Catalytic features of monoclonal antibody i41SL1‐2 subunits

Abstract

A monoclonal antibody (mAb), i41SL1-2, was obtained by immunizing the peptide of complementarity-determining region-1 (CDRL-1: RSSKSLLYSNGNTYLY) of a super catalytic antibody light chain, 41S-2-L, capable of enzymatically destroying the gp41 molecule of the HIV-1 envelope. From the DNA and the deduced amino acid sequences of the light and heavy chain of i41SL1-2 mAb, molecular modeling was conducted that suggested that both subunits of i41SL1-2 mAb possess catalytic triads in their structures. Especially the light chain of i41SL1-2 mAb possesses a characteristic catalytic triad composed of Asp(1), Ser(27A), and His(93), whose positions are identical to the catalytic antibody light chain, VIPase, of S. Paul and colleagues (see text). The antibody gene of i41SL1-2 light chain and VIPase belong to the same germline, bd2, suggesting that the discrete germline inherently possesses catalytic activity. Both light and heavy chains of i41SL1-2 mAb degraded the antigenic peptide CDRL-1 within 47 and 57 h, respectively. The catalytic reaction constant (kcat) of the light and heavy chain was 6.1 x 10(-1) and 6.2 x 10(-1) min(-1), respectively. These are high values for the natural catalytic antibodies reported so far. The catalytic efficiency (kcat/Km) of the light and heavy chain was 3.1 x 10(5) and 4.9 x 10(4) M(-1) min(-1), respectively. The first cleaved bond of the antigenic peptide by subunits of i41SL1-2 mAb was between Arg(1) and Ser(2) in the sequence of CDRL-1, suggesting a serine protease character.

Catalytic antibody light chain capable of cleaving a chemokine receptor CCR‐5 peptide with a high reaction rate constant

“…The value of the light chain was about 10-fold less than that of the heavy chain. Similar phenomena have been observed in many cases, as reported elsewhere (e.g., Hatiuchi et al, 2003;Hifumi et al, 1999Hifumi et al, , 2000aHifumi et al, , 2002Hifumi et al, , 2003Uda et al, 2000).…”

“…Of the six MAbs, ECL2B-2 MAb did not cross-react with other proteins such as KLH, HSA, OVA, and human hemoglobin. Furthermore, the MAbs did not react with some other peptides, such as the gp120 V3 loop of HIV-1, TP41-1 (TPRGPDRPEGIEEEGGERDRD, a peptide of a conserved region of gp41 of HIV-1), RT-2 (KLLRGTKALTEVIPL-TEEAE, a part of the sequence of the reverse transcriptase of HIV-1), CDRL1 of 41S-2 MAb (RSSKSLLYSNGN-TYLY; Hifumi et al, 2003), CDRH2 of 1D3 MAb (RINPYNGATSYNQNFKD; Uda et al, 1993), and CA-2 (RSSKSLLYSNGNTYLYGPRINPYNGATSYNQNFK-DH; Hifumi et al, 2000b).…”

Catalytic Features and Eradication Ability of Antibody Light-chain UA15-L against Helicobacter pylori

“…Cleavage Activity against UreB-Because the epitope of UA15 mAb is not determined, a peptidase activity of UA15-L was examined by using a TP41-1 peptide (TPRGPDRPEGIEEE-GGERDRD), which has been used to monitor the peptidase activity of the catalytic antibody or its subunits (3,12,13,15,16). The degradation of TP41-1 peptide by UA15-L followed the Michaelis-Menten equation, and its kinetic parameters were obtained as k cat ϭ 0.24 min Ϫ1 and K m ϭ 6.2 ϫ 10 Ϫ5 M (see supplemental Appendix S1).…”

“…In our case, UA15-L belonged to bd2 germ line (DDBJ; accession No. AB286872) and possess the identical aa residues to the catalytic triad of VIPase (20), ECL2B-L (13,21), and i41SL2-1-L (15,21). Thus, it was predicted that UA15-L could hydrolyze the antigen UreB.…”

“…Prior to the cleavage test for UreB protein and the purified urease of H. pylori, a peptide, TPRGPDRPEGIEEEGGERDRD (TP41-1), which has mostly been used for monitoring the catalytic activity of the antibody and/or its subunits (3,10,(12)(13)(14), was completely digested by the catalytic reaction of UA15-L. By this reaction, the catalytic activity of UA15-L was held constant, showing no induction time (3,(12)(13)(14)(15). In the cleavage assay of UreB and the urease, the UA15-L mentioned above was used.…”