Protein Engineering, Vol 11, 1249-1256, Copyright © 1998 by Oxford University Press
M Rahman, Z Jia, BR Gabel, SM Marcovina and ML Koschinsky
A number of studies have provided evidence that lipoprotein(a) [Lp(a)]
assembly is a two-step process in which initial non-covalent interactions
between apolipoprotein(a) [apo(a)] and apolipoproteinB-100 (apoB-100)
precede specific disulfide bond formation. We have designed a construct
encoding apo(a) kringle IV type 9 (KIV9) in which the unpaired cysteine at
position 67 in this kringle is replaced with a tyrosine. The single kringle
was expressed in bacteria and purified to homogeneity from cell
homogenates. The purified derivative (designated KIV9deltaCys) was assessed
for its ability to bind to purified human LDL. This interaction was
detected either by ELISA using immobilized LDL or by column chromatography
in which LDL binding to KIV9deltaCys immobilized on Ni2+-Sepharose was
determined. In both cases, the interaction of KIV9deltaCys and LDL was
observed. Further, we demonstrated that the binding interaction was
sensitive to the addition of amino acids including lysine, the lysine
analogue epsilon- aminocaproic acid, arginine, phenylalanine and proline,
with arginine and lysine having the greatest inhibitory effect. Binding of
KIV9deltaCys to an immobilized apoB peptide spanning residues 3732-3745 of
apoB was also demonstrated by ELISA. As was the case for LDL, this binding
interaction was sensitive to the addition of arginine and lysine. Computer
modeling of KIV9 demonstrated an excellent fit with residues 3732-3738
(PSCKLDF) of the apoB peptide. The modeling predicts the presence of
overlapping lysine and phenylalanine-binding pockets in KIV9 which explains
the inhibitory effects of lysine, arginine and phenylalanine which were
observed in the binding assays. In summary, this study represents the first
demonstration that KIV9 can interact directly with LDL through non-covalent
interactions which may contribute to the first step of Lp(a) formation.
ARTICLES
Expression of apolipoprotein(a) kringle IV type 9 in Escherichia coli: demonstration of a specific interaction between kringle IV type 9 and apolipoproteinB-100
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
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