Functional recombinant apolipoprotein A5 that is stable at high concentrations at physiological pH

APOA5 is a low-abundance exchangeable apolipoprotein that plays critical roles in human triglyceride (TG) metabolism. Indeed, aberrations in the plasma concentration or structure of APOA5 are linked to hypertriglyceridemia, hyperchylomicronemia, myocardial infarction risk, obesity, and coronary artery disease. While it has been successfully produced at low yield in bacteria, the resulting protein had limitations for structure-function studies due to its low solubility under physiological buffer conditions. We hypothesized that the yield and solubility of recombinant APOA5 could be increased by: i) engineering a fusion protein construct in a codon optimized expression vector, ii) optimizing an efficient refolding protocol, and iii) screening buffer systems at physiological pH. The result was a high-yield (25 mg/l) bacterial expression system that produces lipid-free APOA5 soluble at concentrations of up to 10 mg/ml at a pH of 7.8 in bicarbonate buffers. Physical characterization of lipid-free APOA5 indicated that it exists as an array of multimers in solution, and far UV circular dichroism analyses show differences in total α-helicity between acidic and neutral pH buffering conditions. The protein was functional in that it bound and emulsified multilamellar dimyristoyl-phosphatidylcholine vesicles and could inhibit postprandial plasma TG accumulation when injected into C57BL/6J mice orally gavaged with Intralipid.

Abstract 199: The Regulation of Intestinal apoC-III by Dietary Fat: Key Differences From Hepatic apoC-III Regulation and Implications for Lipoprotein Synthesis and Secretion

Apolipoprotein C-III (apoC-III) influences both plasma triglycerides and inflammation, and as such is a particularly important target for mediating cardiovascular disease (CVD). ApoC-III is an exchangeable apolipoprotein expressed in both liver and intestine; in humans, apoC-III levels in plasma are an independent predictor of CVD risk. ApoC-III is a potent regulator of plasma triglycerides through effects on intestinal lipid absorption, plasma triglyceride clearance, hepatic lipoprotein uptake, and VLDL secretion. ApoC-III also acts as a signaling molecule modulating vascular function. Hepatic apoC-III is regulated by insulin via the transcription factor, forkhead box protein O1 (FOXO1), and by glucose via carbohydrate-responsive element-binding protein (ChREBP). Though much is known about hepatic apoC-III, little is known about the regulation of intestinal apoC-III (including the primary stimulus for its expression). We have found that apoC-III is secreted from the intestine in response to dietary carbohydrate, and that a fish oil-enriched diet lowers intestinal apoC-III mRNA expression. Our findings suggest that ChREBP is the primary modulator of apoC-III expression in the intestine. While dietary fish oil lowers both hepatic and intestinal apoC-III mRNA levels, it appears to do so through distinctive mechanisms. This is a potentially important difference since apoC-III is a key regulator of post-prandial plasma lipid levels and inflammation.

Lipopolysaccharide binding of an exchangeable apolipoprotein, apolipophorin III, from Galleria mellonella

A new role of apolipophorin III (apoLp-III) as an immune activator has emerged recently. To gain insight into this novel function, the interaction of apoLp-III with lipopoly-saccharide (LPS) was investigated. ApoLp-III fromGalleria mellonellawas incubated with LPS fromEscherichia coliO55:B5, and analyzed by non-denaturing polyacrylamide gel electrophoresis (PAGE). Protein staining showed that apoLp-III mobility was significantly reduced. In addition, silver and LPS fluorescent staining demonstrated that LPS mobility was increased upon incubation with apoLp-III. This result suggests association of apoLp-III with LPS. Sodium dodecyl sulfate (SDS) PAGE analysis showed decreased apoLp-III mobility upon LPS addition, indicative of LPS apoLp-III interaction in the presence of SDS. The unique tyrosine residue that resides in apoLp-III was used to provide additional evidence for LPS binding interaction. In the absence of LPS, apoLp-III tyrosine fluorescence was relatively low. However, LPS addition resulted in a progressive increase in the fluorescence intensity, indicating tertiary rearrangement in the environment of tyrosine 142 upon LPS interaction. Other well-characterized apoLp-IIIs were also examined for LPS binding.Manduca sexta,Bombyx moriandLocusta migratoriaapoLp-III were all able to interact with LPS. The ability of apoLp-III to form complexes with LPS supports the proposed role of apoLp-III in innate immunity.