BMC Cancer 2005, 5:3 http://www.biomedcentral.com/1471-2407/5/3 SFeigquernec3e analysis of five mammalian Hig-2 proteins Sequence analysis of five mammalian Hig-2 proteins. (a) Mul- tiple sequence alignment of Hig2 amino acid sequences of mouse, rat, human, chimpanzee and olive baboon shows that these proteins share strong sequence similarity. (b) A repre- sentative Kyte-Doolittle hydrophobicity plot using the mouse Hig2 amino acid sequence indicates these proteins have hydrophobic N-termini, reminiscent of a signal peptide. these cells contained many very small solid green vesicles moving along the cytoskeleton. These vesicles were approx 1/100 the size of the large vesicles shown in Fig 4A and 4C, and were not observed in cells transfected with either Hig2(Y8V9/D8D9)EGFP or EGFP alone. The rapid- ity of this motion in live cells, even at room temperature, precluded capture of these images but suggested the pos- sibility that measurable amounts of secreted Hig2-EGFP might be found in the culture medium. HEK-293 cells were chosen as they could be transfected at high efficiency (approx 80%), the presence of the green transport vesicles was confirmed, and 48 hours after transfection samples of total cell lysate and conditioned medium were analysed by western blotting. Secreted Hig2-EGFP was detected in the conditioned medium of Hig2-EGFP cells, but not Hig2(Y8V9/D8D9)EGFP cells (Fig 5). Multiple bands were detected in cell lysates for both Hig2-EGFP fusion proteins: whether these represent artifactual degradation products or physiologically relevant biological entities is as yet unknown. Such multiple banding has also been observed with other EGFP-fusion proteins targeted to the secretory pathway (Amphiregulin-EGFP, PK unpublished observations). At least one of the bands may result from internal translation initiation at the consensus Kozak ini- SFmMuiuDgbtucCaertKileolunc4laearllasloncaalylissiastoiofnthoef pauHtaitgi2v-eEsGigFnPalfupseipotnidperionteHinCa1n1dand Subcellular localisation of a Hig2-EGFP fusion protein and mutational analysis of the putative signal peptide in HC11 and MDCK cells. (a and c) Hig2-EGFP is localised to large spher- ical vesicles located in the cytoplasm. (b and d) The intro- duction of two aspartate residues into the putative hydrophobic signal peptide abolishes the vesicular subcellular localisation of the mutant protein. tiation sequence of pEGFP-C1 which is located between the Hig2 and EGFP open reading frames. EGFP was also detected in the conditioned medium. This is consistent with previous reports of GFP secretion via a non-classical Brefeldin A-insensitive pathway [34]. In this study, several cell lines are described (including HEK293) in which wild-type GFP is released from the cell without passing through the golgi apparatus. Thus, it is formally possible that, instead of its secretion being directed by the putative signal peptide, HIG2-EGFP might be released from the cell via this pathway in a manner specifically dependent on the EGFP moiety. The presence of post- translational modifications acquired during endoplasmic reticulum/golgi apparatus mediated secretion would exclude the latter hypothesis. The altered mobility of HIG- 2-EGFP in the medium suggested that it might be glyco- sylated, however the mobility was not changed by treat- ment with the glycosidase PNGaseF, suggesting that this Page 5 of 9 (page number not for citation purposes)