Discrimination of lesion removal of N-methylpurine-DNA glycosylase revealed by a potent neutralizing monoclonal antibody.
N-Methylpurine-DNA glycosylase (MPG), a ubiquitous DNA repair enzyme, initiates excision repair of several N-alkylpurine adducts, induced by alkylating chemotherapeutics, and deaminated and lipid peroxidation-induced purine adducts. We have generated monoclonal antibodies (moAbs) against human MPG. Twelve independent hybridoma clones were characterized, which, except 520-16A, are identical based on epitope exclusion assay. Four moAbs, including 520-2A, 520-3A, 520-16A, and 520-26A, have high affinity (K(D) approximately 0.3-1.6nM), and their subtypes were IgG(2a), IgG(1), IgG(2a), and IgG(2b), respectively. moAb 520-3A recognizes the sequence (52)AQAPCPRERCLGPP(66)T, an epitope exclusively present in the N-terminal extension of human MPG. We found that moAb 520-3A significantly inhibited MPG's enzymatic activity towards different substrates, such as hypoxanthine, 1,N(6)ethenoadenine and methylated bases, which represent different classes of DNA damage, however, with different efficiencies. Real-time binding experiments using surface plasmon resonance (SPR) spectroscopy showed that the pronounced inhibition of activity was not in the substrate-binding step. Single turnover kinetics (STO) revealed that the inhibition was at the catalytic step. Since we found that this antibody has an epitope in the N-terminal tail, the latter appears to have an important role in substrate discrimination, however, with a differential effect on different substrates.
Adhikari
Sanjay
S
Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, United States.
Kennel
Stephen J
SJ
Roy
Gargi
G
Mitra
Partha S
PS
Mitra
Sankar
S
Roy
Rabindra
R
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015153:Blotting, Western; D004249:DNA Damage; D045647:DNA Glycosylases / Q000276:immunology / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D020349:Surface Plasmon Resonance
D000911:Antibodies, Monoclonal; D045647:DNA Glycosylases; C076131:DNA-3-methyladenine glycosidase II