Enzymatically modified low-density lipoprotein is recognized by c1q and activates the classical complement pathway

Abstract

Several studies suggest that the complement system is involved in atherogenesis. To further investigate this question, we have studied the ability of native and modified forms of LDL to bind and activate C1, the complex protease that triggers the classical pathway of complement. Unlike native LDL, oxidized (oxLDL) and enzymatically modified (E-LDL) derivatives were both recognized by the C1q subunit of C1, but only E-LDL particles, obtained by sequential treatment with a protease and then with cholesterol esterase, had the ability to trigger C1 activation. Further investigations revealed that C1q recognizes a lipid component of E-LDL. Several approaches, including reconstitution of model lipid vesicles, cosedimentation, and electron microscopy analyses, provided evidence that C1 binding to E-LDL particles is mediated by the C1q globular domain, which senses unesterified fatty acids generated by cholesterol esterase. The potential implications of these findings in atherogenesis are discussed.

Figure 1

Analysis by surface plasmon resonance spectroscopy of the interaction between C1q and immobilized E-LDL. The E-LDL derivative (14,000 resonance units) was immobilized chemically on the surface of a CM5 sensor chip (GE Healthcare) and allowed to bind to increasing concentrations of soluble C1q (100–600 nM). The K _D value was determined from the ratio of the dissociation and association rate constants (k _off/k _on) (taken from [14], with permission).

Figure 2

C1 activation by E-LDL particle: correlation with the amount of unesterified cholesterol generated. LDL (1 mg/mL) was treated with 20 μg/mL trypsin for 2 h at 37°C and then with 320 milliunits/mL CEase for the indicated periods at 37°C. The lipid fraction from each sample was extracted and incorporated into vesicles. Each vesicle was tested for its C1-activating ability (black bars). The cholesterol content of each lipid fraction (open circles) was determined by reverse-phase HPLC. LDL: native LDL; Try-LDL: trypsin-treated LDL (taken from [15], with permission).

Figure 3

Cosedimentation analysis of the interaction between the C1q globular domain and artificial lipid vesicles. Vesicles were prepared from PC alone, PC : cholesterol (1 : 2, w/w), PC : linoleic acid (1 : 2, w/w), or PC : cholesterol : linoleic acid (1 : 2 : 2, w/w/w). Each type of vesicle was incubated with the C1q globular domain (C1q GR), and binding was measured from the relative amount of C1q GR associated with the vesicles in the ultracentrifugation pellet, as determined by SDS-PAGE analysis of the pellet (P) and supernatant (S) fractions. Chol: cholesterol; LA: linoleic acid; PC: phosphatidylcholine (taken from [15], with permission).

Figure 4

Electron micrographs of E-LDL-bound C1q molecules. (a, b) Examples of C1q molecules interacting through most of their globular domains (a) or only a few (b). (c) Representative example of a free C1q molecule (modified from [15], with permission).