Detailed characterization of the binding site of the lipoprotein lipase-specific monoclonal antibody 5D2.
Monoclonal antibody (MAb) 5D2 recognizes lipoprotein lipases (LPL) from different species but not related lipases. This MAb is a unique reagent, used world-wide, because it differentiates between monomeric inactive and dimeric active LPL, inhibits human LPL enzyme activity, and binds to C-terminal LPL sequences involved in interactions with lipoproteins, lipoprotein receptors, and heparin. In this study we have analyzed the fine specificity of the MAb epitope recognition in order to better understand its functional properties and species-specific LPL immune reactivity. In peptide scan assays, MAb 5D2 reacted with all, except two, 13 amino acid-long peptides located between positions 380 and 410. Peptides from the amino terminal end of this region reacted more strongly than those from the carboxyl terminal end. Furthermore, only a peptide from the amino terminal end competed effectively with the binding of MAb 5D2 to native LPL bound to microtiter plates or nitrocellulose. A systematic peptide mutagenesis study indicated that 8 amino acids of the reactive region, mainly located in the amino terminal end, are critical for binding and probably directly interact with MAb 5D2. The experimentally determined antigenicities of species-specific LPL peptides and of the corresponding denatured full-length LPL proteins on immunoblots were consistent with these findings. According to a proposed 3D-model for LPL, only the amino terminal end of the antigenic region is easily surface-accessible. These data combined with 3D-modelling of monoclonal antibody (MAb)-lipoprotein lipase (LPL) protein interaction provide new insight into the known biological effects of MAb 5D2 on LPL and the antigenic determinants that are recognized.
Chang
S F
SF
Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie, Universität Hamburg, Martinistrasse 52, 20251 Hamburg, Germany.
Reich
B
B
Brunzell
J D
JD
Will
H
H
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D001665:Binding Sites; D001667:Binding, Competitive; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008071:Lipoprotein Lipase / Q000276:immunology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis
D000911:Antibodies, Monoclonal; D008071:Lipoprotein Lipase