Abstract
The interaction of lecithin-cholesterol acyltransferase (LCAT) with apolipoprotein A-I (apoA-I) plays a critical role in high-density lipoprotein (HDL) maturation. We previously identified a highly solvent-exposed apoA-I loop domain (Leu(159)-Leu(170)) in nascent HDL, the so-called "solar flare" (SF) region, and proposed that it serves as an LCAT docking site (Wu, Z., Wagner, M. A., Zheng, L., Parks, J. S., Shy, J. M., 3rd, Smith, J. D., Gogonea, V., and Hazen, S. L. (2007) Nat. Struct. Mol. Biol. 14, 861-868). The stability and role of the SF domain of apoA-I in supporting HDL binding and activation of LCAT are debated. Here we show by site-directed mutagenesis that multiple residues within the SF region (Pro(165), Tyr(166), Ser(167), and Asp(168)) of apoA-I are critical for both LCAT binding to HDL and LCAT catalytic efficiency. The critical role for possible hydrogen bond interaction at apoA-I Tyr(166) was further supported using reconstituted HDL generated from apoA-I mutants (Tyr(166) → Glu or Asn), which showed preservation in both LCAT binding affinity and catalytic efficiency. Moreover, the in vivo functional significance of NO2-Tyr(166)-apoA-I, a specific post-translational modification on apoA-I that is abundant within human atherosclerotic plaque, was further investigated by using the recombinant protein generated from E. coli containing a mutated orthogonal tRNA synthetase/tRNACUA pair enabling site-specific insertion of the unnatural amino acid into apoA-I. NO2-Tyr(166)-apoA-I, after subcutaneous injection into hLCAT(Tg/Tg), apoA-I(-/-) mice, showed impaired LCAT activation in vivo, with significant reduction in HDL cholesteryl ester formation. The present results thus identify multiple structural features within the solvent-exposed SF region of apoA-I of nascent HDL essential for optimal LCAT binding and catalytic efficiency.
Highlights
Tion in LCAT activity have been reported
The presumed functions proposed for the apoA-I residues are speculative and are based on a combination of both site-specific mutagenesis/functional studies and structural models of apoA-I reported in nHDL
In one previous study of combined apoA-I mutants replacing a pair of charged amino acids, both Arg[160] and His[162], with non-polar amino acids (R160V/H162A) produced an apoA-I form that showed a loss of LCAT activation similar to that of R160L and H162Q
Summary
Genetic studies confirm critical roles for both LCAT and apoA-I in lipid transport (8). A functional role for this region as an LCAT interaction site was suggested based upon additional hydrogen-deuterium exchange mass spectrometry results, which showed reductions in deuterium incorporation rate within the overlapping apoA-I peptide region Leu159-Leu[170] of nascent HDL in the presence versus absence of LCAT (24) We called this a “solar flare” (SF) region of apoA-I because it presumably represented a highly solvent-exposed protruding loop on the anti-parallel apoA-I chain in a nascent HDL particle (24). We developed recombinant apoA-I with site-specific 3-nitrotyrosine incorporation only at position 166 of apoA-I using an evolved orthogonal nitro-Tyr-aminoacyl-tRNA synthetase/ tRNACUA pair This enabled in vitro studies with recombinant human apoA-I incorporating this unnatural amino acid exclusively at position 166 to evaluate functional effects of NO2Tyr166-apoA-I within reconstituted HDL and illustrated a reduced capacity to activate LCAT in vitro (26); the impact of this post-translational modification on LCAT-mediated maturation of HDL in vivo has not yet been explored. We explore for the first time the in vivo functional impact of post-translational modification of apoA-I Tyr[166] through nitration, which is enriched in human atherosclerotic lesions, using a humanized mouse model of apoA-I/ LCAT interaction and infusion of endotoxin-free recombinant NO2-Tyr166-apoA-I
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