Davis-Fleische K M, Brigstock D R, Besner G E
Department of Surgery, The Ohio State University and Children's Hospital, Columbus 43205, USA.
Growth Factors. 2001;19(2):127-43. doi: 10.3109/08977190109001081.
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.
肝素结合表皮生长因子样生长因子（HB-EGF）是一种22 kDa的O-糖基化蛋白。用表达人HB-EGF的重组痘苗病毒感染HeLa细胞，产生了一种分泌型生物活性蛋白，其分子量为22,000，经O-聚糖酶处理后降至14,000。利用寡核苷酸和PCR定向技术对HB-EGF cDNA进行定点诱变，将潜在的糖基化位点苏氨酸75和苏氨酸85突变为丙氨酸残基，以防止O-糖基化。对突变蛋白的纯化和表征表明：（i）HB-EGF的两个O-糖基化位点均被利用；（ii）HB-EGF的分泌不需要O-糖基化；（iii）去除O-聚糖不影响HB-EGF前体的蛋白水解切割，也不影响HB-EGF的细胞内运输或亚细胞定位；（iv）HeLa细胞产生的HB-EGF高度唾液酸化。糖基化突变体与野生型HB-EGF之间的比较显示，受体结合活性没有明显差异。
