Two conserved tryptophan residues of tumor necrosis factor and lymphotoxin are not involved in the biological activity

Ostade, Xaveer Van; Tavernier, Jan; Fiers, Walter

doi:10.1016/0014-5793(88)80510-6

Cited by 9 publications

(1 citation statement)

References 15 publications

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“…t Present address: Fermentation Research Institute, Agency of Progress in genetic engineering permitted the production of biologically active hTNF in Escherichia coli (Pennica et al, 1984;Marmennout et al, 1985;Yamada et al, 1985). Some mutant hTNFs have been prepared by site-directed mutagenesis and their properties characterized (Carlino et al, 1987;Creasey et al, 1987;Tsujimoto et al, 1987;Ostade et al, 1988;Kamiljo et al, 1989;Yamamoto et al, 1989). In order to produce a large amount of hTNF for structural studies, e.g., by x-ray crystallography and NMR, we synthesized a gene for hTNF, expressed it in E. coli, and purified the recombinant hTNF.…”

Section: Introductionmentioning

confidence: 99%

27 amino acid residues can be deleted from the N‐terminus of human lymphotoxin without impairment of its cytotoxic activity

Nishikawa

Matsuo

Isaka

et al. 1990

J of Molecular Recognition

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In order to study the relationship between activity and structure of human lymphotoxin (hLT, 171 aa), we synthesized the gene (519 bp) for hLT and expressed it in Escherichia coli. Purification of the recombinant hLT from crude extracts was difficult because of the low level of expression of the gene. To improve the yield of the recombinant protein, we prepared five truncated genes for mutant proteins in which 25, 26, 27, 28 and 37 amino acid residues, respectively, were missing from the N-terminus. All of the genes were efficiently expressed and adequate amounts of mutant proteins were synthesized. The proteins were recovered mainly in the supernatant fractions after disruption of cells, with the exception of LT delta 37N, in which 37 residues were absent from the N-terminal region. Cytotoxic activities against mouse fibroblast L929 cells were detected in supernatant fractions that contained these mutant proteins, except in the case of LT delta 28N, which lacks the first amino acid residue conserved in both hLT and human tumour necrosis factor (hTNF). LT delta 27N, which is the smallest of the active proteins, was purified to homogeneity, and its cytotoxic activity was found to be similar to that of recombinant hTNF.

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Section: Introductionmentioning

confidence: 99%

27 amino acid residues can be deleted from the N‐terminus of human lymphotoxin without impairment of its cytotoxic activity

Nishikawa

Matsuo

Isaka

et al. 1990

J of Molecular Recognition

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Structure-function studies of nerve growth factor: functional importance of highly conserved amino acid residues.

et al. 1990

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Selected amino acid residues in chicken nerve growth factor (NGF) were replaced by site‐directed mutagenesis. Mutated NGF sequences were transiently expressed in COS cells and the yield of NGF protein in conditioned medium was quantified by Western blotting. Binding of each mutant to NGF receptors on PC12 cells was evaluated in a competition assay. The biological activity was determined by measuring stimulation of neurite outgrowth from chick sympathetic ganglia. The residues homologous to the proposed receptor binding site of insulin (Ser18, Met19, Val21, Asp23) were substituted by Ala. Replacement of Ser18, Met19 and Asp23 did not affect NGF activity. Modification of Val21 notably reduced both receptor binding and biological activity, suggesting that this residue is important to retain a fully active NGF. The highly conserved Tyr51 and Arg99 were converted into Phe and Lys respectively, without changing the biological properties of the molecule. However, binding and biological activity were greatly impaired after the simultaneous replacement of both Arg99 and Arg102 by Gly. The three conserved Trp residues at positions 20, 75 and 98 were substituted by Phe. The Trp mutated proteins retained 15‐60% of receptor binding and 40‐80% of biological activity, indicating that the Trp residues are not essential for NGF activity. However, replacement of Trp20 significantly reduced the amount of NGF in the medium, suggesting that this residue may be important for protein stability.

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

et al. 1991

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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.

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Two conserved tryptophan residues of tumor necrosis factor and lymphotoxin are not involved in the biological activity

Cited by 9 publications

References 15 publications

27 amino acid residues can be deleted from the N‐terminus of human lymphotoxin without impairment of its cytotoxic activity

27 amino acid residues can be deleted from the N‐terminus of human lymphotoxin without impairment of its cytotoxic activity

Structure-function studies of nerve growth factor: functional importance of highly conserved amino acid residues.

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

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