The Actinobacillus pleuropneumoniae HMW1C-Like Glycosyltransferase Mediates N-Linked Glycosylation of the Haemophilus influenzae HMW1 Adhesin

The Haemophilus influenzae HMW1 adhesin is an important virulence exoprotein that is secreted via the two-partner secretion pathway and is glycosylated at multiple asparagine residues in consensus N-linked sequons. Unlike the heavily branched glycans found in eukaryotic N-linked glycoproteins, the modifying glycan structures in HMW1 are mono-hexoses or di-hexoses. Recent work demonstrated that the H. influenzae HMW1C protein is the glycosyltransferase responsible for transferring glucose and galactose to the acceptor sites of HMW1. An Actinobacillus pleuropneumoniae protein designated ApHMW1C shares high-level homology with HMW1C and has been assigned to the GT41 family, which otherwise contains only O-glycosyltransferases. In this study, we demonstrated that ApHMW1C has N-glycosyltransferase activity and is able to transfer glucose and galactose to known asparagine sites in HMW1. In addition, we found that ApHMW1C is able to complement a deficiency of HMW1C and mediate HMW1 glycosylation and adhesive activity in whole bacteria. Initial structure-function studies suggested that ApHMW1C consists of two domains, including a 15-kDa N-terminal domain and a 55-kDa C-terminal domain harboring glycosyltransferase activity. These findings suggest a new subfamily of HMW1C-like glycosyltransferases distinct from other GT41 family O-glycosyltransferases

Structural studies of Campylobacter jejuni: Virulence proteins (Cj0977 and JlpA) and novel lipoproteins

Campylobacter jejuni is a major cause of human gastroenteritis worldwide; however, its pathogenesis is not understood well. Cj0977, a cytoplasmic protein regulated by the flagellar promoter s28, and JlpA, a cell-surface exposed lipoprotein adhesin, are virulence factors, but their contributions in pathogenesis of C. jejuni remains elusive. Since there is no clue for these two protein structures, we aimed to discover structure-function relationship of these proteins. My work focused on establishing expression system, purification method, and crystallization method of these proteins, leading up to obtaining high quality crystals for successful X-ray diffraction. Several techniques were employed to yield diffracting crystals: limited proteolysis and stability test for Cj0977 and introduction of two additional Met sites within the JlpA sequence. Here we present the first view of the crystal structures of Cj0977 and JlpA. Cj0977 adopts a ‘hot dog’ fold, a famous protein folds found in numerous CoA derivative binding enzymes. The Cj0977 structure suggests its possible function as an acyl-CoA binding regulatory protein. The JlpA structure reveals a novel fold of unclosed half β-barrel and is reminiscent of other bacterial lipoproteins. The JlpA structure suggests that JlpA may accommodate multiple ligands and that a similar role for JlpA as a carrier of as yet unidentified Campylobacter-specific lipids. The unique fold of JlpA led us to initiate other C. jejuni lipoprotein study. Most of C. jejuni lipoproteins are unknown due to no apparent sequence homology in the sequence databank; however, they are considered to contribute to the pathogenesis. We aimed to uncover the crystal structures of C. jejuni lipoproteins to provide framework for investigating their functions. My work focused on four lipoproteins: Cj0090, Cj1026c, Cj1090c, and Cj1649. Among these, the Cj0090 structure reveals a novel variant of the immunoglobulin fold with β-sandwich architecture, suggesting that Cj0090 may be involved in protein-protein interactions, consistent with a possible role for bacterial lipoproteins. In addition, a phage display technique was performed to identify ligands for our target lipoproteins. The crystal structures revealed in this study contribute to provide clues for possible roles of our target proteins and enable to perform further structure-based functional analyses.Biology and Biochemistry, Department o

N-linked glycosylation of HMW1ct.

<p>The glycosylation reactions were carried out in standard conditions using single (N1348Q, N1352Q, N1366Q) mutants of His-HMW1ct as acceptor proteins and UDP-glucose as donor substrate. (<b>A</b>) After the glycosylation reaction, the samples were separated by SDS-PAGE, and the gel was stained with Coomassie Blue. (<b>B</b>) A duplicate gel was transferred to a PVDF membrane and subjected to a detection reaction using the GlycoProfile III Fluorescent Glycoprotein Detection kit (Sigma). The lanes labeled “Native” and “His-HMW1ct only” are control reaction samples with and without ApHMW1C, respectively. M1 is a pre-staining protein marker (Precision Plus Protein Standards, Bio-Rad), and M2 is a glycosylated protein marker (ProteoProfile PTM Marker, Sigma).</p

Ability of ApHMW1C to complement a deficiency in HMW1C.

<p>Panel <b>A</b> shows Western immunoblots of whole cell sonicates of <i>E. coli</i> BL21(DE3)/pACYC-HMW1ΔC (lane 1), <i>E. coli</i> BL21(DE3)/pACYC-HMW1ΔC + pET45b-HMW1C (lane 2), and <i>E. coli</i> BL21(DE3)/pACYC-HMW1ΔC + pET45b-ApHMW1C (lane 3). Lane 1 contains twice as much protein as loaded in lanes 2 and 3 to increase the visibility of the non-glycosylated HMW1 species. The blot in the upper panel was performed with a guinea pig antiserum reactive with HMW1, and the blot in the lower panel was performed with a guinea pig antiserum reactive with <i>H. influenzae</i> HMW1C. The asterisk indicates the glycosylated HMW1 pro-protein, and the plus sign indicates the non-glycosylated HMW1 pro-protein. The diamond indicates the glycosylated HMW1 mature protein, and the circle indicates the non-glycosylated HMW1 mature protein. Panel <b>B</b> shows <i>in vitro</i> adherence results comparing adherence by <i>E. coli</i> BL21(DE3)/pACYC-HMW1ΔC (<i>hmw1AB</i>), <i>E. coli</i> BL21(DE3)/pACYC-HMW1ΔC + pET45b-HMW1C (<i>hmw1AB</i> + <i>hmw1C</i>), and <i>E. coli</i> BL21(DE3)/pACYC-HMW1ΔC + pET45b-ApHMW1C (<i>hmw1AB</i> + <i>Aphmw1C</i>) to Chang epithelial cells. Bars and error bars represent mean and standard error measurements from a representative assay with measurements performed in triplicate.</p

Initial velocity of ApHMW1C.

<p>(<b>A</b>) Double reciprocal plots of the initial velocity of ApHMW1C as a function of UDP-glucose concentration at the fixed His-HMW1ct concentrations, as indicated (inset). (<b>B</b>) Double reciprocal plots of the initial velocity of ApHMW1C as a function of His-HMW1ct concentration at the fixed UDP-glucose concentrations, as indicated (inset). The true K_m values of UDP-glucose and His-HMW1ct corresponded to 54.5 µM and 2.3 µM, respectively, as obtained by eq. 2.</p

Primers used for cloning, site-directed mutagenesis, and sequence analysis.

<p>Primers used for cloning, site-directed mutagenesis, and sequence analysis.</p

Glycosylation of HMW1ct by ApHMW1C.

<p>To define the donor substrate specificity of ApHMW1C, glycosylation reactions were carried out in the reaction buffer with (R-lanes) or without (C-lanes) ApHMW1C using different UDP (or GDP) activated sugars. HMW1ct (without fusion tag) was used as the acceptor protein (lanes 1, and 3 to 6). As a control, His-tagged HMW1ct (His-HMW1ct) was also tested in a reaction with UDP-glucose as the donor sugar (lanes 2). (<b>A</b>) After the glycosylation reactions, samples were separated by SDS-PAGE, and the gel was stained with Coomassie Blue. (<b>B</b>) In parallel, a duplicate gel was transferred to a PVDF membrane and subjected to a detection reaction using the GlycoProfile III Fluorescent Glycoprotein Detection kit (Sigma). Glycosylated HMW1ct proteins are indicated by arrows: ‘a’ and ‘c’ are glycosylated HMW1ct reacted with UDP-glucose and UDP-galactose, respectively, and ‘b’ is glycosylated His-HMW1ct reacted with UDP-glucose. The lanes labeled “M1,” “M2,” and “HMW1ct only” indicate pre-staining protein markers (Precision Plus Protein Standards, Bio-Rad), glycosylated protein markers (ProteoProfile PTM Marker, Sigma), and HMW1ct only as a control, respectively.</p

Kinetic parameters of ApHMWC and its derivative proteins.

a<p>These are apparent values, determined by varying the concentration of one substrate (sugar donor substrate) at a fixed concentration of the second (protein acceptor).</p

Specificity of HMW1ct glycosylation.

<p>The glycosylation reactions were carried out in standard conditions using His-HMW1ct as acceptor protein and UDP-glucose or UDP-galactose as donor substrate. (<b>A</b>) At each time point, an aliquot of the reaction was stopped by adding an equal volume of 2X SDS-PAGE sample buffer and followed by heating at 95°C for 4 min. (<b>B</b>) At each time point, SDS-PAGE samples were prepared as in A. However, two hrs after reaction with the first donor substrate, the second donor substrate was added to the reaction, as indicated. All samples were separated by 12% SDS-PAGE, and the gel was stained with Coomassie blue. The distinct shifts due to incorporated sugars are indicated by symbols (•, 0 hexose; ▪, 2 hexoses; ⋆, 4 or 5 hexoses; and ⋆’, 5 or 6 hexoses). (<b>C</b>) The glycosylation reactions were carried out in standard conditions using double mutants of His-HMW1ct (N1348Q/N1352Q, N1348Q/N1366Q, and N1352Q/N1366Q) by ApHMW1C using UDP-glucose or UDP-galactose as donor substrates. C1, C2, and C3 indicate control reactions without ApHMW1C. Samples were separated by 12% SDS-PAGE and were stained with Coomassie blue. (<b>D</b>) In parallel, a duplicated gel was transferred to a PVDF membrane and subjected to a detection reaction using the GlycoProfile III Fluorescent Glycoprotein Detection kit (Sigma). The glycosylated proteins by UDP-glucose or by UDP-galactose are indicated by arrows. (<b>E</b>) Model of hexose modifications at Asn-1348, Asn-1352, and Asn-1366.</p