Three arginine residues in apolipoprotein A-I are critical for activation of lecithin:cholesterol acyltransferase

Abstract

Previous studies have suggested that the helical repeat formed by residues 143-164 of apolipoprotein A-I (apoA-I) contributes to lecithin:cholesterol acyltransferase (LCAT) activation. To identify specific polar residues involved in this process, we examined residue conservation and topology of apoA-I from all known species. We observed that the hydrophobic/hydrophilic interface of helix 143-164 contains a cluster of three strictly conserved arginine residues (R149, R153, and R160), and that these residues create the only significant positive electrostatic potential around apoA-I. To test the importance of R149, R153, and R160 in LCAT activation, we generated a series of mutant proteins. These had fluorescence emission, secondary structure, and lipid-binding properties comparable to those of wild-type apoA-I. Mutation of conserved residues R149, R153, and R160 drastically decreased LCAT activity on lipidprotein complexes, whereas control mutations (E146Q, D150N, D157N, R171Q, and A175R) did not decrease LCAT activity by more than 55%. The markedly decreased activities of mutants R149, R153, and R160 resulted from a decrease in the maximal reaction velocity V max because the apparent Michaelis-Menten constant K m values were similar for the mutant and wild-type apoA-I proteins. These data suggest that R149, R153, and R160 participate in apoA-Imediated activation of LCAT, and support the "belt" model for discoidal rHDL. In this model, residues R149, R153, and R160 do not form salt bridges with the antiparallel apoA-I monomer, but instead are pointing toward the surface of the disc, enabling interactions with LCAT.

Πρωτεΐνες Που Ρυθμίζουν Τα Επίπεδα Και Τις Λειτουργίες Της HDL

“…In our case residues R149, R153 and R160 were reported to create a positive electrostatic potential around apoA-I. Mutations in these residues reduced drastically the ability of rHDL particles containing these apoA-I mutants to activate LCAT in vitro (221). Based on the "belt" model for discoidal rHDL, these residues are located on the hydrophilic face of the apoA-I helices and do not form intramolecular salt bridges in the antiparallel apoA-I dimer that covers the fatty acyl chain of the discoidal particle.…”

“…Co-expression of the apoA-I mutant and human LCAT increased and shifted the HDL cholesterol peak towards lower densities, created normal α-HDL subpopulations and generated spherical HDL particles. The apoA-I [218][219][220][221][222] and apoA-I[225-230] mutants had 20% and 31% respectively to promote ABCA1-mediated cholesterol efflux as compared to WT apoA-I. Both mutants had ~65% of normal capacity to activate LCAT in vitro.…”

“…Adenoviruses expressing the WT apoA-I and the apoA-I mutants, apoA-I [218][219][220][221][222], apoA-I[225-230] and apoA-I [218][219][220][221][222][223][224][225][226][227][228][229][230] have been generated as described previously (542). Briefly, the apoA-I mutants were generated by site-directed mutagenesis using the primers described in Table 2.I and the method described in 2.2.1 of the Materials and Methods.…”

“…Briefly, the apoA-I mutants were generated by site-directed mutagenesis using the primers described in Table 2.I and the method described in 2.2.1 of the Materials and Methods. In the case of apoA-I [218][219][220][221][222][223][224][225][226][227][228][229][230] mutant the template was pCDNA3.1apoA-I [218][219][220][221][222]. The mutated apoA-I cDNA was cloned in the shuttle vector pAdTrack-CMV and the recombinant viruses were constructed using the Ad-Easy-1 system where the recombinant adenovirus construct is generated in bacteria BJ-5183 that harbor the required adenoviral genes (543).…”

“…Statistical significant differences in cholesterol, triglyceride, mRNA and protein levels at p<0.05 between mice expressing the WT apoA-I and the apoA-I mutants are indicated by a + symbol and between mice expressing GFP and the apoA-I transgenes are indicated by a ^ symbol. The measurements of Table 3.II A pertinent to the expression of the apoA-I [218][219][220][221][222] in apoA-I -/have been performed by Andreas Kateifides (542).…”