Structural Insights into the Glycosyltransferase Activity of the Actinobacillus pleuropneumoniae HMW1C-like Protein

Abstract

Glycosylation of proteins is a fundamental process that influences protein function. The Haemophilus influenzae HMW1 adhesin is an N-linked glycoprotein that mediates adherence to respiratory epithelium, an essential early step in the pathogenesis of H. influenzae disease. HMW1 is glycosylated by HMW1C, a novel glycosyltransferase in the GT41 family that creates N-glycosidic linkages with glucose and galactose at asparagine residues and di-glucose linkages at sites of glucose modification. Here we report the crystal structure of Actinobacillus pleuropneumoniae HMW1C (ApHMW1C), a functional homolog of HMW1C. The structure of ApHMW1C contains an N-terminal all ␣-domain (AAD) fold and a C-terminal GT-B fold with two Rossmann-like domains and lacks the tetratricopeptide repeat fold characteristic of the GT41 family. The GT-B fold harbors the binding site for UDP-hexose, and the interface of the AAD fold and the GT-B fold forms a unique groove with potential to accommodate the acceptor protein. Structurebased functional analyses demonstrated that the HMW1C protein shares the same structure as ApHMW1C and provided insights into the unique bi-functional activity of HMW1C and ApHMW1C, suggesting an explanation for the similarities and differences of the HMW1C-like proteins compared with other GT41 family members.Glycosylation of proteins is an essential process that plays an important role in protein structure and function. In recent years, glycoproteins have been identified increasingly in prokaryotes, including pathogenic bacteria. Some bacterial species contain complex O-and N-glycosylation pathways encoded by multiple gene clusters (1, 2), and others utilize a glycosyltransferase alone to modify a single target protein. Most bacterial glycoproteins are surface exposed, suggesting that glycosylation may influence interactions with the host (2-5).Non-encapsulated Haemophilus influenzae is a common cause of localized respiratory tract disease in humans and initiates infection by colonizing the upper respiratory tract (6). Approximately 80% of non-encapsulated H. influenzae clinical isolates express two related high molecular weight proteins called HMW1 and HMW2 that mediate high level adherence to respiratory epithelial cells, a critical step in the pathogenesis of H. influenzae disease (7). HMW1 and HMW2 are encoded by homologous genes designated hmw1A and hmw2A, respectively. The hmw1A gene is flanked by the hmw1B and hmw1C accessory genes, and the hmw2A gene is flanked by the hmw2B and hmw2C accessory genes. The hmw1B and hmw2B genes and the hmw1C and hmw2C genes are highly homologous (8, 9).The HMW1 adhesin is synthesized as a pre-pro-protein that contains an atypical signal peptide (amino acids 1-68), an adjacent pro-piece (amino acids 69 -441), and a large exoprotein domain with adhesive activity (amino acids 442-1536) (10 -12). HMW1 undergoes an elaborate maturation process and is ultimately presented on the bacterial surface via the two-partner secretion (TPS) 2 pathway, a common secretion pathway in Gram-negat...

Substrate Specificity of Cytoplasmic N-Glycosyltransferase

“…ApNGT Hydrolyzes Its Sugar Donor Substrates-In previous studies, ApNGT activity was quantified using an indirect assay that measured the formation of UDP as a byproduct of the glycosyltransferase reaction (20,22). We set up a similar approach using purified ApNGT, the previously described A. pleuropneumonaie autotransporter adhesin fragment AtaC 1866 -2428 as substrate protein (23) and UDP-Glc as sugar donor.…”

“…For ApNGT, His 277 has been proposed as a possible catalytic base (22). To further investigate this hypothesis, we made use of the three-dimensional x-ray crystal structure of ApNGT in complex with UDP (PDB code 3Q3H (22)) to identify potentially basic residues near the active site.…”

“…For ApNGT, His 277 has been proposed as a possible catalytic base (22). To further investigate this hypothesis, we made use of the three-dimensional x-ray crystal structure of ApNGT in complex with UDP (PDB code 3Q3H (22)) to identify potentially basic residues near the active site. As the available crystal structures do not reveal the exact location of the sugar moiety or the acceptor peptide, we decided to investigate all basic residues in an 8-Å radius from the (Fig.…”

“…The proteins of Actinobacillus pleuropneumoniae (20,21) and Yersinia enterocolitica (21) have proven N-glycosylation activity. The NGT protein of A. pleuropneumoniae is the best studied example on a biochemical level and its three-dimensional x-ray crystal structure has been determined (22). Detailed analysis of its reaction product has shown that it is an inverting glycosyltransferase that glycosylates the same consensus sequon as eukaryotic OST (NX(S/T), where X P) (21), although with a more relaxed substrate specificity toward nonconsensus sequons (23).…”

Recent developments in bacterial protein glycan coupling technology and glycoconjugate vaccine design

“…The cytoplasmic N-glycosyltransferase (NGT) may prove to be a valuable new tool and an alternative to the current OSTs. Recently, the structure of ApHMW1C has been elucidated (Kawai et al, 2011), thus, revealing further insights into the structure and function of HMW1C-like proteins.…”