Alteration in Apolipoprotein A-I 22-Mer Repeat Order Results in a Decrease in Lecithin:Cholesterol Acyltransferase Reactivity

Abstract

Apolipoprotein A-I contains eight 22-amino acid and two 11-amino acid tandem repeats that comprise 80% of the mature protein. These repeating units are believed to be the basic motif responsible for lipid binding and lecithin:cholesterol acyltransferase (LCAT) activation. Computer analysis indicates that despite a fairly high degree of compositional similarity among the tandem repeats, significant differences in hydrophobic and amphipathic character exist. Our previous studies demonstrated that deletion of repeat 6 (143-164) or repeat 7 (165-186) resulted in a 98 -99% reduction of LCAT activation as compared with wild-type apoA-I. To determine the effects of substituting one of these repeats with a more hydrophobic repeat we constructed a mutant apoA-I protein in which residues 143-164 (repeat 6) were replaced with repeat 10 (residues 220 -241). The cloned mutant protein, 10F6 apoA-I, was expressed and purified from an Sf-9 cell baculoviral system and then analyzed using a number of biophysical and biochemical techniques. Recombinant complexes prepared at a 100: 5:1 molar ratio of L-␣-dimyristoylphosphatidylcholine: cholesterol:wild-type or 10F6 apoA-I showed a doublet corresponding to Stokes diameters of 114 and 108 Å on nondenaturing 4 -30% polyacrylamide gel electrophoresis. L-␣-Dimyristoylphosphatidylcholine 10F6 apoA-I complexes had a 5-6-fold lower apparent V max /apparent K m as compared with wild-type apoA-I containing particles. As expected, monoclonal antibody epitope mapping of the lipid-free and lipid-bound 10F6 apoA-I confirmed that a domain expressed between residues 143 and 165 normally found in wild-type apoA-I was absent. The region between residues 119 and 144 in 10F6 apoA-I showed a marked reduction in monoclonal antibody binding capacity. Therefore, we speculate that the 5-6-fold lower LCAT reactivity in 10F6 compared with wildtype apoA-I recombinant particles results from increased stabilization within the 121-165 amino acid domain due to more stable apoprotein helix phospholipid interactions as well as from conformational alterations among adjacent amphipathic helix repeats.

Structural and functional properties of V156K and A158E mutants of apolipoprotein A-I in the lipid-free and lipid-bound states

“…Recent reports have identified the helix 6 domain, spanning amino acids 143-164 (24,25), and the C-terminal domain as important to LCAT activation (26,27). V156K-rHDL and A158E-rHDL particles served as poor substrates for LCAT activity, evidencing Ͻ 2% of the activity of WT-rHDL.…”

Crystal Structure of C-terminal Truncated Apolipoprotein A-I Reveals the Assembly of High Density Lipoprotein (HDL) by Dimerization

“…Lys 23 and Trp 50 form the C -cation interaction that holds the extended B section of H1 toward the helix bundle as shown in Fig. 2.…”

Quality control in the apoA-I secretory pathway

“…PCR primers (CMV 5 Ј , 5 Ј -GCCTGCAGTCCCCCACGGCCCTT-3 Ј ; CMV 3 Ј , 5 Ј -GCGGATCCCACTTTGGAAACG-3 Ј ) containing embedded Pst I and Bam HI restriction sites, respectively, were used in the preparation of all clones. Introduction of apoA-I mutations was carried out as described previously (29)(30)(31)(32). Constructs lacking the signal peptide sequence were made from human and mutant forms of the human apoA-I cDNA using the 5 Ј primer 5 Ј -GCCTGCAGCGGCCCTTCAGGCGGCATTTCTGGCAG-3 Ј and the same CMV 3 Ј primer listed above.…”

“…ApoA-I helix 6, one of seven proline-punctuated 22 amino acid helices in apoA-I, plays an essential role in determining its lipid-bound conformation (19,23,26,29,31,32). Transgenic mice expressing a helix 6 deletion mutant (⌬6 apoA-I) have remarkably low concentrations of mature HDL.…”