Comparison of haptoglobin and apolipoprotein A-I on biliary lipid particles involved in cholesterol crystallization.
Several proteins are known to modulate cholesterol crystallization. We recently demonstrated that haptoglobin has cholesterol crystallization promoting activity. However, this effect is still not well understood mechanistically. The current study examined the distribution of haptoglobin compared to apolipoprotein A-I (apo A-I) to micelles, vesicles and crystals as an initial step in providing a focus for further studies of the mechanism of cholesterol crystallization activity. Specific protein purification was accomplished by immunoaffinity chromatography. The crystallization-promoting activity of biliary haptoglobin, albumin and commercial apo A-I was measured by a photometric crystal growth assay. The distribution of micelles, vesicles and proteins in model bile was determined by Sepharose CL-6B column chromatography. Detection of the presence of test proteins in cholesterol crystals was determined using specific 125I-radiolabelled proteins. Haptoglobin (20 micrograms/mL) showed a significant crystallization promoting-activity, whereas apo A-I (30 micrograms/mL) only tended to show a slight inhibitory activity. The cholesterol crystal-bound protein in each case was found to be less than 1% of the total concentration of that protein that had been added to the model bile system. The elution profile of commercial apo A-I from a Sepharose CL-6B column was strikingly altered when it was added to model bile prior to elution. In contrast, the column elution profiles for both haptoglobin and albumin were unchanged when model bile was similarly added to the sample. Haptoglobin increased the amount of cholesterol found in the vesicular fraction when compared to apo A-I. Haptoglobin does not bind tightly to either biliary lipid particles or to cholesterol crystals but does increase the amount of cholesterol in vesicles by inducing a shift from micellar cholesterol (P = 0.046). This shift appears to explain in part its promoting effect on cholesterol crystallization.
Yamashita
G
G
Gastrointestinal Research Unit, Cleveland Clinic Foundation, OH 44195-5218, USA.
Secknus
R
R
Chernosky
A
A
Krivacic
K A
KA
Holzbach
R T
RT
D016632:Apolipoprotein A-I / Q000032:analysis*; D001646:Bile / Q000737:chemistry*; D002784:Cholesterol / Q000737:chemistry / Q000502:physiology*; D002850:Chromatography, Gel; D003460:Crystallization; D006242:Haptoglobins / Q000032:analysis*; D006801:Humans; D007158:Immunologic Techniques; D008055:Lipids / Q000737:chemistry*; D008823:Micelles; D010743:Phospholipids / Q000032:analysis
D016632:Apolipoprotein A-I; D006242:Haptoglobins; D008055:Lipids; D008823:Micelles; D010743:Phospholipids; D002784:Cholesterol