Identification of an Antigenic Epitope in Helicobacter pylori Urease That Induces Neutralizing Antibody Production

Abstract

We previously reported a mouse monoclonal antibody (MAb), termed L2, specific for Helicobacter pylori urease strongly inhibited its enzymatic activity. Here, to gain insight into how this antibody affects urease activity, the epitope that was recognized by the antibody was determined. By screening a panel of overlapping synthetic peptides covering the entire sequence of the two subunits (UreA and UreB), we identified a stretch of UreB-derived 19 amino acid (aa) residues (UB-33; aa 321 to 339, CHHLDKSIKEDVQFADSRI) that was specifically recognized by the L2 antibody. Further sequential amino acid deletion of the 19-mer peptide from either end allowed us to determine the minimal epitope as 8 amino acid residues (F8; SIKEDVQF) for L2 reactivity. This epitope appears to lie exactly on a short sequence which formed a flap over the active site of urease, suggesting that binding of the L2 antibody sterically inhibits access of urea, the substrate of urease. Finally, immunization of rabbits with either the 19-mer peptide or the 8-mer minimal epitope resulted in generation of antiurease antibodies that were capable of inhibiting the enzymatic activity. Since urease is critical for virulence of H. pylori, antigenic peptides that induce production of antibodies to inhibit its enzymatic activity may potentially be a useful tool as a vaccine for prevention and treatment of H. pylori infection.

Augmentation of Helicobacter pylori urease activity by its specific IgG antibody: implications for bacterial colonization enhancement

“…As has already described, only L2 showed strong and specifi c neutralizing activity against H. pylori urease (16,21). By screening a panel of overlapping synthetic peptides covering the whole sequence of Ure-B, we identifi ed the minimal linear epitope for L2 as an 8-mer peptide (F8: SIKEDVQF) (16).…”

“…By screening a panel of overlapping synthetic peptides covering the whole sequence of Ure-B, we identifi ed the minimal linear epitope for L2 as an 8-mer peptide (F8: SIKEDVQF) (16). Here, we happened to fi nd that the Ure-A-specifi c MAb, S3, signifi cantly enhanced the enzymatic activity of H. pylori urease in a dose-dependent manner ( Fig.…”

“…pylori urease expressing on the very surface area among the bacterial component seems to be an important virulence factor for the colonization on gastric mucosa (7). We have reported that an IgG monoclonal antibody (MAb) against H. pylori urease, termed L2, recognizing a linear epitope within the large subunit of UreB, strongly inhibited its enzymatic activity, whereas urease-specifi c polyclonal IgG antibodies generated by immunization with purifi ed urease protein did not induce this inhibitory effect at all (16,21). The results suggest that there must be another type of urease-specific IgG that cancels the inhibition by augmenting its enzymatic activity.…”

“…More than 0.1 µg/well of purifi ed S3 MAb significantly augmented the enzymatic activity to catalyze urease. (B) The effect of L2 MAb that inhibited H.pylori urease enzymatic activity (16,21) on the enhancement induced by S3 MAb. The distilled water-extracted urease was incubated overnight with either S3 (1 µg) or L2 (1 µg), or with simultaneous addition of S3 (1 µg) and L2 (1 µg) MAbs at 4°C on 96-well microplates.…”

“…Sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) was blotted onto nitrocellulose-polyvinylidene difluoride (Atto Co., Inc., Tokyo, Japan). The nitrocellulose blots were blocked with 25% Block Ace (Dainihon Seiyaku, Osaka, Japan) in Tris-based solution (TBS) (2 M Tris (pH 8.0), 5 M NaCl, 10% Tween 20) and incubated with murine H. pylori urease-specifi c various MAbs as described previously (16,21). The blots were washed three times with blotting buffer (2 M Tris (pH 8.0), 1.43% glycine, 5% methanol) and incubated with peroxidase-conjugated goat anti-mouse IgG 1 : 500 in PBS with 10% Block Ace for 1 h at 37°C.…”