title: Comparisons of biophysical properties and bioactivities of mono-PEGylated endostatin and an endostatin analog.
abstract: BACKGROUND
  Endostatin (ES) is a well-established potent endogenous antiangiogenic factor. An ES variant, called zinc-binding protein-ES (ZBP-ES), is clinically available; however, its use is limited by rapid renal clearance and short residence time. PEGylation has been exploited to overcome these shortcomings, and mono-PEGylated ES (called M2ES) as well as mono-PEGylated ZBP-ES (MZBP-ES) are developed in our study. This study aimed to compare the biophysical properties and biological effects of M2ES and MZBP-ES to evaluate their druggability.
  
  
  METHODS
  Circular dichroism and tryptophan emission fluorescence were used to monitor the conformational changes of M2ES and MZBP-ES. Their resistance to trypsin digestion and guanidinium chloride (GdmCl)-induced unfolding was examined by Coomassie staining and tryptophan emission fluorescence, respectively. The biological effects of M2ES and MZBP-ES on endothelial cell migration were evaluated using Transwell migration and wound healing assays, and the uptake of M2ES and MZBP-ES in endothelial cells was also compared by Western blotting and immunofluorescence.
  
  
  RESULTS
  Structural analyses revealed that M2ES has a more compact tertiary structure than MZBP-ES. Moreover, M2ES was more resistant to trypsin digestion and GdmCl-induced unfolding compared with MZBP-ES. In addition, although M2ES and MZBP-ES showed comparable levels of inhibiting transwell migration and wound healing of endothelial cells, M2ES displayed an increased ability to enter cells compared with MZBP-ES, possibly caused by the enhanced interaction with nucleolin.
  
  
  CONCLUSIONS
  M2ES has a more compact tertiary structure, is more stable for trypsin digestion and GdmCl-induced unfolding, exhibits increased cellular uptake and shows equivalent inhibitory effects on cell migration relative to MZBP-ES, indicating that M2ES is a more promising candidate for anticancer drug development compared with MZBP-ES.
authors.affiliation: The National Engineering Laboratory for Anti-tumor Protein Therapeutics, School of Life Sciences, Tsinghua University, Beijing, 100084, P. R. China. wangshan24@foxmail.com.|Beijing Key Laboratory for Protein Therapeutics, School of Life Sciences, Tsinghua University, Beijing, 100084, P. R. China. wangshan24@foxmail.com.|Cancer Biology Laboratory, School of Life Sciences, Tsinghua University, Beijing, 100084, P. R. China. wangshan24@foxmail.com.
authors.forename: Shan
authors.initials: S
authors.lastname: Wang
authors.affiliation: The National Engineering Laboratory for Anti-tumor Protein Therapeutics, School of Life Sciences, Tsinghua University, Beijing, 100084, P. R. China. fuyan@tsinghua.edu.cn.|Beijing Key Laboratory for Protein Therapeutics, School of Life Sciences, Tsinghua University, Beijing, 100084, P. R. China. fuyan@tsinghua.edu.cn.|Cancer Biology Laboratory, School of Life Sciences, Tsinghua University, Beijing, 100084, P. R. China. fuyan@tsinghua.edu.cn.
authors.forename: Yan
authors.initials: Y
authors.lastname: Fu
authors.affiliation: The National Engineering Laboratory for Anti-tumor Protein Therapeutics, School of Life Sciences, Tsinghua University, Beijing, 100084, P. R. China. yluo@tsinghua.edu.cn.|Beijing Key Laboratory for Protein Therapeutics, School of Life Sciences, Tsinghua University, Beijing, 100084, P. R. China. yluo@tsinghua.edu.cn.|Cancer Biology Laboratory, School of Life Sciences, Tsinghua University, Beijing, 100084, P. R. China. yluo@tsinghua.edu.cn.
authors.forename: Yongzhang
authors.initials: Y
authors.lastname: Luo
mesh_terms: D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects; D002942:Circular Dichroism; D043169:Endostatins / Q000737:chemistry* / Q000494:pharmacology*; D006801:Humans; D008958:Models, Molecular; D011092:Polyethylene Glycols / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D014364:Tryptophan / Q000737:chemistry
chemical_list: D043169:Endostatins; D011092:Polyethylene Glycols; D014364:Tryptophan