We found that similar levels of FI were detected in permeabilized cells transfected with WT FI and several mutants, both secreted and retained, while no FI could be detected in cells transfected with C4BP as a negative control (Fig. 2A). We further analyzed the subcellular localization of the mutants

by subjecting cell lysates to endoglycosidase H (EndoH) digestion. EndoH cleaves high-mannose oligosaccharides that are present only in the ER and the early Golgi apparatus. Therefore, EndoH sensitivity of a protein indicates an ER and early Golgi localization, while EndoH resistance indicates transport to post-Golgi and secretion. Upon EndoH digestion, a large fraction of WT FI was EndoH-sensitive and displayed faster mobility upon Pirfenidone manufacturer electrophoresis representing protein in ER still undergoing processing, but significant amount

of WT FI was resistant to EndoH (Fig. 2B, band marked with an asterisk). The presence of an EndoH-resistant fraction indicated transport to late secretory compartments and beyond. In contrast, C25F and N133S were entirely sensitive to EndoH digestion (Fig. 2B), indicating that they did not reach the late Golgi compartment and were retained in the ER. In order to investigate the impact of the mutations on the molecular function of FI in more detail, those mutant proteins that were secreted at a sufficient level by the HEK 293 cells were purified. For this purpose, cells were stably transfected and secreted FI was purified from the conditioned medium by affinity chromatography. Using this approach WT FI and six mutant proteins (P32A, M120V, H165R, A222G, selleck products R299W and D501N) were successfully purified. The purified proteins were visualized using Western blotting under non-reducing (Fig.

3A) and reducing (Fig. 3B) conditions. Full-length FI has a molecular weight of 88 kDa, the heavy and light chains contributing 50 and 38 kDa, respectively. It has Pregnenolone been shown earlier that when FI is over-expressed in a cell line, not all of the protein is proteolytically processed. Both the uncleaved polypeptide chain and the mature processed FI protein are simultaneously secreted 33. Importantly, all expressed FI variants examined here were processed with the same efficiency. The FI mutants were analyzed for their ability to degrade C4b (Fig. 4A and B) and C3b (Fig. 4C–H) in the fluid phase. Different concentrations of the WT or mutant FI were mixed with cofactor (C4BP, FH, CR1 (erythrocyte ghosts) or MCP (H2087 cells), C4b/C3b and I125-labeled C4b/C3b, incubated at 37°C and then analyzed by SDS-PAGE. The representative images shown (Fig. 4A and C) display the condition where 5 μg/mL of FI variants was used while in Fig. 4E and G we used 10 or 30 μg/mL FI, respectively. Negative controls show the position of the intact bands for C4b (α′, β, γ; Fig. 4A) and C3b (α′, β; Fig. 4C, E and G). The intensity of the α′-chain band was from three different gels and the mean values for C4b (Fig.