wrote the manuscript; All authors reviewed and approved the manuscript

wrote the manuscript; All authors reviewed and approved the manuscript.. binding on the cell surface, whereas preincubation with EDTA, a divalent cation chelator, completely Chlorpropamide abolished Mg2+ effect, indicating that COM and Mg2+ competitively bind to -enolase. Taken together, we successfully confirmed the role of -enolase as a COM crystal receptor to mediate COM crystal adhesion at apical membrane of renal tubular cells. It may also serve as a target for stone prevention by blocking cell-crystal adhesion and stone nidus formation. Due to the polarized characteristics of renal tubular epithelial cells, their apical membranes directly contact with tubular fluid and thus are involved with COM crystal adhesion, which is one of the initial mechanisms for kidney stone formation1,2. Recently, a number of potential COM crystal-binding molecules and/or proteins expressed on the apical membranes of renal tubular epithelial cells have been identified2,3,4. After renal tubular cell injury by numerous inducers, the injured renal tubular cells showed increased expression of COM crystal-binding molecules/proteins in concordance with the enhanced COM crystal binding on the cell surfaces5. Therefore, identification and characterizations of crystal-binding molecules/proteins on apical membranes of renal tubular epithelial cells may make kidney stone prevention feasible. Our previous expression proteomics study successfully identified a large number of COM crystal-binding proteins isolated from apical membranes of MDCK renal tubular epithelial cells4. Among them, a glycolytic enzyme -enolase was Chlorpropamide also identified by mass spectrometry in the COM crystal-bound fraction. -enolase is a 47-kDa enzyme that plays multiple roles in various cellular processes, including growth control, glycolysis and hypoxic tolerance6. Over the last few years, growing evidence has demonstrated that -enolase is localized not only in cytoplasm but also on the cell surface of a variety of eukaryotic cells at which enzymatic catalytic activity remains7. Surface -enolase also has a role in plasminogen-binding activity and serves as a plasminogen receptor, which is important for the development of some cancers8,9. This protein has increased expression level in the injured and regenerating cells during wound healing process10,11. In kidney stone disease, increasing evidence has pointed out its significance in kidney stone formation. High oxalate and testosterone treatments, both of which are the stone aggravators, increase expression level of -enolase in renal tubular cells12,13, whereas epigallocatechin gallate (EGCG), a stone suppressor from both and studies, decreases -enolase level in renal tubular cells14. Expression and additional data from these studies suggest that -enolase may serve as a potential COM crystal receptor to mediate crystal binding on the cell surface. Nevertheless, the precise role of -enolase as a receptor for COM crystals has not been confirmed. This study thus aimed to validate the role of -enolase as a COM crystal receptor on apical membranes of renal tubular epithelial cells by using Western blotting, immunofluorescence staining, laser-scanning confocal microscopy, cell-crystal adhesion assay, neutralization of surface -enolase by its specific antibody, crystal-protein binding assay, crystal face-specific binding determination, chemico-protein interactions analysis, and competitive binding assay using Mg2+ and divalent cation chelator. Results Western blotting was performed to confirm the presence of -enolase on apical membranes of MDCK renal tubular epithelial cells Chlorpropamide and also in COM crystal-bound fraction. Figure 1 shows that Chlorpropamide -enolase was found in whole cell lysate, apical membrane and COM-bound fractions. Immunofluorescence staining and laser-scanning confocal microscopy were also performed to further validate apical surface localization of -enolase in polarized MDCK cells. Polarized MDCK cells were fixed with 3.7% paraformaldehyde without any permeabilization step (to just demonstrate its surface localization, not the cytoplasmic expression) and incubated with rabbit polyclonal anti–enolase antibody. The confocal micrographs clearly illustrated apical surface localization of -enolase (Fig. 2). These data strengthen the apical membrane localization of -enolase in epithelial cells in addition to cytoplasm, which is its main localization. Open EIF4EBP1 in a separate window Figure 1 Western blot analysis of -enolase.Proteins in whole cell lysate, apical membrane and COM crystal-bound fractions were resolved by 12% SDS-PAGE and subjected to Western blot analysis using rabbit polyclonal anti–enolase (Santa Cruz Biotechnology) as a primary antibody. Coomassie Brilliant Blue G-250-stained gel of the COM-bound fraction was also aligned with the immunoblot. Open in a separate window Figure 2 Confirmation of apical membrane localization of -enolase on polarized MDCK cells.The polarized.