Effects of superoxide on apolipoprotein A-I structure and function.

AIMS: Apolipoprotein A1 (ApoA-I) is the major component of HDL and play crucial roles in all steps of RTC. ApoA-I is susceptible to oxidation which contribute to reduce its cardioprotective effects. It has been reported that hypoclorite can affect ApoA-I structure and the ability of this lipoprotein to promote ABCA1-dependent cholesterol efflux. The effects of Superoxide, a potent oxidant produced in blood vessel, on ApoA-I structure and function have never been reported. The aims of this work was to asses if the ApoA-I function to stimulate the cholesterol esterifying enzime (LCAT) was impaired by incubation with superoxide. Furthermore the Apo-A-I funtion to bind Haptoglobin, (Hpt) a plasma glicoprotein, was tested after incubation by superoxide. METHODS: The superoxide production was monitored by measuring the absorbance at 734 nm of a reaction mixture containing several concentration (10 50 100 µM) of KO2 and ABTS 450 µM and kept at 31°C. The binding experiments were performed by ELISA with coated ApoA-I, several concentration of KO2 (10 50 100 µM) was added, after Hpt in solution and anti-Hpt antibodies was used for detection of bound Hpt. Standard LCAT assay were carried out as published ¹.ApoA-I-containing liposomes was incubated for 1h at 37°C in the presence and absence of superoxide. RESULTS: After affinity chromatography, in ELISA experiments, Hpt from patients binds less efficiently Hb than Hpt from controls. In particular, significant differences were found for samples with 0.1, 0.2 or 0.3µM Hpt (P<0.001). Furthermore, when the same proteins were analyzed for their ability to influence LCAT activity, there are evidence that Hpt from healthy donor bind much better ApoA1 then psoriatic Hpt. The differences between the two Hpt pools in inhibiting LCAT suggest that the protein purified from patients has not the same structure of normal protein. This difference might depend on lower ability of Hpt from patients to limit ApoA1 stimulation of stimulating LCAT activity. DISCUSSION: Here we provide evidence that plasma, in psoriasis, contains different Hpt forms from those circulating in normal condition which might impair the protein function in Hb binding and LCAT regulation. Besides it is possible that Hpt, in psoriasis, exposes different glycan code than normal condition that alters structure and function of it. Nevertheless further analysis of psoriatic Hpt glycosilation is required to support this hypothesis. 1. Chen CH et al., (1982) J Lipid Res. 23, 680-691.