RESUMEN

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.

Asunto(s)

Factor de Crecimiento Epidérmico/química , Factor de Crecimiento Epidérmico/genética , Alanina/química , Sitios de Unión , Western Blotting , Cromatografía Líquida de Alta Presión , ADN Complementario/metabolismo , Electroforesis en Gel de Poliacrilamida , Factor de Crecimiento Epidérmico/metabolismo , Vectores Genéticos , Glicosilación , Células HeLa , Factor de Crecimiento Similar a EGF de Unión a Heparina , Humanos , Péptidos y Proteínas de Señalización Intercelular , Microscopía Fluorescente , Mutagénesis Sitio-Dirigida , Mutación , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/metabolismo , Treonina/química , Virus Vaccinia/metabolismo