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

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].