Interaction of cholesterol-crystallization-promoting proteins with vesicles

Abstract

In this study, the interaction of mucin and concanavalin A-binding proteins isolated from human bile with cholesterol/phospholipid vesicles was investigated. Using resonance energy transfer assays originally developed by Struck, Hoekstra and Pagano [(1981) Biochemistry 20, 4093-4099], no significant protein-induced fusion or aggregation of vesicles was demonstrated. Instead of fusion, these proteins induced destabilization of cholesterol/phospholipid vesicles, as monitored by release of entrapped carboxyfluorescein. A good correlation (rho = 0.81) was obtained between the extent of leakage and the nucleation-promoting activity of the concanavalin A-binding proteins. We conclude that aggregation or fusion of cholesterol/phospholipid vesicles is not an obligatory step in cholesterol crystallization. Biliary protein-induced crystallization seems to be preceded by vesicle disruption.

Mucin-vesicle interactions in model bile: Evidence for vesicle aggregation and fusion before cholesterol crystal formation

“…In a recent study using similar fluorescent assays without validation by microscopy or light scattering, de Bruijn et a1 showed no effect of mucin on vesicle aggregation or fusion. 40 The authors did show that both mucin and concanavalin Abinding glycoproteins were able to induce vesicle disruption. However, these investigators failed to examine vesicle fusion and aggregation in the initial 24 hours, whereas we have shown that the major acceleration of these events by mucin occurs.…”

Quantitative assessment of comparative potencies of cholesterol-crystal-promoting factors: relation to mechanistic characterization

“…The pathogenesis of gallstone disease consists of complex steps involving the presence of cholesterol supersaturation of the gallbladder bile, lipid-vesicle formation and aggregation, which culminates in the phase separation of cholesterol and the nucleation of cholesterol crystals [1][2][3][4][5][6]. Although a variety of factors affect this process, few investigations have compared the potencies of these factors.…”

“…Under our conditions, the physico-chemical state of the MB solution may have been more physiological, suggesting that the failure of CABG to affect lipid distribution represents the physiological state. de Brujin et al [6] reported that the nucleation-promoting action of CABG is mediated, in part, by the vesicle disruption, using a unique vesicle-monitoring system with carboxyfluorescein. Thus the cholesterol-crystallization-promoting action of CABG, which occurs under physiological circumstances, could not be based on the redistribution of lipids among biliary lipid particulate species, but seemingly on the alteration of cholesterol metastability within individual vesicular lipid bilayers Such an alteration in the physico-chemical state of the vesicular lipids could lead to the enhancement of cholesterol-crystal formation.…”

“…Based on video-enhanced microscopy studies of human bile, Halpern et al [3] reported that vesicle aggregation and fusion precede cholesterol nucleation. The crystallization of cholesterol is affected by the presence of various factors in bile, such as biliary proteins [6][7][8][9][10][11], phospholipid species [4,[12][13][14] and bile-salt species [15,16]. The biliary proteins that modulate cholesterol crystallization include concanavalin A (Con A)-binding glycoproteins (CABG), which are promoters, and the apolipoproteins A-1 and A-2, and helix pomacia-bound glycoprotein, which are inhibitors [6][7][8][9][10][11]17].…”