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Localization of the active site of human tumour necrosis factor (hTNF) by mutational analysis

Xaveer Van Ostade, Jan Tavernier UGent, Thierry Prangé and Walter Fiers UGent (1991) EMBO JOURNAL. 10(4). p.827-836
abstract
In order to define the active site(s) of human tumour necrosis factor (hTNF), we mutagenized its gene at random and directly screened the resulting population for loss of cytotoxic activity on L929 cells. Four biologically inactive mutant proteins (Arg32 --> Trp, Leu36 --> Phe, Ser86 --> Phe and Ala84 --> Val) behaved similar to the wild-type in various physico-chemical assays. The residues were positioned on a 3D structural model and were found to cluster together at the base of the molecule at each side of the groove that separates two monomers in the trimeric structure. A very conservative mutation at one of these sites (Ala84 --> Val) almost completely abolished cytotoxic activity. Amino acid alterations in three other residues in close proximity to this receptor binding site were introduced: replacements at positions 29 and 146 clearly reduced cytotoxicity only when non-conservative alterations were introduced (Leu29 --> Ser and Glu146 --> Lys), suggesting an indirect influence on the active site. However, a conservative mutation at position 91 (Val --> Ala) caused a significant drop (500-fold) in bioactivity which suggests that Val91 may also play a direct role in receptor recognition. Our results favor a model in which each TNF molecule has three receptor-interaction sites (between the three subunits), thus allowing signal transmission by receptor clustering.
Please use this url to cite or link to this publication:
author
organization
alternative title
Localization of the active site of human tumor necrosis factor (hTNF) by mutational analysis
year
type
journalArticle (original)
publication status
published
subject
keyword
DNA, CONSTRUCTION, LYMPHOTOXIN, FACTOR TNF, COMPACT TRIMER, CYTO-TOXICITY, MUTAGENESIS, RECEPTOR-BINDING, MOLECULAR-WEIGHT, TUMOR NECROSIS FACTOR, STRUCTURE-FUNCTION ANALYSIS, RANDOM MUTAGENESIS, CYTOTOXICITY, FACTOR-ALPHA, ACTIVE SITE
journal title
EMBO JOURNAL
Embo J.
volume
10
issue
4
pages
827 - 836
Web of Science type
Article
ISSN
0261-4189
language
English
UGent publication?
yes
classification
A1
copyright statement
I have transferred the copyright for this publication to the publisher
id
1925382
handle
http://hdl.handle.net/1854/LU-1925382
date created
2011-10-13 11:16:04
date last changed
2011-11-10 13:23:07
@article{1925382,
  abstract     = {In order to define the active site(s) of human tumour necrosis factor (hTNF), we mutagenized its gene at random and directly screened the resulting population for loss of cytotoxic activity on L929 cells. Four biologically inactive mutant proteins (Arg32 --{\textrangle} Trp, Leu36 --{\textrangle} Phe, Ser86 --{\textrangle} Phe and Ala84 --{\textrangle} Val) behaved similar to the wild-type in various physico-chemical assays. The residues were positioned on a 3D structural model and were found to cluster together at the base of the molecule at each side of the groove that separates two monomers in the trimeric structure. A very conservative mutation at one of these sites (Ala84 --{\textrangle} Val) almost completely abolished cytotoxic activity. Amino acid alterations in three other residues in close proximity to this receptor binding site were introduced: replacements at positions 29 and 146 clearly reduced cytotoxicity only when non-conservative alterations were introduced (Leu29 --{\textrangle} Ser and Glu146 --{\textrangle} Lys), suggesting an indirect influence on the active site. However, a conservative mutation at position 91 (Val --{\textrangle} Ala) caused a significant drop (500-fold) in bioactivity which suggests that Val91 may also play a direct role in receptor recognition. Our results favor a model in which each TNF molecule has three receptor-interaction sites (between the three subunits), thus allowing signal transmission by receptor clustering.},
  author       = {Van Ostade, Xaveer and Tavernier, Jan and Prang{\'e}, Thierry and Fiers, Walter},
  issn         = {0261-4189},
  journal      = {EMBO JOURNAL},
  keyword      = {DNA,CONSTRUCTION,LYMPHOTOXIN,FACTOR TNF,COMPACT TRIMER,CYTO-TOXICITY,MUTAGENESIS,RECEPTOR-BINDING,MOLECULAR-WEIGHT,TUMOR NECROSIS FACTOR,STRUCTURE-FUNCTION ANALYSIS,RANDOM MUTAGENESIS,CYTOTOXICITY,FACTOR-ALPHA,ACTIVE SITE},
  language     = {eng},
  number       = {4},
  pages        = {827--836},
  title        = {Localization of the active site of human tumour necrosis factor (hTNF) by mutational analysis},
  volume       = {10},
  year         = {1991},
}

Chicago
Van Ostade, Xaveer, Jan Tavernier, Thierry Prangé, and Walter Fiers. 1991. “Localization of the Active Site of Human Tumour Necrosis Factor (hTNF) by Mutational Analysis.” Embo Journal 10 (4): 827–836.
APA
Van Ostade, X., Tavernier, J., Prangé, T., & Fiers, W. (1991). Localization of the active site of human tumour necrosis factor (hTNF) by mutational analysis. EMBO JOURNAL, 10(4), 827–836.
Vancouver
1.
Van Ostade X, Tavernier J, Prangé T, Fiers W. Localization of the active site of human tumour necrosis factor (hTNF) by mutational analysis. EMBO JOURNAL. 1991;10(4):827–36.
MLA
Van Ostade, Xaveer, Jan Tavernier, Thierry Prangé, et al. “Localization of the Active Site of Human Tumour Necrosis Factor (hTNF) by Mutational Analysis.” EMBO JOURNAL 10.4 (1991): 827–836. Print.