The AATPAP sequence is a very efficient signal for O-glycosylation in CHO cells.
The peptide signal sequence for protein O-glycosylation is not fully characterized, although a recent in vitro study proposed that the sequence motif, XTPXP, serves as a signal for mucin-type O-glycosylation. Here, we show that the AATPAP sequence acts as an efficient O-glycosylation signal, in vivo. A secreted fibroblast growth factor (secFGF) was used as a model to analyze glycosylation and its effects on the biological activity of FGF. Two constructs encoding [AATPAP]secFGF in which AATPAP was introduced at the N- or C-terminus of secFGF were constructed in an eukaryotic expression vector. [AATPAP]secFGF proteins were then expressed in Chinese hamster ovary (CHO) cells and secreted into the surrounding medium, primarily as modified forms sensitive to sialidase but not to peptide N-glycosidase F. The modifying groups were not seen when the AATPAP sequence was converted to AAAPAP or when [AATPAP]secFGF was expressed in mutant cells incapable of UDP-GalNAc biosynthesis. The results indicate that the modifying groups were mucin-type O-glycans and that the AATPAP served as an efficient O-glycosylation signal sequence. The O-glycosylated forms of [AATPAP]secFGF were as mitogenic toward human vascular endothelial cells as unmodified secFGF, suggesting that introduction of the signal into biologically active polypeptides is a promising approach with which O-glycosylation may be achieved without affecting original activity.
Asada
M
M
Biosignaling Department, National Institute of Bioscience and Human Technology, Tsukuba, Ibaraki, Japan.
Orikasa
N
N
Yoneda
A
A
Oda
Y
Y
Ota
K
K
Imamura
T
T
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004730:Endothelium, Vascular / Q000166:cytology; D005346:Fibroblast Growth Factors / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D006031:Glycosylation; D006801:Humans; D066298:In Vitro Techniques; D009077:Mucins / Q000378:metabolism; D017350:N-Acetylgalactosaminyltransferases; D009439:Neuraminidase / Q000378:metabolism; D017354:Point Mutation; D021382:Protein Sorting Signals / Q000378:metabolism*; D014470:Umbilical Cord / Q000166:cytology; D014536:Uridine Diphosphate N-Acetylgalactosamine / Q000378:metabolism
D009077:Mucins; D021382:Protein Sorting Signals; D005346:Fibroblast Growth Factors; D014536:Uridine Diphosphate N-Acetylgalactosamine; D017350:N-Acetylgalactosaminyltransferases; D009439:Neuraminidase