Detailed characterization of the binding site of the lipoprotein lipase-specific monoclonal antibody 5D2

Monoclonal antibody (MAb) 5D2 recognizes lipoprotein lipases (LPL) from dif ferent species but not related lipases. This MAb is a unique reagent, used world-wide, because it differentiates between monomeric inactive and dimeri c active LPL, inhibits human LPL enzyme activity, and binds to C-terminal L PL sequences involved in interactions with lipoproteins, lipoprotein recept ors, and heparin, In this study we have analyzed the fine specificity of th e MAb epitope recognition in order to better understand its functional prop erties and species-specific LPL immune reactivity, In peptide scan assays, MAb 5D2 reacted with all, except two, 13 amino acid-long peptides located b etween positions 380 and 410, Peptides from the amino terminal end of this region reacted more strongly than those from the carboxyl terminal end. Fur thermore, only a peptide from the amino terminal end competed effectively w ith the binding of MAb 5D2 to native LPL bound to microtiter plates or nitr ocellulose. A systematic peptide mutagenesis study indicated that 8 amino a cids of the reactive region, mainly located in the amino terminal end, are critical for binding and probably directly interact with MAb 5D2, The exper imentally determined antigenicities of species-specific LPL peptides and of the corresponding denatured full-length LPL proteins on immunoblots were c onsistent with these findings. According to a proposed 3D-model for LPL, on ly the amino terminal end of the antigenic region is easily surface-accessi ble. These data combined with SD-modelling of monoclonal antibody (MAb)-lip oprotein lipase (LPL) protein interaction provide new insight into the know n biological effects of MAb 5D2 on LPL and the antigenic determinants that are recognized.