Shotgun proteomics implicates protease inhibition and complement activation in the antiinflammatory properties of HDL

Abstract

HDL lowers the risk for atherosclerotic cardiovascular disease by promoting cholesterol efflux from macrophage foam cells. However, other antiatherosclerotic properties of HDL are poorly understood. To test the hypothesis that the lipoprotein carries proteins that might have novel cardioprotective activities, we used shotgun proteomics to investigate the composition of HDL isolated from healthy subjects and subjects with coronary artery disease (CAD). Unexpectedly, our analytical strategy identified multiple complement-regulatory proteins and a diverse array of distinct serpins with serine-type endopeptidase inhibitor activity. Many acute-phase response proteins were also detected, supporting the proposal that HDL is of central importance in inflammation. Mass spectrometry and biochemical analyses demonstrated that HDL3 from subjects with CAD was selectively enriched in apoE, raising the possibility that HDL carries a unique cargo of proteins in humans with clinically significant cardiovascular disease. Collectively, our observations suggest that HDL plays previously unsuspected roles in regulating the complement system and protecting tissue from proteolysis and that the protein cargo of HDL contributes to its antiinflammatory and antiatherogenic properties.

Figure 1. Global view of biological processes and molecular functions of HDL proteins.

Proteins in total HDL and HDL₃ were identified using LC-ESI-MS/MS (Table 2). Proteins detected in HDL were associated with biological functions using GO process annotations. This approach demonstrated significant overrepresentation of proteins involved in several categories, including lipid metabolism (P = 2 × 10^–27), the acute-phase response (P = 1 × 10^–18), protease inhibitor activity (P = 2 × 10^–6), and complement regulation (P = 5 × 10^–5). apoH, β-2-glycoprotein I; AGT, angiotensinogen; AHSG, α-2-HS-glycoprotein; AMP, bikunin; FGA, fibrinogen; HRP, haptoglobin-related protein; HPX, hemopexin; ITIH4, inter-α-trypsin inhibitor heavy chain H4; KNG1, kininogen-1; LCAT, lecithin-cholesterol acyltransferase; ORM2, α-1-acid glycoprotein 2; PLTP, phospholipid transfer protein; RBP4, retinol binding protein; SERA1, α-1-antitrypsin; SERF1, serpin peptidase inhibitor (clade F, member 1); SERF2, α-2-antiplasmin; TF, transferrin; TTR, transthyretin; VTN, vitronectin.

Figure 2. Mass spectrometric and immunological detection of C3 in HDL₃.

HDL₃ or apoA-I complexes were isolated from plasma of control subjects. In parallel studies, plasma was passed over columns containing albumin or nonspecific immunoglobulin covalently cross-linked to beads. Isolated proteins were separated under denaturing conditions by gel electrophoresis, transferred to nitrocellulose, and analyzed through immunoblotting. (A and B) MS/MS spectra of 2 peptides unique to C3 in HDL₃. (C) Sequence of the C3dg domain of human complement C3. The 3 tryptic digest peptides detected selectively in HDL₃ of CAD subjects are underlined. (D) Detection of proteins reactive with antibody to C3. Lane 1: plasma. Lanes 2 and 3: eluate from control columns (albumin and IgG, respectively). Lane 4: apoA-I complexes. Lanes 5–7: 3 independent preparations of HDL₃ isolated from pooled human plasma.

Figure 3. Immunodetection of vitronectin (A), α-2-HS glycoprotein (B) and α-1–intertrypsin inhibitor (C) in HDL₃.

HDL₃ was isolated from the plasma of 3 subjects by ultracentrifugation. HDL-associated proteins were separated under denaturing conditions by gel electrophoresis, transferred to a PVDF membrane, and subjected to immunoblot analysis. P, plasma.

Figure 4. Peptide number and number of subjects with detectable peptides in HDL₃ isolated from control subjects and CAD subjects.

HDL₃ was isolated from the plasma of 6 control subjects and 7 subjects with established CAD (group 2; Table 1). HDL-associated proteins were subjected to LC-ESI-MS/MS analysis. All protein identifications required detection of at least 2 unique peptides from each protein from at least 2 individuals (Table 1). The P value was assessed by Student’s t test (peptide number) or Fisher’s exact test (subject number). *P < 0.05.

Figure 5. Relative abundance of proteins isolated from HDL₃ of control and CAD subjects.

HDL₃ was isolated from plasma of 6 control subjects and 7 subjects with established CAD. The relative abundance of proteins in the CAD subjects versus the control subjects was assessed by the peptide index, as described in Methods. *P < 0.05 by Student’s t test (peptide number) or Fisher’s exact test (subject number).

Figure 6. Reconstructed ion chromatograms of peptides derived from apoC-IV and apoA-I in HDL₃.

Tryptic digests of HDL₃ isolated from CAD subjects and control subjects were subjected to LC-ESI-MS/MS analysis as described in the legend to Figure 1. Reconstructed ion chromatograms of peptides (arrows) unique to apoC-IV and apoA-I were extracted from the full-scan mass survey spectra of each analysis. cps, counts per second.

Figure 7. Relationship between unique peptides and protein concentration in HDL and HDL₃.

The total number of unique peptides detected by MS in HDL and HDL₃ is as shown in Table 2. The protein concentrations of apoA-I (98 mg/dl), apoA-II (42 mg/dl), apoC-II (4.6 mg/dl), apoB (1.7 mg/dl), and transferrin (0.02 mg/dl) in HDL₃ and HDL isolated by ultracentrifugation are those reported by McPherson et al. (where HDL equals the sum of HDL₂ and HDL₃; ref. 41). The concentration of CETP in HDL₃ (0.1 mg/dl) was estimated as 50% that of plasma CETP (40). Linear regression analysis revealed a strong correlation (r² = 0.98 and 0.88) between protein concentration and unique peptides in HDL and HDL₃, respectively.

Figure 8. Quantification of HDL-cholesterol, apoA-I, apoA-II, and apoE in HDL₃ isolated from control and CAD subjects matched for plasma levels of triglycerides.

Plasma was obtained from 32 apparently healthy individuals and 32 individuals with established coronary artery disease (group 3; Table 1). HDL₃ was isolated from plasma by sequential ultracentrifugation. apoA-I, apoA-II, and apoE were quantified by ELISA. Results are normalized to the protein content of HDL₃. *P = 0.02 by a 2-tailed Student’s t test.