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

Date Published: April 10, 2014

Publisher: Public Library of Science

Author(s): Jessica R. McCann, Joseph W. St. Geme, William E. Goldman.


Partial Text

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.

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 bacteria–host 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 of predicted amino acid sequences, HMW1C-like proteins are prevalent among bacteria in the Pastuereallaceae, Enterobacteriaceae, Neisseriaceae, and Burkholderiaceae families and appear to segregate into two categories [24]. The first category contains enzymes encoded by genes adjacent to predicted tpsA and tpsB genes in apparent TPS systems (Figure 2A). Examples of HMW1C-like proteins that fall into this category are EtpC in enterotoxigenic Escherichia coli (ETEC) [25], RscC in Yersinia enterocolitica[26], and predicted HMW1C-like glycosyltransferases in Y. pestis, Y. pseudotuberculosis, and Burkholderia spp. Among these proteins, only EtpC has been demonstrated to possess glycosyltransferase activity, adding sugar residues to the target EtpA adhesin [25]. In fact, glycosylation of EtpA appears to affect adhesin interaction with host cells, as nonglycosylated EtpA is less adherent to Caco-2 intestinal epithelial cells but hyperadherent to HCT-8 intestinal cells when compared to glycosylated EtpA [25]. By analogy to the HMW1 system and the Etp system, we hypothesize that the HMW1C-like enzymes in this category modify the co-produced TpsA protein.

The crystal structure of ApHMW1C provides some clues as to how HMW1C-like glycosyltransferases are able to decorate asparagines. When overlaid using structural prediction algorithms, HMW1C and ApHMW1C are nearly identical, differing only by a disordered 30-amino acid N-terminal tail that is present in HMW1C and absent in ApHMW1C [19]. Given this level of identity, predictions about structure-function relationships in ApHMW1C are likely to apply to H. influenzae HMW1C and potentially other HMW1C-like proteins. Consistent with this conclusion, mutation of amino acids in HMW1C corresponding to amino acids that line the likely UDP-hexose binding pocket in ApHMW1C eliminates the ability of HMW1C to glycosylate HMW1 or to produce functional HMW1 when expressed in whole bacteria [19]. Examination of the ApHMW1C structure indicates a funnel-shaped groove immediately adjacent to the predicted UDP-hexose binding pocket. When key residues within this groove are mutated in HMW1C, glycosylation of HMW1 is eliminated, indicating that the groove plays an important role in acceptor protein modification [19]. A complete understanding of the specificity and limitations of the HMW1C-like enzyme family may help in an industrial setting, as a single enzyme able to catalyze the first step of N-linked protein glycosylation without a lipid carrier has potential to become a workhorse for in vitro protein production [28].

Despite the relative simplicity of HMW1C-mediated glycosylation, there is still much to learn about how the HMW1C prototype operates and even more to learn about the function and targets of HMW1C homologs in other medically relevant bacteria. First, we do not understand how HMW1C recognizes its target and chooses specific glycosylation sites, as only a subset of NXS/T motifs in HMW1 are decorated based on mass spectrometry analysis. A better understanding of HMW1C target recognition may assist in development of in vitro glycosylation systems for protein manufacture and also increase understanding of protein–protein interactions in bacteria. Second, while it is clear that HMW1C interacts directly with HMW1 [5], the number of HMW1C molecules that bind to HMW1 at any given time and the duration of this interaction in the cytoplasm are still unknown. This information may help to clarify whether HMW1C has both glycosyltransferase activity and chaperone activity to stave off degradation of HMW1 (Figure 1). Third, the glycosylation targets in bacteria that have no co-transcribed tpsA gene alongside the gene that codes for the HMW1C-like enzyme remain to be determined, although progress has been made in identifying potential targets of ApHMW1C [27]. Knowledge of these targets will help to clarify whether there are consensus glycosylation target sequences among HMW1C-like enzymes and whether the cellular location of glycosylation is conserved. Fourth, while it is clear that glycosylation is required for HMW1 tethering to the bacterial surface, the mechanism involved is unknown. Finally, it will also be important to elucidate how HMW1C-mediated glycosylation of target proteins affects bacterial pathogenesis, expanding the limited literature on the role that HMW1C-mediated glycosylation may play in bacteria–host interactions [19], [25], [26], [29].




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