Secondary Structure Changes in ApoA-I Milano (R173C) Are Not Accompanied by a Decrease in Protein Stability or Solubility

Abstract

Apolipoprotein A-I (apoA-I) is the main protein of high-density lipoprotein (HDL) and a principal mediator of the reverse cholesterol transfer pathway. Variants of apoA-I have been shown to be associated with hereditary amyloidosis. We previously characterized the G26R and L178H variants that both possess decreased stability and increased fibril formation propensity. Here we investigate the Milano variant of apoAI (R173C; apoAI-M), which despite association with low plasma levels of HDL leads to low prevalence of cardiovascular disease in carriers of this mutation. The R173C substitution is located to a region (residues 170 to 178) that contains several fibrillogenic apoA-I variants, including the L178H variant, and therefore we investigated a potential fibrillogenic property of the apoAI-M protein. Despite the fact that apoAI-M shared several features with the L178H variant regarding increased helical content and low degree of ThT binding during prolonged incubation in physiological buffer, our electron microscopy analysis revealed no formation of fibrils. These results suggest that mutations inducing secondary structural changes may be beneficial in cases where fibril formation does not occur.

Dual Actions of Apolipoprotein A-I on Glucose-Stimulated Insulin Secretion and Insulin-Independent Peripheral Tissue Glucose Uptake Lead to Increased Heart and Skeletal Muscle Glucose Disposal

“…Expression and Purification of Recombinant Human ApoA-I Human apoA-I containing a His tag at the N terminus was expressed in bacteria and purified using immobilized metal affinity chromatography followed by tobacco etch virus protease treatment to remove the His tag, as previously described (10,11). ApoA-I proteins were produced either in-house or at the Lund University Protein Production Platform (LP3).…”

Plasma stem cell factor levels are associated with risk of cardiovascular disease and death

“…Copperoxidized LDL and Apo AI were prepared as previously described [23][24][25]. The leucocytes were mixed with equal volume of PBS before overlaid over 15 mL Ficoll-Paque (GE Healthcare).…”

“…Before stimulation, hCaSMC and HUVEC cells were starved in serum-free medium for 24 h. Cells were then stimulated for 24 h at 37°C in 5% CO 2 with either glucose (5-35 mmol L À1 ), ApoA1 (10-50 lg mL À1 ), copperoxidized low-density lipoprotein (LDL) (10-50 lg mL À1 ), soluble Fas ligand (2.5 lg mL À1 ), TNF (10 ng mL À1 ) or IL1b (10 ng mL À1 ; Peprotech, Rocky Hill, NJ, USA) in complete RPMI. Copperoxidized LDL and Apo AI were prepared as previously described [23][24][25]. To assess the SCF secretion in all cultures, medium was analysed using SCF ELISA (BioLegend) according to the company's instructions.…”

Anti‐ApoA‐I IgG antibodies are not associated with carotid artery disease progression and first‐time cardiovascular events in middle‐aged individuals

“…To measure IgG antibodies against ApoA‐I in human serum, we used recombinant human ApoA‐I as capture antigen in an ELISA assay. Human ApoA‐I containing a His‐tag at the N‐terminus was expressed in bacteria and purified using immobilized metal affinity chromatography followed by tobacco etch virus protease treatment to remove the His‐tag as previously described . Protein purity was analysed by SDS‐PAGE, and the concentration was determined using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), which was based on the extension coefficient (32 490 L mol −1 cm −1 ) and molecular weight (28 279 Da) of ApoA‐I.…”