Site-directed mutagenesis of heparin-binding EGF-like growth factor (HB-EGF): analysis of O-glycosylation sites and properties.
Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a 22 kDa, O-glycosylated protein. HeLa cells infected with a recombinant vaccinia virus expressing human HB-EGF produced a secreted, bioactive protein, with Mr 22,000 that was decreased to 14,000 by treatment with O-glycanase. Site-directed mutagenesis of HB-EGF cDNA using oligonucleotide- and PCR-directed techniques was performed to change the potential glycosylation sites, Thr75 and Thr85, to alanine residues to prevent O-glycosylation. Purification and characterization of the mutant proteins demonstrated that: (i) both O-glycosylation sites of HB-EGF are utilized, (ii) HB-EGF secretion does not require O-glycosylation, (iii) removal of O-glycans does not affect proteolytic cleavage of the HB-EGF precursor, nor does it influence HB-EGF intracellular trafficking or subcellular localization, and (iv) HB-EGF produced by HeLa cells is heavily sialylated. Comparisons between glycosylation mutants and wild-type HB-EGF revealed no significant apparent differences in receptor binding activity.
Davis-Fleische
K M
KM
Department of Surgery, The Ohio State University and Children's Hospital, Columbus 43205, USA.
Brigstock
D R
DR
Besner
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GE
D000409:Alanine / Q000737:chemistry; D001665:Binding Sites; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors; D006031:Glycosylation; D006367:HeLa Cells; D066257:Heparin-binding EGF-like Growth Factor; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D013912:Threonine / Q000737:chemistry; D014616:Vaccinia virus / Q000378:metabolism
D018076:DNA, Complementary; C587883:HBEGF protein, human; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D011994:Recombinant Proteins; D013912:Threonine; D004815:Epidermal Growth Factor; D000409:Alanine