Interaction of apolipoprotein[a] with apolipoproteinB-100 Cys3734 region in lipoprotein[a] is confirmed immunochemically

Monospecific polyclonal antibodies (MPAbs) to apoB-100 regions Cys3734 and Cys4190 were isolated by affinity chromatography using the synthetic polypeptides, Q₃₇₃₀VPSSKLDFREIQIYKK₃₇₄₆ and G₄₁₈₂IYTREELSTMFIREVG₄₁₉₈, respectively, coupled to a hydrophilic resin. Molecular modeling and fluroescence labeling studies have suggested that Cys67 located in kringle type 9 (LPaK9, located between residues 3991 and 4068 of the apo[a] sequence inferred by cDNA) of the apo[a] molecule is disulfide linked to Cys3734 of apoB-100 in human lipoprotein[a] (Lp[a]). This possibility has been further explored with MPAbs. Four species of MPAbs directed to a Cys3734 region of apoB-100 (3730–3746) were isolated from goat anti-human LDL serum by a combination of synthetic peptide (Q₃₇₃₀VPSSKLDFREIQIYKK₃₇₄₆) affinity chromatography and preparative electrophoresis (electrochromatography). MPAbs to the Cys4190 region of apoB-100, a second or alternative disulfide link-site between apo[a] and apoB-100, were also isolated using a synthetic peptide (G₄₁₈₂IYTREELSTMFIREVG₄₁₉₈) affinity resin. Results of immunoassays showed that binding of these four MPAbs to Lp[a] was significantly lower than to LDL. In contrast, MPAbs to the apoB-100 region 4182–4198 which contains Cys4190, a second or alternative disulfide link-site between apo[a] and apoB-100, displayed a less significant difference in binding to Lp[a] and LDL. These results provide additional evidence that the residues 3730–3746 of apoB-100 interact significantly with apo[a] in Lp[a], and that Cys3734 is a likely site for the disulfide bond connecting apo[a] and apoB-100.Abbreviations amino acids single letter, e.g., alanine, A, etc. - BSA bovine serum albumin - d density (g/ml) - aca -aminocaproic acid - ELISA enzyme-linked immunosorbant assay - DTT dithiothreitol - HRP horseradish peroxidase - MAb monoclonal antibody - MPAb monospecific polyclonal antibody - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecyl sulfate - Na₂EDTA sodium ethylenediaminetetraacetate - NaN₃ sodium azide - TRIS (hydroxymethyl)aminomethane