Biliary secretory immunoglobulin A is a major constituent of the new group of cholesterol crystal-binding proteins  Norbert Busch, Frank Lammert, Siegfried Matern  Gastroenterology  Volume 115, Issue 1, Pages 129-138 (July 1998) DOI: 10.1016/S0016-5085(98)70374-9 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Effect of crystal-binding proteins purified by preparative gel electrophoresis on cholesterol crystal growth in supersaturated model bile (cholesterol saturation index, 1.3; total lipid concentration, 125 g/L; bile salt/phospholipid molar ratio, 4.0) at different protein concentrations (2.5–10 μg/mL). (A) Twenty-eight–kilodalton, (B) 63-kilodalton, and (C) 74-kilodalton crystal-binding protein. Crystal concentrations are proportional to turbidities of the samples. Graphs were normalized by setting the plateau turbidity of each individual control of the independent experiments to unity. Protein concentrations: ■, 2.5 μg/mL; ▾, 5.0 μg/mL; ●, 10.0 μg/mL. Control curve (....) is given as mean ± SD (n = 3); each experimental curve is given as mean (n = 2). The three crystal-binding protein fractions inhibit kinetics of crystallization as well as final crystal concentrations in comparison with control curves. The inhibiting effects increase with higher protein concentrations and are strongest for the 28-kilodalton crystal-binding protein. Differences between control and experimental curves are statistically significant (P < 0.05) except for the 63-kilodalton protein at a concentration of 2.5 μg/mL. Gastroenterology 1998 115, 129-138DOI: (10.1016/S0016-5085(98)70374-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Effect of crystal-binding proteins purified by preparative gel electrophoresis on cholesterol crystal growth in supersaturated model bile (cholesterol saturation index, 1.3; total lipid concentration, 125 g/L; bile salt/phospholipid molar ratio, 4.0) at different protein concentrations (2.5–10 μg/mL). (A) Twenty-eight–kilodalton, (B) 63-kilodalton, and (C) 74-kilodalton crystal-binding protein. Crystal concentrations are proportional to turbidities of the samples. Graphs were normalized by setting the plateau turbidity of each individual control of the independent experiments to unity. Protein concentrations: ■, 2.5 μg/mL; ▾, 5.0 μg/mL; ●, 10.0 μg/mL. Control curve (....) is given as mean ± SD (n = 3); each experimental curve is given as mean (n = 2). The three crystal-binding protein fractions inhibit kinetics of crystallization as well as final crystal concentrations in comparison with control curves. The inhibiting effects increase with higher protein concentrations and are strongest for the 28-kilodalton crystal-binding protein. Differences between control and experimental curves are statistically significant (P < 0.05) except for the 63-kilodalton protein at a concentration of 2.5 μg/mL. Gastroenterology 1998 115, 129-138DOI: (10.1016/S0016-5085(98)70374-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Effect of crystal-binding proteins purified by preparative gel electrophoresis on cholesterol crystal growth in supersaturated model bile (cholesterol saturation index, 1.3; total lipid concentration, 125 g/L; bile salt/phospholipid molar ratio, 4.0) at different protein concentrations (2.5–10 μg/mL). (A) Twenty-eight–kilodalton, (B) 63-kilodalton, and (C) 74-kilodalton crystal-binding protein. Crystal concentrations are proportional to turbidities of the samples. Graphs were normalized by setting the plateau turbidity of each individual control of the independent experiments to unity. Protein concentrations: ■, 2.5 μg/mL; ▾, 5.0 μg/mL; ●, 10.0 μg/mL. Control curve (....) is given as mean ± SD (n = 3); each experimental curve is given as mean (n = 2). The three crystal-binding protein fractions inhibit kinetics of crystallization as well as final crystal concentrations in comparison with control curves. The inhibiting effects increase with higher protein concentrations and are strongest for the 28-kilodalton crystal-binding protein. Differences between control and experimental curves are statistically significant (P < 0.05) except for the 63-kilodalton protein at a concentration of 2.5 μg/mL. Gastroenterology 1998 115, 129-138DOI: (10.1016/S0016-5085(98)70374-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 Immunoblots after SDS-PAGE under reducing conditions. Immunostain with anti–κ-light-chain, anti–IgA-heavy-chain, and anti–secretory-component antibodies. 28 kD, 28-kilodalton crystal-binding protein; IgA, IgA-bound fraction; Helix, Helix-bound fraction; 63 kD, 63-kilodalton crystal-binding protein; 74 kD, 74-kilodalton crystal-binding protein. Immunoblotting provides evidence for the 28-kilodalton protein to be the light chain (lanes 1–3), the 63-kilodalton protein to be the heavy chain (lanes 4–6), and the 74-kilodalton crystal-binding protein to be the secretory component of human IgA (lanes 7–9). Gastroenterology 1998 115, 129-138DOI: (10.1016/S0016-5085(98)70374-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 SDS-PAGE of crystal-binding proteins and biliary IgA under reducing conditions without and after enzymatic deglycosylation (degly). Silver stain. STD, standard; 74 kD, 74-kilodalton crystal-binding protein (3 μg) before and after treatment with N-glycosidase F; 63 kD, 63-kilodalton crystal-binding protein (3 μg) before and after treatment with N-glycosidase F; IgA, biliary IgA (5 μg) before and after treatment with N-glycosidase F, O-glycosidase, and neuraminidase. The 74-kilodalton protein (lanes 2 and 3) and the 63-kilodalton protein (lanes 4 and 5) display sugar contents of 16% and 8% of their total molecular weights, respectively. For comparison, biliary IgA is shown in adjacent lanes 6 and 7. Gastroenterology 1998 115, 129-138DOI: (10.1016/S0016-5085(98)70374-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 Effect of biliary IgA purified by immunoaffinity chromatography on cholesterol crystal-growth curves in supersaturated model bile (cholesterol saturation index, 1.3; total lipid concentration, 125 g/L; bile acid/phospholipid molar ratio, 4.0). Crystal concentrations are proportional to turbidities of the samples. Graphs were normalized by setting the plateau turbidity of the control to unity. IgA concentrations: ■, 1.0 μg/mL; ▾, 10.0 μg/mL; ●, 50.0 μg/mL; ×, 100.0 μg/mL. Control curve (.....) is given as mean ± SD (n = 3); each experimental curve is given as mean (n = 2). Purified biliary IgA inhibits cholesterol crystallization significantly at concentrations of ≤100 μg/mL (P< 0.05). The inhibiting effect is enhanced with increasing IgA concentrations, and the strongest effect occurs at 50 μg/mL. Further increase of IgA concentration to 100 μg/mL weakens the inhibiting effect. At 300 μg/mL, no significant effects are detected in the crystal-growth assay. Gastroenterology 1998 115, 129-138DOI: (10.1016/S0016-5085(98)70374-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions