Face-specific incorporation of osteopontin into urinary and inorganic calcium oxalate monohydrate and dihydrate crystals

Abstract

Our aim was to examine the attachment to, and incorporation of intact, highly phosphorylated osteopontin (OPN) into inorganic (i) and urinary (u) calcium oxalate monohydrate (COM) and dihydrate (COD) crystals. uCOM and uCOD crystals were precipitated from ultrafiltered (UF) urine containing human milk OPN (mOPN) labelled with Alexa Fluor 647 fluorescent dye at concentrations of 0.1-5.0 mg/L. iCOM and iCOD crystals were generated in aqueous solutions at concentrations of 0.01-0.5 mg/L. Crystals were demineralised with EDTA and the resulting extracts analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis and western blotting, or examined by fluorescent confocal microscopy and field emission scanning electron microscopy before and after washing to remove proteins bound reversibly to the crystal surfaces. Binding of mOPN to pre-formed iCOM crystals was also studied in phosphate-buffered saline (PBS) and ultrafiltered (UF) urine. mOPN attached to the {100} faces and to the {010} sides of the {100}/{010} edges of iCOM crystals was removed by washing, indicating that it was not incorporated into the mineral bulk. In both PBS and urine, mOPN was attached to the {021} faces of pre-formed iCOM crystals as well as to the {100}/{010} edges, but was concentrated at the intersection points of the {100} and {121} faces at the crystal tips. Attachment in UF urine appeared to be greater than in PBS and stronger at higher calcium concentrations than lower calcium concentrations. In uCOM crystals, the distribution of fluorescence and patterns of erosion after washing suggested attachment of mOPN to the four end faces, followed by interment within the mineral phase. Fluorescence distributions of mOPN associated with both iCOD and uCOD crystals were consistent with uniform binding of the protein to all equivalent {101} faces and concentration along the intersections between them. Persistence of fluorescence after washing indicated that most mOPN was incarcerated within the mineral phase. We concluded that attachment of mOPN to calcium oxalate crystals is face-specific and depends upon the anatomical and genetic source of the protein and whether the crystals are (1) COM or COD; (2) pre-formed or precipitated from solution, and (3) precipitated from urine or aqueous solutions. Our findings emphasise the need for caution when drawing conclusions about possible roles of OPN or other proteins in urolithiasis from experimental data obtained under inorganic conditions.