Insights into the C-terminal domain of apolipoprotein E from chimera studies with apolipophorin III.

Apolipoprotein E3 (apoE) is a critical cholesterol transport protein in humans and is composed of two domains: a well characterized N-terminal (NT) domain that harbors the low-density lipoprotein LDL receptor, and a less understood C-terminal (CT) domain that is the site of protein oligomerization and initiation of lipid binding. To better understand the domain structure of apoE, the CT domain was fused to apolipophorin III (apoLp-III), a single-domain, monomeric apolipoprotein of insect origin, to yield a chimeric protein, apoLp-III/CT-apoE. Recombinant apoLp-III/CT-apoE maintained an overall helical content similar to that of the parent proteins, while chemical induced unfolding studies indicated that its structural integrity was not compromised. Analysis using 1-anilinonaphthalene-8-sulfonic acid (ANS), a sensitive fluorescent indicator of exposed hydrophobic sites and protein folding, demonstrated that whereas apoLp-III provided few ANS binding sites, apoLp-III/CT-apoE harbored an abundance of ANS binding sites. Thus, this indicated tertiary structure formation in CT-apoE when part of the chimera. Size-exclusion chromatography and chemical crosslinking analysis demonstrated that while apoLp-III is monomeric, the chimeric protein formed large oligomeric complexes, similar to native apoE3. Compared to apoLp-III, the chimera showed a two-fold enhancement in phospholipid vesicle solubilization rates and a significantly improved ability to bind to lipolyzed low-density lipoprotein, preventing the onset of lipoprotein aggregation at concentrations comparable to that of parent CT-apoE. These results confirm that high lipid binding and self-association sites are located in the CT domain of apoE, and that these properties can be transferred to an unrelated apolipoprotein, demonstrating that these properties operate independently from the NT domain.

Fragments of Locusta migratoria apoLp-III provide insight into lipid binding.

Apolipophorin III (apoLp-III) from Locusta migratoria is an exchangeable apolipoprotein with a critical role in lipid transport in insects. The protein is composed of a bundle of five amphipathic α-helices which undergo a large conformational change upon lipid binding. To better understand the apoLp-III lipid binding interaction, the protein was cleaved by cyanogen bromide upon introduction of a S92M mutation, generating an N-terminal fragment corresponding to the first three helices (NTH1-3) and a C-terminal fragment of the last two helices (CTH4-5). MALDI-TOF analysis of the HPLC purified fragments provided masses of 9863.8 Da for NTH1-3 and 7497.0 Da for CTH4-5 demonstrating that the intended fragments were obtained. Circular dichroism spectra revealed a decrease in helical content from 82% for the intact protein to 57% for NTH1-3 and 41% for CTH4-5. The fragments adopted considerably higher α-helical structure in the presence of trifluoroethanol or phospholipids. Equimolar mixing of the two fragments did not result in changes in helical content or tryptophan fluorescence, indicating recombination into the native protein fold did not occur. The rate of protein induced dimyristoylphosphatidylcholine vesicle solubilization increased 15-fold for NTH1-3 and 100-fold for CTH4-5 compared to the intact protein. Despite the high activity in phospholipid vesicle interaction, CTH4-5 did not protect phospholipase-treated low-density lipoprotein from aggregation. In contrast, NTH1-3 provided protection to lipoprotein aggregation similar to the intact protein, indicating that specific amino acid residues in this part of apoLp-III are essential for lipoprotein binding interaction.

Expression of the C-terminal domain of human apolipoprotein A-I using a chimeric apolipoprotein.

Human apolipoprotein A-I (apoA-I) is the most abundant protein in high-density lipoprotein, an anti-atherogenic lipid-protein complex responsible for reverse cholesterol transport. The protein is composed of an N-terminal helix bundle domain, and a small C-terminal (CT) domain. To facilitate study of CT-apoA-I, a novel strategy was employed to produce this small domain in a bacterial expression system. A protein construct was designed of insect apolipophorin III (apoLp-III) and residues 179-243 of apoA-I, with a unique methionine residue positioned between the two proteins and an N-terminal His-tag to facilitate purification. The chimera was expressed in E. coli, purified by Ni-affinity chromatography, and cleaved by cyanogen bromide. SDS-PAGE revealed the presence of three proteins with masses of 7 kDa (CT-apoA-I), 18 kDa (apoLp-III), and a minor 26 kDa band of uncleaved chimera. The digest was reloaded on the Ni-affinity column to bind apoLp-III and uncleaved chimera, while CT-apoA-I was washed from the column and collected. Alternatively, CT-apoA-I was isolated from the digest by reversed-phase HPLC. CT-apoA-I was α-helical, highly effective in solubilizing phospholipid vesicles and disaggregating LPS micelles. However, CT-apoA-I was less active compared to full-length apoA-I in protecting lipolyzed low density lipoproteins from aggregating, and disrupting phosphatidylglycerol bilayer vesicles. Thus the novel expression system produced mg quantities of functional CT-apoA-I, facilitating structural and functional studies of this critical domain of apoA-I.

Transfer of C-terminal residues of human apolipoprotein A-I to insect apolipophorin III creates a two-domain chimeric protein with enhanced lipid binding activity.

Apolipophorin III (apoLp-III) is an insect apolipoprotein (18kDa) that comprises a single five-helix bundle domain. In contrast, human apolipoprotein A-I (apoA-I) is a 28kDa two-domain protein: an α-helical N-terminal domain (residues 1-189) and a less structured C-terminal domain (residues 190-243). To better understand the apolipoprotein domain organization, a novel chimeric protein was engineered by attaching residues 179 to 243 of apoA-I to the C-terminal end of apoLp-III. The apoLp-III/apoA-I chimera was successfully expressed and purified in E. coli. Western blot analysis and mass spectrometry confirmed the presence of the C-terminal domain of apoA-I within the chimera. While parent apoLp-III did not self-associate, the chimera formed oligomers similar to apoA-I. The chimera displayed a lower α-helical content, but the stability remained similar compared to apoLp-III, consistent with the addition of a less structured domain. The chimera was able to solubilize phospholipid vesicles at a significantly higher rate compared to apoLp-III, approaching that of apoA-I. The chimera was more effective in protecting phospholipase C-treated low density lipoprotein from aggregation compared to apoLp-III. In addition, binding interaction of the chimera with phosphatidylglycerol vesicles and lipopolysaccharides was considerably improved compared to apoLp-III. Thus, addition of the C-terminal domain of apoA-I to apoLp-III created a two-domain protein, with self-association, lipid and lipopolysaccharide binding properties similar to apoA-I. The apoA-I like behavior of the chimera indicate that these properties are independent from residues residing in the N-terminal domain of apoA-I, and that they can be transferred from apoA-I to apoLp-III.

Backbone and side chain chemical shift assignments of apolipophorin III from Galleria mellonella.

Apolipophorin III, a 163 residue monomeric protein from the greater wax moth Galleria mellonella (abbreviated as apoLp-IIIGM), has roles in upregulating expression of antimicrobial proteins as well as binding and deforming bacterial membranes. Due to its similarity to vertebrate apolipoproteins there is interest in performing atomic resolution analysis of apoLp-IIIGM as part of an effort to better understand its mechanism of action in innate immunity. In the first step towards structural characterization of apoLp-IIIGM, 99 % of backbone and 88 % of side chain (1)H, (13)C and (15)N chemical shifts were assigned. TALOS+ analysis of the backbone resonances has predicted that the protein is composed of five long helices, which is consistent with the reported structures of apolipophorins from other insect species. The next stage in the characterization of apoLp-III from G. mellonella will be to utilize these resonance assignments in solving the solution structure of this protein.