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. 2010 Sep;51(9):2664-71.
doi: 10.1194/jlr.M007021. Epub 2010 Jun 16.

Distinctive structure and interfacial activity of the human apolipoprotein A-IV 347S isoprotein

Richard B Weinberg  1 , Victoria R Cook
Affiliations

Distinctive structure and interfacial activity of the human apolipoprotein A-IV 347S isoprotein

Richard B Weinberg et al. J Lipid Res. 2010 Sep.

Abstract

The T347S polymorphism in the human apolipoprotein (apo) A-IV gene is present at high frequencies among all the world's populations. Carriers of a 347S allele exhibit faster clearance of triglyceride-rich lipoproteins, greater adiposity, and increased risk for developing atherosclerosis, which suggests that this conservative amino acid substitution alters the structure of apo A-IV. Herein we have used spectroscopic and surface chemistry techniques to examine the structure, stability, and interfacial properties of the apo A-IV 347S isoprotein. Circular dichroism spectroscopy revealed that the 347S isoprotein has similar alpha-helical structure but lower thermodynamic stability than the 347T isoprotein. Fluorescence spectroscopy found that the 347S isoprotein exhibits an enhanced tyrosine emission and reduced tyrosine-->tryptophan energy transfer, and second derivative UV absorption spectra noted increased tyrosine exposure, suggesting that the 347S isoprotein adopts a looser tertiary conformation. Surface chemistry studies found that although the 347S isoprotein bound rapidly to the lipid interface, it has a lower interfacial exclusion pressure and lower elastic modulus than the 347T isoprotein. Together, these observations establish that the T347S substitution alters the conformation of apo A-IV and lowers its interfacial activity-changes that could account for the effect of this polymorphism on postprandial lipid metabolism.

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Figures

Fig. 1.
Fig. 1.
SDS-PAGE of purified apo A-IV 347S and apo A-IV 347T. Lane 1, apo A-IV 347T; lane 2, molecular weight standards; lane 3, apo A-IV 347S. Abbreviation: Apo, apolipoprotein.
Fig. 2.
Fig. 2.
CD spectra of apo A-IV 347S and apo A-IV 347T. The mean residue ellipticity of 3 μM solutions of apo A-IV 347S (▽) and apo A-IV 347T (○) was recorded at 25°C as a function of wavelength from 190 nm to 250 nm. Abbreviations: Apo, apolipoprotein; CD, circular dichroism.
Fig. 3.
Fig. 3.
Thermal denaturation of apo A-IV 347S and apo A-IV 347T. The fractional unfolding of 1 µM solutions of apo A-IV 347S and apo A-IV 347T was monitored at 222 nm at 2 sec intervals as the cuvette temperature was raised from 25 to 75°C. Inset: Van't Hoff plots of the data. Lines are linear curve fits of the data for apo A-IV 347S (dash-dot line) and apo A-IV 347T (solid line). The X-axis intercepts of the lines yield the reciprocals of the transition temperature midpoints. Abbreviation: Apo, apolipoprotein.
Fig. 4.
Fig. 4.
Fluorescence spectra of apo A-IV 347S and apo A-IV 347T. The fluorescence emission of 3 µM solutions of apo A-IV 347S and apo A-IV 347T excited at 280 nm (solid line) and 295 nm (tryptophan emission, dash-dot line) was monitored from 290 nm to 400 nm. The tyrosine fluorescence emission (dotted line) was deconvoluted from the 280 nm and 295 nm spectra as described in the text. Abbreviation: Apo, apolipoprotein.
Fig. 5.
Fig. 5.
Second derivative UV absorption spectra of apo A-IV 347S and apo A-IV 347T. The absorption of 8 µM solutions of apo A-IV isoproteins was monitored from 270 nm to 300 nm. Five individual spectra were averaged, smoothed, and differentiated using a filter length of 5. Apo A-IV 347S (solid line); apo A-IV 347T (dashed line). Abbreviation: Apo, apolipoprotein.
Fig. 6.
Fig. 6.
Interfacial exclusion pressure of apo A-IV 347S and apo A-IV 347T. Apo A-IV 347S (▽) and apo A-IV 347T (•) were injected beneath EPC monolayers spread at increasing initial surface pressures, and the change in surface pressure was determined. Data points are the means ± SE of two separate experiments; the solid lines are linear regressions of the data. Extrapolation of the lines to the X axis yields exclusion pressures of 26.5 ± 0.2 mN/m for apo A-IV 347S and 28.7 ± 0.2 for apo A-IV 347T. Abbreviations: Apo, apolipoprotein; EPC, egg phosphatidylcholine.
Fig. 7.
Fig. 7.
Binding of apo A-IV 347S and apo A-IV 347T) to the triolein/water interface. Triolein drops (10 μl) were injected into the optical cuvette of a ITC Tracker® tensiometer containing 25 μg/ml solutions of apo A-IV 347S (▽) and apo A-IV 347T (•), and the surface tension was continuously monitored. Data points are the means ± SE of two separate experiments. Abbreviation: Apo, apolipoprotein.
Fig. 8.
Fig. 8.
Elastic behavior of apo A-IV 347S and apo A-IV 347T at the triolein/water interface. Triolein drops (10 μl) were injected into the optical cuvette of a ITC Tracker® tensiometer containing 25 μg/ml solutions of apo A-IV 347S (▽) and apo A-IV 347T (•). After the surface tension had stabilized, the drop volume was sinusoidally oscillated ± 5 μl at six cycles/min, and the change in surface tension was continuously monitored as a function of drop surface area. Abbreviation: Apo, apolipoprotein.
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