Abstract
We confirm the single site observation of decreased sialylation and abnormal glycosylation of Tamm-Horsfall protein in patients with interstitial cystitis compared to control subjects. Urine samples from 41 controls and 48 patients with interstitial cystitis from a total of 5 North American sites were obtained in blinded fashion as to participant status. Tamm-Horsfall protein was isolated from urine samples by salt precipitation. Protein content was determined by size exclusion chromatography and normalized to creatinine. Sialic acid was quantified by 1,2-diamino-4,5-methylene dioxybenzene (Sigma®) high performance liquid chromatography with fluorescence detection. Neutral and amino sugars were determined by high pH anion exchange chromatography with pulsed amperometric detection. N-glycans were labeled with 2-aminobenzamide and profiled using high pH anion exchange chromatography with fluorescence detection. Samples were also analyzed by matrix assisted laser desorption/ionization-time of flight mass spectrometry. Permethylated N-glycans were analyzed in the mass-to-charge ratio range of 3,000 to 6,000. There was no difference in the protein-to-creatinine ratio of Tamm-Horsfall protein from patients with interstitial cystitis vs controls (49.12 vs 46.4 mg/gm, p = 0.26). Sialic acid content (67 vs 77 nmol/mg Tamm-Horsfall protein, p = 0.025) and total monosaccharide content (590.9 vs 680.6 nmol/mg Tamm-Horsfall protein, p = 0.003) were significantly decreased in patients with interstitial cystitis vs controls. Results were supported by 2-aminobenzamide N-glycan profiling and mass spectrometry, which showed a 45% decrease in a major tetra-sialylated peak (mass-to-charge ratio 4,590) in Tamm-Horsfall protein from patients with interstitial cystitis compared to controls. These multisite data validate that abnormal glycosylation of Tamm-Horsfall protein occurs in patients with interstitial cystitis and may have a role in interstitial cystitis causation.
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