Apolipoprotein modulation of streptococcal serum opacity factor activity against human plasma high-density lipoproteins

Abstract

Human plasma HDL are the target of streptococcal serum opacity factor (SOF), a virulence factor that clouds human plasma. Recombinant (r) SOF transfers cholesteryl esters (CE) from approximately 400,000 HDL particles to a CE-rich microemulsion (CERM), forms a cholesterol-poor HDL-like particle (neo HDL), and releases lipid-free (LF) apo A-I. Whereas the rSOF reaction requires labile apo A-I, the modulation effects of other apos are not known. We compared the products and rates of the rSOF reaction against human HDL and HDL from mice overexpressing apos A-I and A-II. Kinetic studies showed that the reactivity of various HDL species is apo-specific. LpA-I reacts faster than LpA-I/A-II. Adding apos A-I and A-II inhibited the SOF reaction, an effect that was more profound for apo A-II. The rate of SOF-mediated CERM formation was slower against HDL from mice expressing human apos A-I and A-II than against WT mice HDL and slowest against HDL from apo A-II overexpressing mice. The lower reactivity of SOF against HDL containing human apos is due to the higher hydropathy of human apo A-I, particularly its C-terminus relative to mouse apo A-I, and the higher lipophilicity of human apo A-II. The SOF-catalyzed reaction is the first to target HDL rather than its transporters and receptors in a way that enhances reverse cholesterol transport (RCT). Thus, effects of apos on the SOF reaction are highly relevant. Our studies show that the "humanized" apo A-I-expressing mouse is a good animal model for studies of rSOF effects on RCT in vivo.

Fig. 1

SDS-PAGE and Western blots of HDL preparations. A. Coomasie blue staining of LpA-I and LpA-I/A-II as labeled in non-reduced and reduced form following LpA-I and LpAI/A-II isolation by covalent chromatography with TPS. B. Coomasie blue staining of HDL isolated from wild type mice and mice overexpressing human ApoA-I, ApoA-II (Lo), and ApoA-II (Hi) C. Immunoblotting of WT and gene-altered mice as labeled using antibodies to human apo A-I and apo A-II.

Fig. 2

SEC of human HDL subfractions pre and post rSOF treatment. A. Total HDL; B. LpA-I; C. LpA-I/A-II. Pre rSOF (grey fill); post rSOF (−). After rSOF treatment, 47%, 42% and 51% of HDL protein was released as LF apo A-I in A, B and C, respectively.

Fig. 3

Kinetics of Opacification of LpA-I/A-II and LpA-I. The rate constants for opacification of LpA-I (upper curve) and LpA-I/A-II (lower curve) were k = (28.73 ± 0.28) × 10⁻³ sec⁻¹ and (11.57 × ± 0.08) × 10⁻³ sec⁻¹, respectively. Data (gray symbols); fitted curve (−).

Fig. 4

Analysis of the effects rSOF on apo-enriched HDL. HDL (0.5 mg/mL) was incubated for 30 minutes at 25 °C with LF apos A-I or A-II and then treated with rSOF (1 µg/mL) for 18 h at 37 °C. The reaction products were separated by SEC and fractions of major peaks were analyzed by immunoblotting. Pre rSOF (grey fill) and upper immunoblot panels; post rSOF (−) and lower immunoblot panels. A. HDL; B. HDL + 0.05 mg/mL apo A-I. C. HDL + 0.23 mg/mL apo A-I; D. HDL + 0.05 mg/mL apo A-II; E. HDL + 0.23 mg/mL apo A-II. The indicated fractions in A, D and E were immunoblotted for apo A-I. Fractions in B and C were immunoblotted for apo A-II. Addition of apoA-I was analyzed for its ability to dissociate ApoA-II from the HDL particle and vice versa.

Fig. 5

Kinetics of opacification of apo-enriched HDL. Opacification kinetics of HDL (0.5 mg/mL) treated with increasing concentrations of (A) apos A-I and (B) A-II. The concentrations of added apos were 0.0, 0.11, 0.23 and 0.67 mg/mL for curves a, b, c and d respectively. Grey points are raw data, black lines are fits to the data using the equation given in the Methods. C. Effect of increasing apo concentration on the rate constant for opacification. For 0.0 to 0.23 mg/mL added apo, the reduction in opacification rate was −7.2 × 10⁻³ sec⁻¹mg⁻¹ for apo A-II and −3.8 × 10⁻³ sec⁻¹mg⁻¹ for apo A-I. D. Maximum opacification of HDL in the presence of added apo A-I or A-II relative to control HDL. For C and D, open circles are addition of apo A-I, closed circles are apo A-II.

Fig. 6

SEC analysis of the effects rSOF on HDL from WT and transgenic mice expressing human apos A-I and A-II. rSOF (1 µg/mL) was incubated with HDL (0.25 mg/mL) for 18 h at 37 °C and analyzed by SEC. Pre rSOF (gray-filled curves); post rSOF (−). A. WT; dashed line is SEC trace of rSOF + human HDL from (Figure 2). B. Apo A-I (+); C. Apo A-II (Lo); D. Apo A-II (Hi).

Fig. 7

Kinetics of opacification of HDL from WT and transgenic mice expressing human apos A-I and A-II. A. Turbidimetric kinetics for mouse HDL (0.5 mg/mL) treated with 1 µg/mL of rSOF as labeled. B. Opacification rate constants for mouse HDL treated with rSOF. C. Maximum opacification relative to WT HDL (= 100%).

Fig. 8

Summary of Rate Data. Arrows denote samples rich in human apo A-II. Data compiled from kinetic curves in Figures 3, 5, and 7.