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.
Guevara
J
J
Department of Medicine, Baylor College of Medicine, Houston, Texas 77030, USA.
Valentinova
N V
NV
Garcia
O
O
Gotto
A M
AM
Yang
C Y
CY
Legal
S
S
Gaubatz
J
J
Sparrow
J T
JT
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology / Q000378:metabolism; D053299:Apolipoprotein B-100; D001053:Apolipoproteins / Q000378:metabolism*; D001055:Apolipoproteins B / Q000276:immunology / Q000378:metabolism*; D053241:Apoprotein(a); D001665:Binding Sites / Q000235:genetics; D002845:Chromatography / Q000379:methods; D002846:Chromatography, Affinity; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004586:Electrophoresis / Q000379:methods; D006801:Humans; D017270:Lipoprotein(a); D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology; D011485:Protein Binding
D000906:Antibodies; D053299:Apolipoprotein B-100; D001053:Apolipoproteins; D001055:Apolipoproteins B; D004220:Disulfides; D017270:Lipoprotein(a); D010455:Peptides; D053241:Apoprotein(a)