Aspartic acid 50 and tyrosine 108 are essential for receptor binding and cytotoxic activity of tumour necrosis factor beta (lymphotoxin)

Single amino acid substitutions were generated in predictedhydrophilic loop regions of the human tumour necrosis factorbeta (TNF-ß) molecule, and the mutant proteins wereexpressed in Escherichia coli and purified. Mutants with singleamino acid changes at either of two distinct loop regions, atpositions aspartic acid 50 or tyrosine 108, were found to havegreatly reduced receptor binding and cytotoxic activity. Thesetwo regions in TNF-ß correspond to known loop regionswhere mutations also result in loss of biological activity ofTNF–, a related cytokine which shares the same cellularreceptors with TNF-ß. The two distinct loops at positions31-34 and 84-89 in the known three-dimensional structure ofTNF- (equivalent to positions 46–50 and 105–110respectively in TNF-ß), lie on opposite sides of theTNF- monomer. When the TNF-a monomer forms a trimer, the twoloops, each from a different subunit of the trimer, come togetherand lie in a cleft between adjacent subunits. Together, thesefindings suggest that a TNF receptor binds to a cleft betweensubunits via surface loops at amino acid residues 31–34and 84–89 in TNF–, and similarly via surface loopsincluding amino acids aspartic acid 50 and tyrosine 108 in TNF–ß.     

Histidine-15: an important role in the cytotoxic activity of human tumor necrosis factor

The amino acids that are required for the cytotoxic activityof recombinant human tumor necrosis factor- (TNF) were investigatedby chemical modification and oligonucleotide-directed site-specificmutagenesis. TNF contains three histidine residues, locatedat positions 15, 73 and 78. The histidine-specific reagent diethylpyrocarbonate(DEP) was used to chemically modify TNF. The chemical inactivationof the in vitro cytotoxic activity of this lymphokine (usingmurine L929 target cells) was found to be time- and dose-dependent.Inactivated TNF failed to compete with fully bioactive [¹²⁵I]TNFfor human MCF-7 target cell receptors. Mutant polypeptides ofTNF were genetically engineered by oligonucoleotide-directedsite-specific mutagenesis. The cytotoxicity of a double histidinemutant, in which histidine-73 and histidine-78 were replacedwith glutamine, was not altered and was chemically inactivatedby DEP. Substituting glutamine for histidine-15 resulted in10–15% of the wild-type bioactivity. Replacing histidine-15with either asparagine, lysine or glycine resulted in a biologicallyinactive molecule. The data show that the histidine residueat position 15 is an amino acid that is required for the cytotoxicactivity of TNF.     

A mutational analysis of receptor binding sites of interleukin-1{beta}:differences in binding of human interleukin-1{beta} muteins to human and mouse receptors

The 3-D crystal structure of interleukin-1ß(IL-1ß)has been used to define its receptor binding surface by mutationalanalysis. The surface of IL-1ß was probed by site-directedmutagenesis. A total of 27 different IL-1ß muteinswere constructed, purified and analyzed. Receptor binding measurementson mouse and human cell lines were performed to identify receptoraffinities. IL-1ß muteins with modified receptor affinitywere evaluated for structural integrity by CD spectroscopy orX-ray crystallography. Changes in six surface loops, as wellas in the C- and N-termini, yielded muteins with lower bindingaffinities. Two muteins with intact binding affinities showed10- to 100-fold reduced biological activity. The surface regioninvolved in receptor binding constitutes a discontinuous areaof 1000 Å2 formed by discontinuous polypeptide chain stretches.Based on these results, a subdivision into two distinct localareas is proposed. Differences in receptor binding affinitiesfor human and mouse receptors have been observed for some muteins,but not for wild-type IL-1ß. This is the first timea difference in binding affinity of IL-1ß muteinsto human and mouse receptors has been demonstrated     

Diphtheria toxin receptor-binding domain substitution with interleukin 6: genetic construction and interleukin 6 receptor-specific action of a diphtheria toxin-related interleukin 6 fusion protein

We have genetically replaced that portion of the diphtheriatoxin structural gene which encodes the native receptor-bindingdomain with a synthetic gene encoding the cytokine interleukin6 (IL-6/IFN-ß2/BSF-2). The resulting gene fusion encodesthe chimeric toxin DAB₃₈₉-IL-6. Following expression and purification,we demonstrate that DAB₃₈₉-IL-6 is selectively cytotoxic foreukaryotic cells bearing the interleukin 6 receptor. In addition,the cytotoxic action of DAB₃₈₉-IL-6 is shown to require bindingto the IL-6 receptor, internalization by receptor-mediated endocytosisand passage through an acidic compartment. Following the deliveryof the catalytically active fragment A to the cytosol of targetcells, cellular protein synthesis is inhibited by the ADP-ribosylationof elongation factor 2. While eukaryotic cells which are devoidof the IL-6 receptor are uniformly resistant to the action ofthis fusion toxin, the data presented suggest that a minimalnumber of IL-6 receptors may be necessary to mediate the internalizationof sufficient levels of DAB₃₈₉-IL-6 to result in the intoxicationof target cells.     

Structural and functional domains in human tumour necrosis factors

Human tumour necrosis factors (hTNFs) and ß are relatedpleiotropic cytokines which share many activities and competewith each other for binding to two receptor components on manycell types. Although structural and biological data indicatethat the active form of hTNF- may be a symmetrical trimer, themanner in which hTNFs interact with their receptors to triggera myriad of cell type-dependent responses is not clear. A combinationof chemical modification, epitope mapping and site-directedmutagenesis approaches suggest that at least four distinct peptidesequences are Important for the biological activity of hTNF-.In particular, certain peptide sequences between amino acidpositions 11 and 35 in hTNF- appear to be critical for receptorbinding and triggering biological responses. The recent cloningof the two hTNF-/ß receptors opens the way for precisemapping of the functional domains in hTNFs     

Expression of deletion constructs of bovine {beta}-1, 4-galactosyltransferase in Escherichia coli: importance of Cysl34 for its activity

Bovine ß-1, 4-galactosyltransferase (ß-1,4-GT; EC 2.4.1.90 [EC] ) belongs to the glycosyltransferase familyand as such shares a general topology: an N-terminal cytoplasmictail, a signal anchor followed by a stem region and a catalyticdomain at the C-tenninal end of the protein. cDNA constructsof the N-terminal deleted forms of ß-1, 4-GT wereprepared in pGEX-2T vector and expressed in E.coli as glutathione-S-transferase(GST) fusion proteins. Recombinant proteins accumulated withininclusion bodies as insoluble aggregates that were solubilizedin 5 M guanidine HCl and required an ‘oxido-shuffling’reagent for regeneration of the enzyme activity. The recombinant(ß-1, 4-GT, devoid of the GST domain, has 30–85%of the sp. act. of bovine milk ß-1, 4-GT with apparentK_ms for N-acetylglucosamine and UDP-galactose similar to thoseof milk enzyme. Deletion analysesshow that both (ß-1,4-GT and lactose synthetase activities remain intact even inthe absence of the first 129 residues (pGT-dl29). The activitiesare lost when either deletions extend up to residue 142 (pGT-dl42)or Cysl34 is mutatedto Ser (pGT-dl29C134S). These results suggestthat the formation of a disulfide bond involving Cysl34 holdsthe protein in a conformation that is required for enzymaticactivity.     

A new protein conjugate that replaces the use of secondary antibodies engineered from the two staphylococcal enzymes protein A and 6-phospho-{beta}-galactosidase

The lacG gene encoding the 6-phospho-ß-galactosidase(E.C.3.2.1.85) of Staphylococcus aureus was fused to the proteinA gene in the plasmid pRIT2T. Escherichia coli cells containingthis plasmid produce a fusion protein with both IgG bindingand 6-phospho-ß-galactosidase activities after heatinduction. The recombinant gene was overexpressed and the hybridprotein was purified to homogeneity in high yield. The chimericprotein was shown to have almost identical enzymatic characteristicsto pure 6-phospho-ß-galactosidase. This result leadsto the conclusion that a free N-terminus of the 6-phospho-ß-galactosidaseis not required for biological activity. The hybrid proteinof protein A and 6-phospho-ß-galactosidase was usedas an enzyme conjugate in enzyme-linked immunosorbent assays(ELISA). The experiments presented demonstrate that the 6-phospho-ß-galactosidaseis a suitable fusion partner in various diagnostic applicationswhere an unique biological activity is required.     

Inhibition of cell growth by a fused protein of human ribonuclease 1 and human basic fibroblast growth factor

Pancreatic-type RNases are considered to have cytotoxic potentialdue to their ability to degrade RNA molecules when they enterthe cytosol. However, most of these RNases show little cytotoxicitybecause cells have no active uptake mechanism for these RNasesand because the ubiquitous cytoplasmic RNase inhibitor is consideredto play a protective role against the endocytotic leak of RNasesfrom the outside of cells. To study the cytotoxic potentialof RNase toward malignant cells targeting growth factor receptors,the C-terminus of human RNase 1 was fused to the N-terminusof human basic fibroblast growth factor (bFGF). This RNase–FGFfused protein effectively inhibited the growth of mouse melanomacell line B16/BL6 with high levels of cell surface FGF receptor.This effect appeared to result from prolongation of the overallcell cycle rather than the killing of cells or specific arrestin a particular phase of the cell cycle. Thus, human RNase 1fused to a ligand of cell surface molecules, such as the FGFreceptor, is shown to be an effective candidate for a selectivecell targeting agent with low toxic effects on normal cell types.     

Permuteins of interleukin 1{beta}--a simplified approach for the construction of permutated proteins having new termini

A technique for the rapid and simple generation of permutatedversions of the interleukin-1ß (IL-1ß) geneis described. In this method, the human IL-1ß cDNAis twice amplified by the polymerase chain reaction (PCR) andthe resulting DNA fragments are ligated in tandem. Between thetwo genes, the DNA sequence encodes a short four amino acidloop to link the native N- and C-terminal ends of the IL-1ßprotein. By using PCR amplification from this starting template,a new version of the IL-1ß cDNA was obtained thatencodes a permutated form of the IL-1ß protein wherethe new N- and C-terminal amino acids correspond to residues65 and 64 of the native IL-1ß sequence, respectively.The name ‘permutein’ is proposed to describe proteinsgenerated by this technology. The molecular profile (IL-1 receptorbinding, biologic activity and solution properties) of the IL-1permutein produced by this technology, permutein 65/64, is shownto be identical to that of native IL-1ß The approachshould be useful to define further the structural features ofthis protein that are important for its function.     

Study of cysteine residues in the {alpha} subunit of Escherichia coli tryptophan synthase. 2. Role in enzymatic function

To understand the functional roles of Cys residues in the subunitof tryptophan synthase from Escherichia coli, single mutantsof the subunit, in which each of the three Cys residues wassubstituted with Ser, Gly, Ala or Val, were constructed by site-directedmutagenesis. The effects of the substitutions on the functionof tryptophan synthase were investigated by activity measurements,calorimetric measurements of association with the ßsubunit and steadystate kinetic analysis of catalysis. Althoughthe three Cys residues are located away from the apparentlyimportant parts for enzymatic activity, substitutions at position81 by Ser, Ala or Val caused decreases in the intrinsic activityof the subunit. Furthermore, Cys81Ser and Cys81Val reducedstimulation activities in the and ß reactions dueto formation of a complex with the ß subunit. Thelower stimulation activities of the mutant proteins were notcorrelated with their abilities to associate with the ßsubunit but were correlated with decreases in k_cat. The presentresults suggest that position 81 plays an indirectly importantrole in the activity of the subunit itself and the mutual activationmechanism of the complex.