tpsB gene). HMW1 and HMW2 are TpsA proteins and are encoded by hmw1A and hmw2A, respectively, and HMW1B and HMW2B are the cognate TpsB proteins and are encoded by hmw1B and hmw2B, respectively [3,4]. The hmw1A-hmw1B and hmw2A-hmw2B gene clusters have a similar configuration and are located in physically separate regions of the H. influenzae chromosome. A distinctive feature of the HMW1 and HMW2 systems is the presence of a third protein, called HMW1C in the HMW1 system and HMW2C in the HMW2 system. HMW1C and HMW2C are highly homologous glycosyltransferases [5,6] that are responsible for adding sugar moieties to HMW1 and HMW2 and are encoded by the hmw1C and hmw2C genes, located downstream of hmw1B and hmw2B, respectively. Since the HMW1 and HMW2 systems have similar properties [7], in this review we will confine our discussion to the HMW1 system. The HMW1 adhesin is presented on the bacterial surface via a multistep process that requires HMW1C-mediated glycosylation (reviewed in [8]). As shown schematically in Figure 1, HMW1 is synthesized and glycosylated in the cytoplasm and is directed to the Sec translocase in the inner membrane via an extended Nterminal signal sequence [9]. The signal sequence is cleaved by signal peptidase I, and nascent HMW1 is then directed to its cognate HMW1B b-barrel pore in the outer membrane [9]. The initial interaction between HMW1 and HMW1B occurs via the N-terminal TPS secretion domain in the HMW1 pro-piece and the periplasmic domain in HMW1B [10]. The HMW1 pro-piece spans amino acids 69 441 and is cleaved during or following secretion through the HMW1B pore [9]. HMW1 is ultimately tethered to the bacterial surface via a noncovalent interaction that requires the C-terminal 20 amino acids of the protein and is dependent upon disulfide bond formation between two conserved cysteine residues in this region (cysteines 1518 and 1528). Immunolabeling studies have demonstrated that the immediate C terminus of HMW1 is inaccessible to surface labeling, suggesting that it remains in the periplasm or is buried in the HMW1B pore [5,11]. Elimination of HMW1C results in degradation of HMW1 in bacterial lysates, indicating that glycosylation is required for HMW1 stability. Any remaining nonglycosylated HMW1 is released into the culture supernatant, indicating that glycosylation is also required for HMW1 tethering to the bacterial surface [5]. Manual analysis of mass spectra of HMW1 was required to recognize that glycan structures are present at asparagine residues in conserved NXS/T motifs, reflecting the fact that the modifying carbohydrates are mono-hexose or dihexose groups rather than complex polysaccharides [12,13]. There are at least 31 residues that are modified with glucose, galactose, glucose-glucose, or glucose-galactose residues in the mature surfacelocalized HMW1 protein [12,13]. Based on biochemical analysis and examination of the crystal structure of the HMW1 propiece, the pro-piece is nonglycosylated, perhaps because glycosylation would interfere with cleavage of this fragment, which occurs by an undefined mechanism (Figure 1) [9,12,14]. HMW1C Is the Prototype Member of a New Subfamily of Glycosyltransferases Protein glycosylation occurs in all kingdoms of life and is thought to influence protein folding, stability, and function [15,16]. Some bacteria produce complex O-linked or N-linked glycosyltransferase systems. These systems have been studied in pathogenic bacteria and glycosylate proteins that are typically surface exposed, suggesting a role for glycosylation in bacteriahost interactions [17]. However, none of the previously studied bacterial glycosyltransferase pathways operates like HMW1C, which is capable by itself of forming both N-linked carbohydrate bonds to the HMW1 polypeptide and O-glycosidic bonds between hexose sugars [12,18,19]. Based on homology analysis and molecular modeling, HMW1C belongs to the GT41 family of glycosyltransferases, a family that otherwise contains O-GlcNAc transferases. HMW1C consists of three discrete domains, including an a-helical AAD domain at the N terminus and two Rossman-like domains that create a GT-B fold at the C terminus. Interestingly, the AAD fold in HMW1C differs from the socalled tetratricopeptide repeats (TPR) fold that is characteristic of the GT41 family, and the contacts between the AAD domain and the GT-B domain in HMW1C create a unique groove that is absent in other known members of the GT41 family. Thus, the HMW1C protein represents a novel glycosyltransferase subfamily [13]. Among the best-described bacterial Nlinked glycosyltransferase systems is the Pgl system in Campylobacter jejuni [2022]. While both the Pgl system and HMW1C affix sugars to their target proteins at asparagines, the similarities end there. First, the Pgl system consists of at least ten proteins encoded by a gene cluster [23], rather than a single protein like HMW1C. Second, the Pgl enzymes are active in the periplasm, while HMW1C acts in the cytoplasm [5]. Third, the Pgl enzymes add a heptasaccharide that is most likely formed in the cytoplasm on a lipid carrier that is then flipped into the periplasm. In contrast, HMW1C adds single UDP-linked sugars to HMW1 without the contribution of a lipid carrier [12]. The HMW1C-Like Glycosyltransferases Segregate into Two Subsets Based on homology analysis of predicted amino acid sequences, HMW1C-like proteins are prevalent among bacteria in the Pastuereallaceae, Enterobacteriaceae, Neisseriaceae, an (...truncated)