Apolipoprotein A-I Helix 6 Negatively Charged Residues Attenuate Lecithin−Cholesterol Acyltransferase (LCAT) Reactivity

Abstract

Apolipoprotein A-I (apoA-I), the major protein in high density lipoprotein (HDL) regulates cholesterol homeostasis and is protective against atherosclerosis. An examination of the amino acid sequence of apoA-I among 21 species shows a high conservation of positively and negatively charged residues within helix 6, a domain responsible for regulating the rate of cholesterol esterification in plasma. These observations prompted an investigation to determine if charged residues in helix 6 maintain a structural conformation for protein-protein interaction with lecithin-cholesterol acyltransferase (LCAT) the enzyme for which apoA-I acts as a cofactor. Three apoA-I mutants were engineered; the first, (3)/(4) no negative apoA-I, eliminated 3 of the 4 negatively charged residues in helix 6, no negative apoA-I (NN apoA-I) eliminated all four negative charges, while all negative (AN apoA-I) doubled the negative charge. Reconstituted phospholipid-containing HDL (rHDL) of two discrete sizes and compositions were prepared and tested. Results showed that LCAT activation was largely influenced by both rHDL particle size and the net negative charge on helix 6. The 80 A diameter rHDL showed a 12-fold lower LCAT catalytic efficiency when compared to 96 A diameter rHDL, apparently resulting from an increased protein-protein interaction, at the expense of lipid-protein association on the 80 A rHDL. When mutant apoproteins were compared bound to the two different sized rHDL, a strong inverse correlation (r = 0.85) was found between LCAT catalytic efficiency and apoA-I helix 6 net negative charge. These results support the concept that highly conserved negatively charged residues in apoA-I helix 6 interact directly and attenuate LCAT activation, independent of the overall particle charge.

The impact of glycation on apolipoprotein A-I structure and its ability to activate lecithin:cholesterol acyltransferase

“…* p<0.05 compared with unmodified (A-I)rHDL. † p<0.05 compared with (A-I)rHDL incubated for 1 h with 3 mmol/l MG § p<0.01 compared with (A-I)rHDL incubated for 1 h with 3 mmol/l MG LCAT activation [46]. The present results suggest that this may also be the case when positively charged residues are modified.…”

Structure and Stability of Apolipoprotein A-I in Solution and in Discoidal High-Density Lipoprotein Probed by Double Charge Ablation and Deletion Mutation

“…However, even with this considerable amount of accumulated data, the structural details of the lipid-free and lipid-bound conformation are not fully defined and experimental results leading to definition of residues involved in stabilizing the protein lipid-free and lipid-bound conformations are not sufficient. So far, there have been no reported experimental data demonstrating that electrostatic interactions between specific basic and acidic residues located in two anti-parallel apoA-I molecules on the edge of rHDL are essential for the stabilization of the discs, as predicted by the double belt model (27).Studies of bioengineered mutant apoA-I forms remain efficient to obtain information that leads to detailed understanding of the conformation and structure-function relationships of apoA-I (5,(7)(8)(9)(10)(11)(12)(13)(14)(15)24,29,30). Our earlier study (29) using mutation to probe the conformation and stability of the C-terminus of apoA-I suggested the presence of stable helical structure in the region between residues 165 and 243 and a close proximity of the N-and C-termini in the lipid-free protein.…”

“…Studies of bioengineered mutant apoA-I forms remain efficient to obtain information that leads to detailed understanding of the conformation and structure-function relationships of apoA-I (5,(7)(8)(9)(10)(11)(12)(13)(14)(15)24,29,30). Our earlier study (29) using mutation to probe the conformation and stability of the C-terminus of apoA-I suggested the presence of stable helical structure in the region between residues 165 and 243 and a close proximity of the N-and C-termini in the lipid-free protein.…”

Ratio determination of plasma wild-type and L159R apoA-I using mass spectrometry: tools for studying apoA-IFin

“…In previous studies, the Escherichia coli IMPACT TM system from New England Biolabs has been used routinely in our laboratory for the expression and purification of wild-type apoA-I and numerous mutant forms of apoA-I (14,15). However, our initial attempts to express L159R apoA-I using this system were unsuccessful because of the extensive degradation of the target protein in the bacteria.…”

“…However, our initial attempts to express L159R apoA-I using this system were unsuccessful because of the extensive degradation of the target protein in the bacteria. This problem was overcome by inducing expression at a higher temperature (i.e., 30jC) rather than at the 15jC used previously for apoA-I mutant protein expression (14,15). Instead of degradation of the L159R apoA-I, expression at 30jC resulted in the formation of inclusion bodies within the bacteria.…”