This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Two structurally related glycoproteins, the uroplakins (UP) Ia and Ib, interact with UP II and III, to form 16 nm particles hexagonally packed to form 2D crystals that cover almost the entire apical surface of mammalian bladder epithelium. It has been proposed that glycosylation patterns of the UPs determine the binding efficiency of bacteria that cause urinary tract infections. A rapid and sensitive MS strategy has been utilized in this study for the structural determination of the glycans and the identification of occupied glycosylation sites. The results should contribute to a better understanding of the mechanism of urinary tract infection and to improvements in its diagnosis and treatment. Murine and bovine UPs Ia and Ib were purified by SDS-PAGE. The excellent resolution between murine 24k-Da UP Ia and 29k-Da UP Ib contrasted with the poor resolution between bovine 27-kDa UP Ia and 28k-Da UP Ib. Bands of interest were excised and deglycosylated in-gel with PNGase F, and the extracted glycans were subjected to permethylation. Tryptic digestion of the proteins was performed in-gel, after release of the N-glycans. The peptides and the permethylated oligosaccharides were characterized using a Bruker Reflex IV matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer, and further analyzed using a QSTAR Pulsar i quadrupole-orthogonal TOF mass spectrometer (QoTOF MS). The carbohydrates and peptides of interest were sequenced by MS/MS. Using a combination of glycosidase and protease in-gel digestion, MALDI MS, and ESI MS/MS, we verified the amino acid sequences of the proteins and determined the pattern of glycoform heterogeneity at the single glycosylation site in UPs Ia and Ib from bovine and murine samples. Bovine UP Ia/Ib were found to contain a series of high mannose type N-linked glycans at Asn131 of UP Ib and Asn170 of UP Ia. The N-linked glycan population at Asn169 in murine UP Ia was determined to be a series of high mannose glycans, while murine UP Ib was found to contain a series of multiple-antennary complex, high mannose, and hybrid N-linked glycans at Asn131. The permethylated glycan pool generated in this study allowed relative quantification of glycan constituents. The survey on the distribution of glycoforms in UP Ia and Ib was carried out using MALDI-TOF MS. The main glycoforms of murine UP Ia were found to be the high mannose glycans, while those of murine UP Ib were found to be mainly complex glycans (more than 85% of the glycoforms), along with small amounts of high mannose and hybrid glycans. MALDI MS profiles of the native and permethylated glycans from bovine UP Ia/Ib suggested that the observed profile of native glycans is in good agreement with the results obtained after permethylation, in terms of both the identities and distributions of glycoforms. These results are consistent with the electrophoretic mobility changes of UPs Ia and Ib after they are treated with endo H and F glycosidases. Our results provide a biochemical explanation for the observation that the type 1-fimbriated, uropathogenic E. coli bacteria bind to murine uroplakin 1a, but not to the closely related murine uroplakin Ib.
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