Distinct Proteomic Signatures in 16 HDL (High-Density Lipoprotein) Subspecies

Abstract

Objective- HDL (high-density lipoprotein) in plasma is a heterogeneous group of lipoproteins typically containing apo AI as the principal protein. Most HDLs contain additional proteins from a palate of nearly 100 HDL-associated polypeptides. We hypothesized that some of these proteins define distinct and stable apo AI HDL subspecies with unique proteomes that drive function and associations with disease. Approach and Results- We produced 17 plasma pools from 80 normolipidemic human participants (32 men, 48 women; aged 21-66 years). Using immunoaffinity isolation techniques, we isolated apo AI containing species from plasma and then used antibodies to 16 additional HDL protein components to isolate compositional subspecies. We characterized previously described HDL subspecies containing apo AII, apo CIII, and apo E; and 13 novel HDL subspecies defined by presence of apo AIV, apo CI, apo CII, apo J, α-1-antitrypsin, α-2-macroglobulin, plasminogen, fibrinogen, ceruloplasmin, haptoglobin, paraoxonase-1, apo LI, or complement C3. The novel species ranged in abundance from 1% to 18% of total plasma apo AI. Their concentrations were stable over time as demonstrated by intraclass correlations in repeated sampling from the same participants over 3 to 24 months (0.33-0.86; mean 0.62). Some proteomes of the subspecies relative to total HDL were strongly correlated, often among subspecies defined by similar functions: lipid metabolism, hemostasis, antioxidant, or anti-inflammatory. Permutation analysis showed that the proteomes of 12 of the 16 subspecies differed significantly from that of total HDL. Conclusions- Taken together, correlation and permutation analyses support speciation of HDL. Functional studies of these novel subspecies and determination of their relation to diseases may provide new avenues to understand the HDL system of lipoproteins.

Keywords: apolipoproteins; cardiovascular diseases; enzyme-linked immunosorbent assay; humans; lipoproteins, HDL; peptides; proteomics.

Figure 1.

The novel sandwich ELISA procedure, using apoA-I of HDL with and without apoC-III as an example

Figure 2.

The apoA-I concentration in plasma and in each HDL subspecies by race. White (n=10 pools, n=59 individuals), Black (n=4 pools, n=13 individuals), Hispanic (n=3 pools, 8 individuals). Each data point is the average of the pools (mean ± sd). To the right of each is the overall mean percentage of apoA-I associated with each HDL protein. ApoAII HDL = HDL containing apoA-II; apoCI HDL = HDL containing apoC-I; and so forth. PON1 = paraoxonase-1; A1AT = alpha-1-anti-trypsin; PLMG = plasminogen; FBG = fibrinogen; CoC3 = complement C3; HP = haptoglobin; CP = ceruloplasmin.

Figure 3.. Protease inhibition.

Proteomics of apoA-I HDL subspecies that are defined by proteins involved in protease inhibition. Data are mean of 3 pools representing 18 individuals with standard error bars. Missing bars indicate that protein was not detected in any of three replicates of that HDL subspecies. Graphs are in logarithmic scale. Proteins are expressed relative to the total HDL proteome with each protein’s spectral counts normalized by apoA-I spectral count within each pool. Enrichment relative to total HDL is defined as a mean value greater than 1 whose standard error range does not include 1, while depletion is defined as mean value less than 1 whose standard error range does not include 1.

Figure 4.. Hemostasis.

Proteomics of apoA-I HDL subspecies that are defined by proteins involved in hemostasis. See Figure 3 for experimental details and explanation.

Figure 5.. Lipid Metabolism.

Proteomics of apoA-I HDL subspecies that are defined by proteins involved in lipid metabolism. See Figure 3 for experimental details and explanation.

Figure 6.. Antioxidant.

Proteomics of apoA-I HDL subspecies that are defined by proteins involved in antioxidant functions. See Figure 3 for experimental details and explanation.

Figure 7.. Immune Function.

Proteomics of apoA-I HDL subspecies that are defined by proteins involved in immune functions (apoL-I) or the complement system (complement C3). See Figure 3 for experimental details and explanation.

Figure 8.. Network plot linking HDL subspecies.

Like-colored HDL subspecies were hypothesized to have similar functionality. Lines link subspecies with strong correlation (r>0.55, p<0.0004) with weight of line proportional to strength of correlation. Blue lines link subspecies that were hypothesized to have similar functionality. Red lines link subspecies that were not hypothesized to have similar functionality. Grey patches represent revised groupings of subspecies with similar purported functionality based on proteomic profile results.