Changes in the hemagglutination activity of different concentration of rPnxIIIA with sheep erythrocytes (E). When compared with the domains in the HMM database, several PnxIIIA domains have large repeat sequences that contain the

hemagglutinin repeat in the primary sequence. rPnxIIIA was subjected to a hemagglutination assay with washed sheep erythrocytes. Figure 3E shows the results of the hemagglutination assay with rPnxIIIA. Hemagglutination of sheep erythrocytes was observed at rPnxIIIA concentrations exceeding 12.5 μg/ml, indicating that rPnxIIIA participates in the hemagglutination of sheep erythrocytes. We also measured the hemoglobin released from the sheep erythrocytes when they were VRT752271 ic50 cultured with rPnxIIIA; however, rPnxIIIA did not exhibit typical hemolytic activity, indicating that rPnxIIIA is less MK5108 molecular weight involved in hemolysis. Characterization of deletion mutants of rPnxIIIA variants To clarify

the role of large repeat sequences in the functions of PnxIIIA, we generated soluble rPnxIIIA and deletion mutants of rPnxIIIA variants. rPnxIIIA, rPnxIIIA209, rPnxIIIA197, and rPnxIIIA151 essentially contained 255 kDa, 209 kDa, 197 kDa, and 151 kDa of the parent PnxIIIA, respectively (Additional file 3A). To compare the binding ability of the rPnxIIIA variants, we performed binding assays with collagen type I coated on the 96-well plate when 10 μg/ml of the rPnxIIIA variants were applied. The A620 of wild-type rPnxIIIA was 0.55 ± 0.05, compared to 0.30 ± 0.06, Ribonucleotide reductase 0.27 ± 0.01, and 0.26 ± 0.04 for that of rPnxIIIA209, rPnxIIIA197, and rPnxIIIA151, respectively (Additional file 3B). Almost all A620s of the deletion mutant proteins were lower than that of the parent rPnxIIIA. These results indicate that rPnxIIIA can bind to ECMs and that its lack of repeat sequences reduces its ability to bind ECMs.

We subjected the rPnxIIIA variants to a hemagglutination assay with washed sheep erythrocytes. Although the deletion mutant protein rPnxIII209 promoted hemagglutination at the same concentration as that of rPnxIIIA, more than 25 μg/ml of both rPnxIIIA197 and rPnxIIIA151 were required for hemagglutination (Additional file 3C). Although exact differentiation among the rPnxIIIA variants was not observed in hemagglutination, these results indicate that rPnxIIIA plays a role in hemagglutination and that the repeat sequences located in the C-terminal portion are necessary for enhanced hemagglutination. Localization and interaction of PnxIIIA Figure 4A shows the results of the Poziotinib supplier Western blotting analysis of fractionated P. pneumotropica ATCC 35149 cells with anti-rPnxIIIA rabbit IgG. Signals of proteins of approximately 250 kDa in size were detected in all fractions; however, in the case of the OM fraction, the intensity of the signal was strong and located above the 250-kDa marker and other fractions.