The Binding Activity of the Macrophage Lipoprotein(a)/Apolipoprotein(a) Receptor Is Induced by Cholesterol via a Post-translational Mechanism and Recognizes Distinct Kringle Domains on Apolipoprotein(a)

Abstract

Elevated plasma levels of lipoprotein(a) (Lp(a)) can be a risk factor for atherosclerosis, and the interaction of Lp(a) with cholesterol-loaded macrophages (foam cells) in atheromata may be important in Lp(a)-induced atherogenesis. We have previously shown that when cultured macrophages are loaded with cholesterol, they acquire the ability to internalize and lysosomally degrade Lp(a) via interaction between a novel cell-surface receptor activity and the apolipoprotein(a) (apo(a)) moiety of Lp(a). Herein we explore the cell-surface binding of recombinant apo(a) (r-apo(a)) by foam cells. Whereas the induction of degradation of r-apo(a) by cholesterol loading of macrophages depended on new protein synthesis, the induction of binding of r-apo(a) did not. Furthermore, J774 macrophages bound r-apo(a) in a cholesterol-regulatable and specific manner but degraded r-apo(a) poorly. Thus, the binding and internalization/ degradation functions of the receptor activity are distinct. To explore which domains on r-apo(a) interact with the foam cell receptor, we conducted a series of competitive and direct binding and degradation experiments using 12 r-apo(a) constructs that differed in their content of specific kringle subtypes. These data, as well as complementary data with anti-apo(a) monoclonal antibodies, indicated that the region centered around kringle type IV, subtypes 6-7 (KIV 6 -7 ) is important in receptor binding. Remarkably, a cholesterol-induced receptor activity with similar structural specificity was also found on Chinese hamster ovary cells. In conclusion, the foam cell Lp(a)/apo(a) receptor consists of a cholesterol-regulatable binding activity and a shortlived component necessary for internalization or lysosomal degradation; the binding activity interacts with a distinct region of apo(a) that is different from that involved in competition for plasminogen binding. Lp(a)1 is an LDL-like lipoprotein in which the apoB-100 moiety of LDL is covalently attached to a glycoprotein called apo(a) (1, 2). Apo(a) consists of multiple domains called kringles, which are regions of protein folds each stabilized by three disulfide bonds (3). Apo(a) shares 80% homology with another kringle-containing protein, plasminogen (3). Although the physiological role of Lp(a) is not known, elevated levels of this lipoprotein in certain human populations are often associated with increased risk for atherosclerotic coronary artery disease and stroke (4). Furthermore, Lp(a) transgenic mice (5, 6) and, in one report ( (7); cf. Ref. 8), apo(a) transgenic mice have been found to have accelerated atherosclerosis.The mechanism of Lp(a)-induced atherosclerosis is not known. Several groups of investigators have postulated that the ability of Lp(a) to compete for plasminogen binding sites on cells is important in certain potentially atherogenic processes, such as decreased fibronolysis (9 -11) and increased smooth muscle cell proliferation (12). Another possible clue to the potential atherogenicity of Lp(a) comes from the observation that Lp(a) an...

Identification of a Critical Lysine Residue in Apolipoprotein B-100 That Mediates Noncovalent Interaction with Apolipoprotein(a)

“…A 17-kringle (17K)-containing form of r-apo(a) and an apo(a) derivative containing KIV [5][6][7][8] were constructed and expressed as previously described (14,15). Proteins were purified from the conditioned medium (CM) of stably expressing human embryonic kidney HEK 293 (16) cell lines by lysine-Sepharose affinity chromatography (Amersham Pharmacia Biotech) (8).…”

“…Protein-containing fractions were pooled, dialyzed against HBS, and concentrated using polyethylene glycol 20000 (Fluka). Protein concentrations for all r-apo(a) derivatives were obtained by absorbance measurements at 280 nm (corrected for Rayleigh scattering) using previously determined extinction coefficients (14,15).…”

Sequences Within the Amino Terminus of ApoB100 Mediate Its Noncovalent Association With Apo(a)

“…20,21 The generation of expression plasmids encoding apoB100 and carboxyl-terminal truncations of apoB100, including apoB94, 22 apoB42, apoB37, and apoB29 23 as well as apoB18 24 and apoB15, 25 has been previously described as indicated. All apoB constructs were used to stably transfect the rat hepatoma cell line McA-RH7777 (American Type Culture Collection, Rockville, Md; No.…”

“…21 CM harvested from stably expressing cell lines was loaded onto 50-mL lysine-Sepharose CL-4B columns equilibrated with PBS, pH 7.4. Columns were washed with PBS containing 0.5 mol/L NaCl, pH 7.4, and eluted with 0.2 mol/L ⑀-aminocaproic acid (⑀-ACA) in the same buffer.…”

Lipoprotein(a) Concentration and Apolipoprotein(a) Size

“…These events may include uptake of native Lp(a) by macrophages, which is mediated by apo(a) kringle IV types 6 and 7. 19 Kronenberg and colleagues, in the stimulating Discussion section, provide a clear and concise evaluation of the possible mechanisms by which Lp(a) may be involved in the early stages of atherosclerosis.…”