N-homocysteinylation of apolipoprotein A-I impairs the protein’s antioxidant ability but not its cholesterol efflux capacity

Abstract

A high homocysteine (Hcy) level is a risk factor for atherosclerosis. Hcy can be added to proteins through a process known as N-homocysteinylation. This is thought to be a potential cause of atherosclerosis induction. We previously reported that N-homocysteinylated apolipoprotein A-I (N-Hcy-apoA-I) was identified in normal human plasma. In this study, the effect of N-homocysteinylation on the functions of apoA-I was examined. A kinetic study using dimyristoyl phosphatidylcholine (DMPC) liposomes indicated that N-Hcy-apoA-I showed increased lipid-binding activity compared to wild-type apoA-I. Two reconstituted high-density lipoprotein (rHDL) particles of different sizes (approximately 8.2 nm and 7.6 nm in diameter) were produced by mixing apoA-I and 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC). However, an increased ratio of large to small particles was found in rHDL prepared with N-Hcy-apoA-I. The normal apoA-I antioxidant ability, estimated by the suppression of conjugated diene formation in low-density lipoprotein (LDL) induced by copper sulfate oxidation, was considerably impaired when using N-Hcy-apoA-I. Although N-Hcy-apoA-I functioned as an oxidant, no significant difference was observed in the cholesterol efflux capacity from THP-1 macrophages between wild-type apoA-I and N-Hcy-apoA-I. These results suggest that N-Hcy-apoA-I might be proatherogenic due to its oxidative behavior but not an attenuation of cholesterol efflux capacity.

Cholesterol Efflux Capacity of Apolipoprotein A-I Varies with the Extent of Differentiation and Foam Cell Formation of THP-1 Cells

“…Only a few studies have been reported about the relationship between Hcy and cholesterol efflux capacity [32, 33]. We previously reported that the cholesterol efflux capacity of apoA-I was not significantly reduced with N -homocysteinylation [15]. In the previous study the period of PMA stimulation was 3 days, indicating that the cells might be in the state of excessive differentiation to consider the possible effect of N -homocysteinylation.…”

“…Using these different states of cells, we examined the effect of N -homocysteinylation on cholesterol efflux capacity of apoA-I. N -Hcy apoA-I was detected as a more positively charged band than intact apoA-I (Figure 6) and was defined as a percentage to total apoA-I, as described previously [15]. The variation in cholesterol efflux capacities of both apoA-I and N -Hcy apoA-I using THP-1 cells treated with PMA in different time intervals indicates similar trends, as compared to the values that reached the peaks at day 2 and then gradually decreased (Figure 7(a)).…”

“…N -Homocysteinylation of apoA-I was performed according to the previously reported method with a slight modification [15]. The purified apoA-I (70  μ mol/L) was incubated with or without 10 mmol/L of HcyT (MP Biomedicals) at 37°C for 6 hours.…”

“…It is known that apoA-I, which plays a central role in cholesterol efflux capacity, is modified by various reactions such as oxidation, glycation, and homocysteinylation [11–15]. These modifications of apoA-I, presumed to lead to the development of atherosclerosis, have been the focal point of many studies investigating dysfunctionalities in cholesterol efflux.…”

A fast semi-quantitative LC–MS method for measurement of intact apolipoprotein A-I reveals novel proteoforms in serum

“…Some of the measured proteoforms of ApoA-I resulting from single modifications will likely prove to have pathobiological and diagnostic significance as further clinical studies are performed. Apart from the mentioned ApoA-I proteoforms we also have searched for possible singly or multiply modified proteoforms of ApoA-I unequivocally resulting from nitration, chlorination and N-homocysteinylation, described to occur in a number of amino acid residues of ApoA-I [27,28]. These modifications of ApoA-I have been described to negatively affect the potency of ApoA-I in supporting the RCT pathway or its anti-inflammatory action and could serve as future clinically useful markers in classifying the percentage of crippled ApoA-I occurring in a specific sample.…”