Catalytic features and eradication ability of antibody light-chain UA15-L against Helicobacter pylori.

We have successfully developed a catalytic antibody capable of degrading the active site of the urease of Helicobacter pylori and eradicating the bacterial infection in a mouse stomach. This monoclonal antibody UA15 was generated using a designed recombinant protein UreB, which contained the crucial region of the H. pylori urease beta-subunit active site, for immunization. The light chain of this antibody (UA15-L) by itself showed a proteolytic activity to substantially degrade both UreB and the intact urease. Oral administration of UA15-L also significantly reduced the number of H. pylori in a mouse stomach. This is the first example of a monoclonal catalytic antibody capable of functioning in vivo, and such an antibody may have a therapeutic utility in the future.

Specific degradation of H. pylori urease by a catalytic antibody light chain.

Catalytic antibodies capable of digesting crucial proteins of pathogenic bacteria have long been sought for potential therapeutic use. Helicobacter pylori urease plays a crucial role for the survival of this bacterium in the highly acidic conditions of human stomach. The HpU-9 monoclonal antibody (mAb) raised against H. pylori urease recognized the alpha-subunit of the urease, but only slightly recognized the beta-subunit. However, when isolated both the light and the heavy chains of this antibody were mostly bound to the beta-subunit. The cleavage reaction catalyzed by HpU-9 light chain (HpU-9-L) followed the Michaelis-Menten equation with a K(m) of 1.6 x 10(-5) m and a k(cat) of 0.11 min(-1), suggesting that the cleavage reaction was enzymatic. In a cleavage test using H. pylori urease, HpU-9-L efficiently cleaved the beta-subunit but not the alpha-subunit, indicating that the degradation by HpU-9-L had a specificity. The cleaved peptide bonds in the beta-subunit were L121-A122, E124-G125, S229-A230, Y241-D242, and M262-A263. BSA was hardly cleaved by HpU-9-L, again indicating the digestion by HpU-9-L was specific. In summary, we succeeded in the preparation of a catalytic antibody light chain capable of specifically digesting the beta-subunit of H. pylori urease.

Endopeptidase character of monoclonal antibody i41-7 subunits.

We prepared six anti-idiotypic monoclonal antibodies (mAbs) against parent 41S-2 mAb whose light chain is a super catalytic antibody (41S-2-L) capable of degrading targeted HIV-1gp41 molecule. Out of the obtained six mAbs, i41-7 mAb showed the strongest affinity to the parent 41S-2 mAb. The three dimensional structure of i41-7 mAb was created by molecular modeling using the deduced amino acid sequence of the light and heavy chain of i41-7 mAb. It suggests that the light and heavy chain possess catalytic triad-like structure composed of Ser, His and Asp in their conformations. Both chains of i41-7 mAb could cleave peptide bond of some peptides such as a polypeptide, TP41-1 (TPRGPDRPEGIEEEGGERDRD), as anticipated. The cleaving reaction advanced in accordance with Michaelis-Menten equation. The catalytic efficiency (kcat/Km) of light and heavy chain was 9.1 x 10(3) and 1.7 x 10(4) M(-1) x min(-1), respectively, while the intact i41-7 mAb did not exhibit any catalytic activity. The first cleaved bond of the TP41-1 peptide by the light chain was between 14E and 15G in the sequence. It was revealed that both light and heavy chains had endopeptidase characteristics.