Characterization of site-specific N -glycosylation signatures of isolated uromodulin from human urine.
Tianhai LinZhuo ChenMengqi LuoYang ZhaoWenjuan ZengShanshan ZhengTao SuYi ZhongShisheng WangYoumei JinLiqiang HuWanjun ZhaoJiaxu LiXuanyi WangChangwei WuDapeng LiFang LiuGuisen LiHao YangYong ZhangPublished in: The Analyst (2023)
Uromodulin (Umod, Tamm-Horsfall protein) is the most abundant urinary N -glycoprotein produced exclusively by the kidney. It can form filaments to antagonize the adhesion of uropathogens. However, the site-specific N -glycosylation signatures of Umod in healthy individuals and patients with IgA nephropathy (IgAN) remain poorly understood due to the lack of suitable isolation and analytical methods. In this study, we first presented a simple and fast method based on diatomaceous earth adsorption to isolate Umod. These isolated glycoproteins were digested by trypsin and/or Glu-C. Intact N -glycopeptides with or without HILIC enrichment were analyzed using our developed EThcD-sceHCD-MS/MS. Based on the optimized workflow, we identified a total of 780 unique intact N -glycopeptides (7 N -glycosites and 152 N -glycan compositions) from healthy individuals. As anticipated, these glycosites exhibited glycoform heterogeneity. Almost all N -glycosites were modified completely by the complex type, except for one N -glycosite (N275), which was nearly entirely occupied by the high-mannose type for mediating Umod's antiadhesive activity. Then, we compared the N -glycosylation of Umod between healthy controls ( n = 9) and IgAN patients ( n = 9). The N -glycosylation of Umod in IgAN patients will drastically decrease and be lost. Finally, we profiled the most comprehensive site-specific N -glycosylation map of Umod and revealed its alterations in IgAN patients. Our method provides a high-throughput workflow for characterizing the N -glycosylation of Umod, which can aid in understanding its roles in physiology and pathology, as well as serving as a potential diagnostic tool for evolution of renal tubular function.