Engineering neoglycoproteins with multiple O-glycans using repetitive pentapeptide glycosylation units.
Controlled protein remodeling with O-linked glycans has been limited by our incomplete understanding of the process of glycosylation. Here we describe a secretable fibroblast growth factor (FGF) with multiple mucin-type O-glycans produced by introducing a minimum pentapeptide glycosylation unit in a decarepeat format at its N- or C-terminus. Expressed in Chinese hamster ovary cells, chemical and biochemical analyses of the resultant proteins (Nm10-FGF and Cm10-FGF, respectively) demonstrated that all O-glycosylation units were glycosylated and the dominant structure was sialylated Gal[beta1-3]GalNAc. This indicates that minimum O-glycosylation unit in multirepeat format serves as a remarkably efficient acceptor in CHO cells. The Nm10-FGF and Cm10-FGF proteins maintained the mitogenic activity to vascular endothelial cells. In addition, intact Cm10-FGF and its desialylated form interacted with several lectins in the same way as mucin-type glycoproteins. The intact Cm10-FGF with multiple sialylated O-glycans exhibited a longer lifetime in circulating blood, whereas the Cm10-FGF with desialylated O-glycans exhibited a shorter lifetime than the deglycosylated form of Cm10-FGF. Our approach would thus appear to be highly effective for engineering neoglycoproteins, the characteristics of which are determined by their multiple mucin-type O-glycans.
Yoneda
A
A
Gene Discovery Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
Asada
M
M
Yamamoto
S
S
Oki
J
J
Oda
Y
Y
Ota
K
K
Ogi
Y
Y
Fujishima
S
S
Imamura
T
T
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002846:Chromatography, Affinity; D006224:Cricetinae; D004727:Endothelium / Q000166:cytology / Q000187:drug effects; D016220:Fibroblast Growth Factor 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D006023:Glycoproteins / Q000737:chemistry*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D051379:Mice; D008934:Mitogens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D015202:Protein Engineering; D013997:Time Factors; D014162:Transfection
D006023:Glycoproteins; D008934:Mitogens; D010455:Peptides; D016220:Fibroblast Growth Factor 1; D006026:Glycoside Hydrolases; D019158:N-Acetylneuraminic Acid