Apolipoprotein A-V N-terminal Domain Lipid Interaction Properties in Vitro Explain the Hypertriglyceridemic Phenotype Associated with Natural Truncation Mutants

Kasuen Wong-Mauldin,Vincent Raussens,Trudy M Forte,Robert O Ryan

doi:10.1074/jbc.m109.040972

Kasuen Wong-Mauldin, Vincent Raussens + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m109.040972

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Abstract

The N-terminal 146 residues of apolipoprotein (apo) A-V adopt a helix bundle conformation in the absence of lipid. Because similarly sized truncation mutants in human subjects correlate with severe hypertriglyceridemia, the lipid binding properties of apoA-V(1-146) were studied. Upon incubation with phospholipid in vitro, apoA-V(1-146) forms reconstituted high density lipoproteins 15-17 nm in diameter. Far UV circular dichroism spectroscopy analyses of lipid-bound apoA-V(1-146) yielded an alpha-helix secondary structure content of 60%. Fourier transformed infrared spectroscopy analysis revealed that apoA-V(1-146) alpha-helix segments align perpendicular with respect to particle phospholipid fatty acyl chains. Fluorescence spectroscopy of single Trp variant apoA-V(1-146) indicates that lipid interaction is accompanied by a conformational change. The data are consistent with a model wherein apoA-V(1-146) alpha-helices circumscribe the perimeter of a disk-shaped bilayer. The ability of apoA-V(1-146) to solubilize dimyristoylphosphatidylcholine vesicles at a rate faster than full-length apoA-V suggests that N- and C-terminal interactions in the full-length protein modulate its lipid binding properties. Preferential association of apoA-V(1-146) with murine plasma HDL, but not with VLDL, suggests that particle size is a determinant of its lipoprotein binding specificity. It may be concluded that defective lipoprotein binding of truncated apoA-V contributes to the hypertriglyceridemia phenotype associated with truncation mutations in human subjects.

Highlights

Grant HL-073061. 1 Recipient of a Gates Millennium Scholarship. 2 Senior Research Associate for the National Fund for Scientific Research (Belgium). 3 To whom correspondence should be addressed: CHORI, 5700 Martin Luther in alternate lipid-free and lipid-bound states
Truncated apoA-V proteins in this size range have been reported in human subjects with severe hypertriglyceridemia (HTG) [9, 10]
Stock solutions of protein and lipid vesicles were prepared in 50 mM citrate, pH 3.0, 150 mM NaCl. 500 ␮g of DMPC was incubated in the absence or presence of 100 ␮g of apolipoprotein at 23 °C in a thermostatted cell holder

Summary

EXPERIMENTAL PROCEDURES

Site-directed Mutagenesis and Recombinant Protein Expression—Recombinant human apoA-V and CT truncation variants were produced in Escherichia coli and isolated as NOVEMBER 27, 2009 VOLUME 284 NUMBER 48. Protein (0.5 ␮g/␮l) was dissolved in 20 mM sodium phosphate, pH 7.4, 150 mM NaCl. The samples were excited at 295 nm, and emission was collected from 300 to 450 nm (slit width, 2.0 nm). For Trp fluorescence quenching studies DMPC-bound apoA-V(1–146) samples (200 ␮g of protein in 400 ␮l of total volume) were excited at 295 nm, and emission was monitored at their ␭max (slit width, 2.0 nm). Stock solutions of protein and lipid vesicles were prepared in 50 mM citrate, pH 3.0, 150 mM NaCl. 500 ␮g of DMPC was incubated in the absence or presence of 100 ␮g of apolipoprotein (total volume, 400 ␮l) at 23 °C in a thermostatted cell holder. Isolated VLDL (d Ͻ 1.006 g/ml) or HDL (d ϭ 1.063–1.21 g/ml) was incubated with recombinant apoA-V(1–146) (weight ratio, 10:1, lipoprotein protein:apoA-V(1–146)) for 1 h at 22 °C in Tris-buffered saline. The film was developed using a Kodak M35A X-OMAT processor

RESULTS

Nonpolar face

Ksvb KI Acrylamide

DISCUSSION