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

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

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

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

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

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