Quality control in the apoA-I secretory pathway

Abstract

From a total of 47 known apolipoprotein A-I (apoA-I) mutations, only 18 are linked to low plasma HDL apoA-I concentrations, and 78% of these map to apoA-I helices 6 and 7 (residues 143-186). Gene transfer and transgenic mouse studies have shown that several helix 6 apoA-I mutations have reduced hepatic HDL production. Our objective was to examine the impact of helix 6 modifications on intracellular biosynthetic processing and secretion of apoA-I. Cells were transfected with wild-type or mutant apoA-I, radiolabeled with [ 35 S]Met/Cys, and then placed in unlabeled medium for up to 4 h. Results show that Ͼ 90% of newly synthesized wild-type apoA-I was secreted by 60 min. Over the same length of time, only 20% of helix 6 deletion mutant ( ⌬ 6 apoA-I) was secreted, whereas 80% remained cell associated. Microscopic and biochemical studies revealed that cell-associated ⌬ 6 apoA-I was located predominantly within the cytoplasm as lipid-protein inclusions, whereas wild-type apoA-I was localized in the endoplasmic reticulum/Golgi. Results using other helix deletions or helix 6 substitution mutations indicated that only complete removal of helix 6 resulted in massive cytoplasmic accumulation. These data suggest that alterations in native apoA-I conformation can lead to aberrant trafficking and accumulation of apolipoprotein-phospholipid structures. Thus, conformation-dependent alterations in intracellular trafficking and turnover may underlie the reduced plasma HDL concentrations observed in individuals harboring deletion mutations within helix 6. -Bhat, S., M. Zabalawi, M. C. Willingham, G. S. Shelness, M. J. Thomas, and M. G. Sorci-Thomas. Quality control in the apoA-I secretory pathway: deletion of apoA-I helix 6 leads to the formation of cytosolic phospholipid inclusions.

The Conformation of Lipid-Free Human Apolipoprotein A-I in Solution

“…The coding sequence for wild-type and mutant apoA-I was cloned from the CMV5 vector (28) by Custom DNA Constructs (see Supplemental Material), amplified by PCR as described (29) and inserted into the pTYB11 vector (30). Expression and purification of the cysteine containing apoA-I mutants from inclusion bodies were conducted as previously described (28).…”

Dysfunctional HDL containing L159R ApoA-I leads to exacerbation of atherosclerosis in hyperlipidemic mice

“…Following weaning of founder mice, a mouse tail biopsy of approximately 1 cm in length was used for DNA analysis by polymerase chain reaction for the presence of the mutant apoA-I transgene. All mice were maintained in specific pathogen-free barrier facilities in Microisolater TM caging and were fully crossed into the LDLr receptor and apoA-I double knockout (LDLr −/− , apoA-I −/− or DKO) background for > 9 generations to obtain FIN-DKO mice [30, 34, 36]. Transgenic human apoA-I WT mice were obtained from Charles River Laboratories (Bar Harbor, ME) and were also crossed into the DKO background to obtain WT-DKO mice.…”

“…Total protein was determined by the method of Lowry [38]. To assess the VLDL/LDL, LDL/HDL and HDL fractions apolipoprotein composition, an equivalent of 10 µg protein of chow and diet pooled fractions were run on 12% SDS-PAGE as previously described [36]. …”

“…The pulse was carried out as previously described [36] except that 200 µCi of 35 S Met/Cys was added to each 60 mm plate containing 6 × 10 5 cells for 45 min. The chase was initiated by aspirating the labeling medium and then adding 1 mL of methionine-free DMEM-FBS.…”

“…After incubating overnight at 4°C on a rotating wheel, immune complexes were recovered by centrifugation at 12,000 rpm for 5 min. ApoA-I was eluted from the Protein G-Sepharose beads then separated by 12% SDS-PAGE as previously described [36, 43]. Gels containing 35 S-labeled apoA-I were dried and the radioactivity associated with each band determined using a FUJIFILM BAS-5000 Imaging Plate Scanner (Stamford, CT).…”