Lipoprotein(a) assembly. Quantitative assessment of the role of apo(a) kringle IV types 2-10 in particle formation.
We have developed a system for the quantitative assessment of the efficiency of lipoprotein(a) [Lp(a)] formation in vitro. Amino-terminally truncated derivatives of a 17-kringle form of recombinant apo(a) [r-apo(a)] were transiently expressed in human embryonic kidney cells. Equimolar amounts of r-apo(a) derivatives were incubated with a fourfold molar excess of purified human low density lipoprotein, and r-Lp(a) formation was assessed by densitometric analysis of Western blots. Although r-Lp(a) formation was observed with each r-apo(a) derivative, both the rate and extent of particle formation were greatly lower on removal of kringle IV type 7. Additional substantial decreases in these parameters were observed on removal of kringle IV type 8, thereby suggesting a major role for these two kringles in Lp(a) assembly. We directly demonstrated that the lysine-binding sites (LBSs) within kringle IV types 5-9 are "masked" in the context of the Lp(a) particle and are consequently unavailable for interaction with lysine-Sepharose. Using site-directed mutagenesis, we also demonstrated that the previously described LBS in kringle IV type 10 is not required for r-Lp(a) formation: r-Lp(a) formation using a mutated form of apo(a) that lacks this LBS is comparable in efficiency to that of wild-type r-apo(a) and can be inhibited to a similar extent by epsilon-amino-n-caproic acid. In summary, the results of our study indicate that apo(a) kringle IV types 7 and 8 are required for maximal efficiency of Lp(a) formation, likely by virtue of their ability to mediate lysine-dependent non-covalent interactions with apoB-100 that precede disulfide bond formation.
Gabel
B R
BR
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
May
L F
LF
Marcovina
S M
SM
Koschinsky
M L
ML
D001483:Base Sequence; D002460:Cell Line; D018014:Gene Transfer Techniques; D006801:Humans; D018082:Kringles; D017270:Lipoprotein(a) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D017270:Lipoprotein(a); D011994:Recombinant Proteins