Abstract
Although it has been shown that mouse uroplakin (UP) Ia, a major glycoprotein of urothelial apical surface, can serve as the receptor for the FimH lectin adhesin of type 1-fimbriated Escherichia coli, the organism that causes a great majority of urinary tract infections, the glycan structure of this native receptor was unknown. Using a sensitive approach that combines in-gel glycosidase and protease digestions, permethylation of released glycans, and mass spectrometry, we have elucidated for the first time the native glycoform structures of the mouse UPIa receptor and those of its non-binding homolog, UPIb, and have determined the glycosylation site occupancy. UPIa presents a high level of terminally exposed mannose residues (located on Man(6)GlcNAc(2) to Man(9)GlcNAc(2)) that are capable of specifically interacting with FimH. We have shown that this property is conserved not only in the mouse uroplakins but also in cattle and, even more importantly, in human UPIa, thus establishing the concept that UPIa is a major urothelial receptor in humans and other mammals for the mannose-specific FimH variant. In contrast, our results indicate that most terminally exposed glycans of mouse UPIb are non-mannose residues, thus explaining the failure of FimH to bind to this UPIb. In cattle, on the other hand, complex carbohydrates constituted only about 20% of the UPIb N-linked glycans. Human UPIa contained exclusively high mannose glycans, and human UPIb contained only complex glycans. The drastically different carbohydrate processing of the UPIa and UPIb proteins, two closely related members of the tetraspanin family, may reflect differences in their folding and masking due to their interactions with their associated proteins, UPII and UPIIIa, respectively. Results from this study shed light on the molecular pathogenesis of urinary tract infections and may aid in the design of glyco-mimetic inhibitors for preventing and treating this disease.
Highlights
The essential first step in mucosal infection is for the microorganisms to latch onto the mucosal surfaces to escape being expelled by physical forces and other innate host defenses [1]
Mono-mannose proteins do not exist in nature, and erythrocytes and yeast are not involved in E. coli adhesion to the urothelial surface
uroplakin Ia (UPIa) could be quantitatively converted to a lower Mr form by endoglycosidase H (Endo H) (Fig. 1B, lanes 4 and 5), only about 30 – 40% of the UPIb glycans were Endo H-sensitive, and the rest of the glycoprotein required PNGase F for complete deglycosylation (Fig. 1B, lanes 10 –12)
Summary
Reagents—The chemicals and their sources are listed below. Enzymes including recombinant, glycerol-free PNGase F, Endo H (New England Biolabs Inc, Beverly, MA), and trypsin (Promega Corp., San Luis Obispo, CA) were used in digestions. After cooling to room temperature and removal of the supernatant, the gel pieces were incubated at 45 °C for 45 min in the dark in 25 l of 50 mM fresh iodoacetamide in 50 mM NH4HCO3, washed in 50 l of 50 mM NH4HCO3, 100% CH3CN, and dried using a SpeedVac to remove SDS, reducing, and alkylation reagents. Oligosaccharides and Peptides Extraction—Glycans were extracted from the gel pieces by removing the incubation buffer, which already may have contained some sugars, and using three exchanges of 100 l water, with sonication for 30 min each time. One l of sample solution was mixed with the MALDI matrix ␣-cyano-4-hydroxycinnamic acid (10 mg/ml in acetonitrile:water:trifluoroacetic acid (50/50/0.1 v/v/v)), deposited on a MALDI target, and observed as [MϩH]ϩ in the positive ion spectra. Peaks whose observed mass suggested that they contained potential glycosylation sites were selected for MS/MS analyses
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
