Characterization of a human UT‐B urea transporter antibody (897.3)

Abstract

Urea entry into the gastrointestinal tract is facilitated by the urea transporter UT‐B1 (Stewart 2011). This process is crucial to our intestinal wellbeing as it is the first step in the urea nitrogen salvaging mechanism, which is necessary for maintaining healthy gut bacterial populations. In a previous study using a bovine UT‐B antibody, a 35kDa human UT‐B1 protein was found to be highly expressed in the ascending colon (Collins et al., 2010). The aim of this study was to produce a reliable human UT‐B antibody to be used for further investigations of human UT‐B expression. Initial PCR analysis using primers designed along the UT‐B gene confirmed UT‐B1 as the major isoform expressed at the RNA level in all human gastrointestinal tissues. Three polyclonal human UT‐B antibodies ‐ one designed against the N‐terminal (UT‐B#N) and two against the C‐terminal (UT‐B#C and UT‐Bc19) ‐ were made according to the UT‐B1 amino acid sequence and extensive characterization was carried out using Western blot analysis. While both C‐terminal antibodies detected UT‐B1 in the membrane of erythrocytes as a large glycosylated signal at 45‐60kDa as expected, the UT‐B#N antibody failed to do so and was rejected. As UT‐B transporters are glycoproteins, deglycosylation experiments were performed on UT‐B1 protein using PNGaseF enzyme. While UT‐Bc19 successfully detected 36 kDa deglycosylated UT‐B1, UT‐B#C failed to do so and was also rejected. Next, β‐mercaptoethanol was used to reduce UT‐B1 from its native multimer to a monomer and UT‐Bc19 successfully detected a big shift from a smeared 40‐100kDa signal to a neat 40kDa signal. Finally, UT‐Bc19 also detected a strong 40‐70kDa glycosylated UT‐B1 signal in human ileum protein. In conclusion, these data have shown that UT‐Bc19 is the only consistent human UT‐B antibody. Future investigations of UT‐B1 expression will therefore utilise UT‐Bc19 with confidence and help establish the physiological significance of UT‐B1 in the human gastrointestinal tract.Grant Funding Source: Supported by The Wellcome Trust and Science Foundation Ireland.