A Look at the Proteome of Lp(a) Isolated by Immunoprecipitation

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Introduction Lipoprotein [Lp)(a)] is composed of an apoB100 containing lipoprotein bound to a glycoprotein, apolipoprotein (a). Epidemiological, genome-wide association, and Mendelian randomization studies show high Lp(a) plasma levels as causal in the development of atherosclerotic cardiovascular disease (ASCVD). Plasma Lp(a) levels are 70-90% determined by the number of DNA-base repeats coding a highly polymorphic sequence of the LPA gene called Kringle IV Type 2 (KIV-2). Most individuals have two Lp(a) isoforms that differ in apo(a) size. The pathways regulating Lp(a) clearance and secretion in humans are not well understood and are currently being studied by our group using stable isotopes. The latter may be regulated by the different proteins carried by the Lp(a) particle. However, these studies have concentrated on small cohorts and subjects with high levels of plasma Lp(a). Our goal was to examine the effect of Lp(a) levels (low vs. high) on Lp(a) protein composition, and in addition examine if the proteins carried by large size apo(a) isoforms (associated with low Lp(a) levels) and low size apo(a) isoforms (associated with high Lp(a) levels) were similar. We hypothesize that the protein expression of isolated Lp(a) particles from subjects with high and low Lp(a) will be enriched with different proteins. Methods: We enrolled 13 healthy volunteers (7 male, 6 female), obtained fasting bloods and isolated plasma samples from EDTA-containing tubes. We measured plasma Lp(a) levels via a validated and standardized ELISA assay. Isoform sizes were obtained via SDS-page electrophoresis and standardized to a known KIV-2 repeat in 9 of the 13 subjects. We compared the proteomic profiles of isolated Lp(a) in 6 subjects with high Lp(a) levels (>75nmol/L) to that of 7 subjects with low Lp(a) levels (<40nmol/L). Lp(a) was isolated from 500uL of plasma by immunoprecipitation. The proteome of eluent was obtained by gel-based protein identification. The use of proteome software Scaffold4 enabled the detection of protein biomarkers that may be related to functional abnormalities involved in the pathophysiology of Lp(a).Results: We found similar protein expression in subjects with high and low levels of Lp(a), these proteins are listed in Table 1. Additionally, protein expression counts for apo(a) and apoB100 were higher in subjects with high Lp(a) levels when compared with subjects with low Lp(a) levels. Lp(a) levels are inversely correlated to isoform size, i.e. subjects with small isoforms have high levels and vice versa. We examined the protein composition of Lp(a) in a small sub-cohort of subjects expressing >24 repeats (low Lp(a) levels), n=6 and <24 (high Lp(a) levels), n=3. We found that subjects with >24 repeats (Table 2-A) presented with higher protein expression counts of apoE and Von W factor while subjects with <24 (Table 2 -B) presented with higher protein expression of complement C3. Conclusion: We did not find differences in the proteome of subjects with high and low levels of Lp(a). Although protein expressions are similar across Lp(a) levels, Lp(a) circulating isoforms may be linked to higher or lower levels of certain proteins. The latter may link isoforms to specific roles in Lp(a) disease.