Abstract
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.
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Abbreviations
- amino acids:
-
single letter, e.g., alanine, A, etc.
- BSA:
-
bovine serum albumin
- d :
-
density (g/ml)
- εaca:
-
ε-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
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Guevara, J., Valentinova, N.V., Garcia, O. et al. Interaction of apolipoprotein[a] with apolipoproteinB-100 Cys3734 region in lipoprotein[a] is confirmed immunochemically. J Protein Chem 15, 17–25 (1996). https://doi.org/10.1007/BF01886807
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DOI: https://doi.org/10.1007/BF01886807