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A Systematic Investigation of Structure/Function Requirements for the Apolipoprotein A-I/Lecithin Cholesterol Acyltransferase Interaction Loop of High-density Lipoprotein Public Deposited

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  • The interaction of lecithin-cholesterol acyltransferase (LCAT) with apolipoprotein A-I (apoA-I) plays a critical role in highdensity lipoprotein (HDL) maturation.Wepreviously identified a highly solvent-exposed apoA-I loop domain (Leu¹⁵⁹–Leu¹⁷⁰) 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 (Pro¹⁶⁵, Tyr¹⁶⁶, Ser¹⁶⁷, and Asp¹⁶⁸) 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 Tyr¹⁶⁶ was further supported using reconstituted HDL generated from apoA-I mutants (Tyr¹⁶⁶→Glu or Asn), which showed preservation in both LCAT binding affinity and catalytic efficiency. Moreover, the in vivo functional significance of NO₂-Tyr¹⁶⁶-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-Tyr¹⁶⁶-apoA-I, after subcutaneous injection into hLCATᵀᵍ/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.
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  • Gu, X., Wu, Z., Huang, Y., Wagner, M. A., Baleanu-Gogonea, C., Mehl, R. A., ... & Hazen, S. L. (2016). A systematic investigation of structure/function requirements for the apolipoprotein AI-lecithin cholesterol acyltransferase interaction loop of HDL. Journal of Biological Chemistry, 291(12), 6386-6395. doi:10.1074/jbc.M115.696088
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  • 291
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  • 12
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  • This work was supported by National Institutes of Health Grants P01 HL076491 and R01 HL119962 (to J. S. P.). S. L. H. is named as co-inventor on pending patents held by the Cleveland Clinic relating to cardiovascular and inflammation diagnostics. S. L. H. reports having been paid as a consultant for the following companies: Esperion and P&G. S. L. H. reports receiving research funds from Abbott, Astra Zeneca, P&G, Pfizer Inc., Roche, and Takeda. S. L. H. Hazen reports having the right to receive royalty payments for inventions or discoveries related to cardiovascular diagnostics or therapeutics from the following companies: Cleveland Heart Lab, Siemens, Esperion, Frantz Biomarkers, LLC.
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