Objective: Pronucleating nonmucin glycoproteins in human cholesterol and black gallstones were isolated and identified. Summary Background Data: Gallbladder bile contains nonmucin glycoproteins that are pronucleating of cholesterol monohydrate crystals. Little is known about the presence or activity of these proteins within gallstones. Methods: Nonmucin glycoproteins were isolated from single cholesterol (n = 8), multiple cholesterol (n = 8), and black pigment (n = 8) gallstones by concanavalin A lectin-affinity chromatography. The proteins were separated by sodium dodecyl sulfate gradient electrophoresis. Western blot analysis was performed for Fab immunoglobulin fragments, and heavy chains from the immunoglobulin G, A, E, and M subclasses. A crystal observation time assay was performed on the combination of isolated nonmucin glycoproteins from gallstones and isolated Fab fragments. Results: Nonmucin glycoproteins of molecular weights 10, 15, 17, 22, 28, and 208 kD were identified in gallstones. These six nonmucin glycoproteins shortened the crystal observation time by more than 50% (p < 0.01) compared with model bile. Western blot analysis confirmed the identity of the 22 and 28-kD proteins as immunoglobulin Fab fragments. These were seen in all gallstones, irrespective of the gallstone type. The isolated Fab 28- kD fragment from the gallstones of 23 patients shortened the extrapolated crystal observation time by 78% (p < 0.01). However, commercially available Fab fragments had no effect on either cholesterol crystal appearance or growth. Conclusions: Nonmucin glycoproteins that are pronucleating for cholesterol monohydrate crystals are also found in human cholesterol and black pigment gallstones. Fab immunoglobulin fragments were found in all gallstones irrespective of the gallstone type. Fab immunoglobulin fragments from gallstones shortened the crystal observation time but not crystal growth or total crystal content compared with model bile or commercially available Fab fragments. These data suggest that an antigen immune (Fab) complex may contribute to cholesterol crystal function.
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