Bacterial expression and characterization of human recombinant apolipoprotein(a) kringle IV type 9.
Elevated plasma lipoprotein(a) [Lp(a)] is an independent risk factor for several vascular diseases. Lp(a) particles are generated through the formation of a disulfide bond between Cys4057 of kringle IV type 9, (KIVt9), of the multikringle apolipoprotein(a) [apo(a)] and a cysteine in apoB-100 low-density lipoprotein (LDL). To better understand this interaction, we have expressed and purified KIVt9 from Escherichia coli as a His-Tag fusionprotein. Dithiothreitol (DTT)-treated purified KIVt9 migrated as a single approximately 17. 3-kDa band on SDS-PAGE gels. Without DTT, an additional band twice the molecular weight of KIVt9 was observed. The double-size band presumably resulted from dimerization of individual kringles, through their unpaired cysteine residues, since a mutation Cys4057 --> Ser ([Ser4057]KIVt9) abolished dimer formation. Using a gel-shift assay, we showed that KIVt9 could couple to 14-amino-acid apoB-100 synthetic peptides (apoB3732-3745 and apoB4319-4332) containing Cys3734 or Cys4326. Both of these apoB-100 cysteines have been reported to associate with apo(a) to generate Lp(a). In the presence of either apoB-100 peptide, KIVt9 was shifted to a higher molecular weight that was consistent with the covalent addition of a 1.2-kDa apoB-100 peptide. Identical apoB-100 peptides in which the cysteine residues were replaced by alanine ([Ala3734]apoB3732-3745 and [Ala4326]apoB4319-4332) had no effect in the gel-shift assay. Furthermore, [Ser4057]KIVt9 did not covalently interact with apoB3732-3745 or apoB4319-4332. These results indicated that KIVt9 couples to the Cys-apoB-100 peptides through a disulfide linkage. This system may be suitable for further investigating the apo(a)/apoB-100 coupling reaction and the structure of KIVt9 through X-ray crystallographic studies.
Chung
F Z
FZ
Department of Molecular Biology, Department of Medicinal Chemistry, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Co., 2800 Plymouth Road, Ann Arbor, Michigan 48105, USA.
Wu
L H
LH
Lee
H T
HT
Mueller
W T
WT
Spahr
M A
MA
Eaton
S R
SR
Tian
Y
Y
Settimi
P D
PD
Oxender
D L
DL
Ramharack
R
R
D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000378:metabolism; D003545:Cysteine / Q000235:genetics; D004220:Disulfides / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D018082:Kringles; D017270:Lipoprotein(a) / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism*
D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D004220:Disulfides; D017270:Lipoprotein(a); D010446:Peptide Fragments; D010455:Peptides; D011993:Recombinant Fusion Proteins; D003545:Cysteine