Postgestational lymphotoxin/lymphotoxin beta receptor interactions are essential for the presence of intestinal B lymphocytes

Lymphotoxin (LT), a cytokine belonging to the TNF family, has established roles in the formation of secondary lymphoid structures and in the compartmentalization of T and B lymphocyte areas of the spleen. In this study, we examine the role of LT in directing the composition of intestinal lymphocytes. We report that mice deficient in LT have a normal composition of intestinal lamina propria (LP) T lymphocytes, and an absence of intestinal LP B lymphocytes. We further refine this observation to demonstrate that the interaction of LT with the LTbetaR is essential for the presence LP B lymphocytes. The LT/LTbetaR-dependent events relevant for the presence of LP B lymphocytes occur after birth, do not require the presence of Peyer's patches, lymph nodes, or the spleen; and therefore, are distinct and independent from the previously identified roles of LT/LTbetaR. The LT-dependent signal relevant for the presence of LP B lymphocytes is optimally supplied by a LT-sufficient B lymphocyte, and requires a LTbetaR-sufficient radio-resistant, non-bone marrow-derived cell. Based upon the severity of the deficit of LP B lymphocytes we observed, these novel LT/LTbetaR-dependent events are of primary importance in directing the entry and residence of LP B lymphocytes.     

Aspartic acid 252 and asparagine 185 are essential for activity of lipid N-acetylglucosaminylphosphate transferase

A key step in the assembly of oligosaccharide-lipid intermediatesin N-linked glycosylation is the transfer of N-acetylglucosamine1-phosphate to dolichyl phosphate, catalyzed by the enzyme UDP-N-acetylglucosaminyl:dolichylphosphate N-acetylglucosaminyl phosphoryl transferase (L-G1PT).Comparison of the amino acid sequences of L-G1PT from five diversespecies showed 75 amino acids identical in all five proteins.Using site-directed mutagen-esis, we analyzed the importanceof a number of these conserved residues to the enzymatic activityof L-G1PT using a plasmid shuffling procedure in Schizosaccharomycespombe. S.pombe cells containing a chromosomal deletion of theessential gpt⁺ gene are rescued by a plasmid containing theS.pombe gpt open reading frame. Replacement of that plasmidby a plasmid encoding a mutated hamster L-G1PT cDNA sequenceindicated that the mutated protein provided sufficient enzymeactivity to permit cell growth. Mutations of aspartic acid 252and asparagine 185 did not allow plasmid shuffling, indicatingthese residues were essential for activity. A combination ofmutations at asparagine 182 and tryptophan 122 did not allowplasmid shuffling, although the single mutations did. Overexpressionof the mutant proteins in S.pombe conferred tunicamycin (TM)resistance, indicating that the mutant proteins had a conformationnecessary for binding TM, a substrate analog. The mutant proteinswere also detected in Western blots and were correctly localizedto the membrane fractions. However, the overexpressed proteinsdid not increase the endogenous level of enzymatic activityin these cells, indicating they were enzymatically inactive. N-acetylglucosaminylphosphate transferase mutagenesis plasmid shuffling Schizosaccharomyces pombe tunicamycin     

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

Each of the two highly conserved tryptophan residues in hTNF (positions 28 and 114) was converted into phenylalanine by site-directed mutagenesis and the mutant proteins were partially purified. A cytotoxicity assay on mouse L929 cells showed only a slight reduction in biological activity, strongly suggesting that neither of the two amino acids is involved in the active site.     

Natural human tumor necrosis factor beta (lymphotoxin). Variable O-glycosylation at Thr7, proteolytic processing, and allelic variation.

Natural human tumor necrosis factor beta (TNF-beta) purified from supernatants of a human B-lymphoblastoid cell line was found to be heterogeneous in molecular mass, with seven components resolved by gel electrophoresis. All components are N-glycosylated at Asn62; N-glycosylation does not contribute to heterogeneity. In addition, part of the molecules are O-glycosylated at Thr7; O-glycosylation is heterogeneous due to variable decoration with neuraminic acid. The four lower molecular mass forms are derived from the full-length protein by trypsin-like proteolytic cleavage in the N-proximal region; these clipped molecules lack O-linked carbohydrates. Two allelic variants differing in amino acid position 26 (threonine/asparagine) were identified.     

Aspartic acid residues at positions 190 and 192 of rat DNA polymerase beta are involved in primer binding

The sequence Gly-Asp-Met-Asp, spanning positions 189-192 of rat DNA polymerase beta, is similar to the sequence motif Gly-Asp-Thr-Asp that is highly conserved in a number of replicative DNA polymerases from eukaryotic cells, viruses, and phages. The role of this sequence in the catalytic function of rat DNA polymerase beta was investigated by individually changing each amino acid in this region by site-directed mutagenesis. The mutant enzymes DE190 and DE192, in which aspartic acid residues at positions 190 and 192, respectively, were replaced by glutamic acid, showed about 0.1% activity of the wild-type enzyme. On the other hand, the replacement of Gly-189 by alanine or Met-191 by isoleucine or threonine only slightly affected the enzyme activity. A gel mobility shift assay showed that DNA complexes with enzyme DE190 and especially with DE192 were less stable than the corresponding complex with the wild-type enzyme. Kinetic analysis with these mutant enzymes indicate that their Km's for primer DNA were about 10-fold higher than that of the wild type, while Km's for deoxyribonucleoside triphosphate were not changed. Since neither DE190 nor DE192 had any significant alteration in secondary structure, our results suggest that both Asp-190 and Asp-192 are located in the active site and are involved in the interaction of DNA polymerase beta with primer.     

Conserved residues of tumour necrosis factor and lymphotoxin constitute the framework of the trimeric structure

Four distinct areas of primary sequence conservation between known tumour necrosis factor and lymphotoxin polypeptides from various species can be recognized. When these amino acid sequences are highlighted in the three-dimensional structure, all are found in the same region, constituting the framework of the trimeric structure.     

Probing the diphosphoglycerate binding pocket of HbA and HbPresbyterian (beta 108Asn --> Lys).

HbPresbyterian (beta 108Asn --> Lys, HbP) contains an additional positive charge (per alpha beta dimer) in the middle of the central cavity and exhibits a lower oxygen affinity than wild-type HbA in the presence of chloride. However, very little is known about the molecular origins of its altered functional properties. In this study, we have focused on the beta beta cleft of the Hb tetramer. Recently, we developed an approach for quantifying the ligand binding affinity to the beta-end of the Hb central cavity using fluorescent analogues of the natural allosteric effector 2, 3-diphosphoglycerate (DPG) [Gottfried, D. S., et al. (1997) J. Biol. Chem. 272, 1571-1578]. Time-correlated single-photon counting fluorescence lifetime studies were used to assess the binding of pyrenetetrasulfonate to both HbA and HbP in the deoxy and CO ligation states under acidic and neutral pH conditions. Both the native and mutant proteins bind the probe at a weak binding site and a strong binding site; in all cases, the binding to HbP was stronger than to HbA. The most striking finding was that for HbA the binding affinity varies as follows: deoxy (pH 6.35) > deoxy (pH 7.20) > CO (pH 6.35); however, the binding to HbP is independent of ligation or pH. The mutant oxy protein also hydrolyzes p-nitrophenyl acetate, through a reversible acyl-imidazole pathway linked to the His residues of the beta beta cleft, at a considerably higher rate than does HbA. This implies a perturbation of the microenvironment of these residues at the DPG binding pocket. Structural consequences due to the presence of the new positive charge in the middle of the central cavity have been transmitted to the beta beta cleft of the protein, even in its liganded conformation. This is consistent with a newly described quaternary state (B) for liganded HbPresbyterian and an associated change in the allosteric control mechanism.     

Analysis of cytostatic/cytotoxic lymphokines: relationship of natural killer cytotoxic factor to recombinant lymphotoxin, recombinant tumor necrosis factor, and leukoregulin

Previous results that were obtained by using supernatants from the co-culture of human peripheral blood lymphocytes and the natural killer susceptible cell line K562 strongly inhibited the growth of various tumor cell lines. No correlation was observed between the susceptibility of the target cell lines to growth inhibition and to lysis by natural killer cells. Rather the spectrum of cytostatic activity and the characteristics of the soluble factor were similar to those of leukoregulin (LRG), a recently described lymphokine. Because of the recent availability of recombinant tumor necrosis factor (TNF) and lymphotoxin (LT), we compare the target selectivity and mechanism of action of these (TNF, LT, LRG) factors with natural killer cytotoxic factor (NKCF). The pattern of target cell susceptibility to growth inhibition or cytolysis by the factors were quite distinct from the pattern observed when cells were exposed to NKCF. Furthermore, antibodies to rLT or rTNF had no effect on LRG cytostasis or NKCF lysis, arguing against a requirement for or synergistic interaction with low levels of LT or TNF. Some of the targets susceptible to LRG were growth inhibited but were not lysed, thereby distinguishing it from NKCF. Furthermore, LRG cytostasis was not inhibited by mannose-6-PO4 or rabbit antibodies to granule cytolysin, both of which block natural killer cytotoxic factor. Therefore, LRG appears to be a cytostatic factor produced by large granular lymphocytes in response to K562 that is distinct from NKCF, TNF, and LT. In addition, NKCF, rLT, rTNF, and LRG, although having cytotoxic/cytostatic activity, are distinct functional factors and may represent a family of lytic factors.     

Kinase activity and calmodulin binding are essential for growth signaling by the phytosulfokine receptor PSKR1

The cell growth‐promoting peptide phytosulfokine (PSK) is perceived by leucine‐rich repeat (LRR) receptor kinases. To elucidate PSK receptor function we analyzed PSKR1 kinase activity and binding to Ca²⁺ sensors and evaluated the contribution of these activities to growth control in planta. Ectopically expressed PSKR1 was capable of auto‐ and transphosphorylation. Replacement of a conserved lysine within the ATP‐binding region by a glutamate resulted in the inhibition of auto‐ and transphosphorylation kinase activities. Expression of the kinase‐inactive PSKR1(K762E) receptor in the pskr null background did not restore root or shoot growth. Instead, the mutant phenotype was enhanced suggesting that the inactive receptor protein exerts growth‐inhibitory activity. Bioinformatic analysis predicted a putative calmodulin (CaM)‐binding site within PSKR1 kinase subdomain VIa. Bimolecular fluorescence complementation analysis demonstrated that PSKR1 binds to all isoforms of CaM, more weakly to the CaM‐like protein CML8 but apparently not to CML9. Mutation of a conserved tryptophan (W831S) within the predicted CaM‐binding site strongly reduced CaM binding. Expression of PSKR1(W831S) in the pskr null background resulted in growth inhibition that was similar to that of the kinase‐inactive receptor. We conclude that PSK signaling requires Ca²⁺/CaM binding and kinase activity of PSKR1 in planta. We further propose that the inactivated kinase interferes with other growth‐promoting signaling pathway(s).     

Recombinant 55-kDa tumor necrosis factor (TNF) receptor. Stoichiometry of binding to TNF alpha and TNF beta and inhibition of TNF activity.

The extracellular domain of the 55-kDa TNF receptor (rsTNFR beta) has been expressed as a secreted protein in baculovirus-infected insect cells and Chinese hamster ovary (CHO)/dhfr- cells. A chimeric fusion protein (rsTNFR beta-h gamma 3) constructed by inserting the extracellular part of the receptor in front of the hinge region of the human IgG C gamma 3 chain has been expressed in mouse myeloma cells. The recombinant receptor proteins were purified from transfected cell culture supernatants by TNF alpha- or protein G affinity chromatography and gel filtration. In a solid phase binding assay rsTNFR beta was found to bind TNF alpha with high affinity comparable with the membrane-bound full-length receptor. The affinity for TNF beta was slightly impaired. However, the bivalent rsTNFR beta-h gamma 3 fusion protein bound both ligands with a significantly higher affinity than monovalent rsTNFR beta reflecting most likely an increased avidity of the bivalent construct. A molecular mass of about 140 kDa for both rsTNFR beta.TNF alpha and rsTNFR beta.TNF beta complexes was determined in analytical ultracentrifugation studies strongly suggesting a stoichiometry of three rsTNFR beta molecules bound to one TNF alpha or TNF beta trimer. Sedimentation velocity and quasielastic light scattering measurements indicated an extended structure for rsTNFR beta and its TNF alpha and TNF beta complexes. Multiple receptor binding sites on TNF alpha trimers could also be demonstrated by a TNF alpha-induced agglutination of Latex beads coated with the rsTNFR beta-h gamma 3 fusion protein. Both rsTNFR beta and rsTNFR beta-h gamma 3 were found to inhibit binding of TNF alpha and TNF beta to native 55- and 75-kDa TNF receptors and to prevent TNF alpha and TNF beta bioactivity in a cellular cytotoxicity assay. Concentrations of rsTNFR beta-h gamma 3 equimolar to TNF alpha were sufficient to neutralize TNF activity almost completely, whereas a 10-100-fold excess of rsTNFR beta was needed for similar inhibitory effects. In view of their potent TNF antagonizing activity, recombinant soluble TNF receptor fragments might be useful as therapeutic agents in TNF-mediated disorders.     

The genes for tumor necrosis factor (TNF-alpha) and lymphotoxin (TNF-beta) are tandemly arranged on chromosome 17 of the mouse

We have isolated clones containing the gene for tumor necrosis factor (TNF-alpha) from a mouse genomic library. Four out of five clones containing the TNF-alpha gene also hybridized to a human lymphotoxin (TNF-beta) probe. We constructed a restriction enzyme cleavage map of a 6.4 kb region from one of the genomic clones. From partial sequencing data and hybridizations with exon-specific oligonucleotide probes, we conclude that this region contains the mouse TNF-alpha and TNF-beta genes in a tandem arrangement, that they are separated by only about 1100 bases, and that their intron-exon structure is very similar to that seen in man. We probed genomic blots of DNA from human/mouse hybrids containing single mouse chromosomes for the presence of the mouse TNF genes. The results show that the genes are located on mouse chromosome 17, which also contains the major histocompatibility complex. Therefore, both the mouse and the human TNF genes are tandemly arranged and located on the same chromosome as the MHC.     

Receptor tyrosine phosphatase beta (RPTPbeta) activity and signaling are attenuated by glycosylation and subsequent cell surface galectin-1 binding

O-Mannosyl-linked glycosylation is abundant within the central nervous system, yet very few glycoproteins with this glycan modification have been identified. Congenital diseases with significant neurological defects arise from inactivating mutations found within the glycosyltransferases that act early in the O-mannosyl glycosylation pathway. The N-acetylglucosaminyltransferase known as GnT-Vb or -IX is highly expressed in brain and branches O-mannosyl-linked glycans. Our results using SH-SY5Y neuroblastoma cells indicate that GnT-Vb activity promotes the addition of the O-mannosyl-linked HNK-1 modification found on the developmentally regulated and neuron-specific receptor protein-tyrosine phosphatase beta (RPTPbeta). These changes in glycosylation accompany decreased cell-cell adhesion and increased rates of migration on laminin. In addition, we show that expression of GnT-Vb promotes its dimerization and inhibits RPTPbeta intrinsic phosphatase activity, resulting in higher levels of phosphorylated beta-catenin, suggesting a mechanism by which GnT-Vb glycosylation couples to changes in cell adhesion. GnT-Vb-mediated glycosylation of RPTPbeta promotes galectin-1 binding and RPTPbeta levels of retention on the cell surface. N-Acetyllactosamine, but not sucrose, treatment of cells results in decreased RPTP retention, showing that galectin-1 binding contributes to the increased retention after GnT-Vb expression. These results place GnT-Vb as a regulator of RPTPbeta signaling that influences cell-cell and cell-matrix interactions in the developing nervous system.     

Methylation and proteolysis are essential for efficient membrane binding of prenylated p21K-ras(B).

Plasma membrane targeting of p21K-ras(B) requires a CAAX motif and a polybasic domain. The CAAX box directs a triplet of post-translational modifications: farnesylation, proteolysis of the AAX amino acids and methylesterification. These modifications are closely coupled in vivo. However, in vitro translation of mRNA in rabbit reticulocyte lysates produces p21K-ras(B) proteins which are arrested in processing after farnesylation. Intracellular membranes are then required both for proteolytic removal of the AAX amino acids and methylesterification of farnesylated p21K-ras(B). Binding of p21K-ras(B) to plasma membranes in vitro can then be shown to depend critically on AAX proteolysis and methylesterification since p21K-ras(B) which is farnesylated, but not methylated, binds inefficiently to membranes.     

Multiple autophosphorylation sites of the epidermal growth factor receptor are essential for receptor kinase activity and internalization. Contrasting significance of tyrosine 992 in the native and truncated receptors.

The role of epidermal growth factor (EGF) receptor autophosphorylation sites in the regulation of receptor functions has been studied using cells transfected with mutant EGF receptors. Simultaneous point mutation of 4 tyrosines (Y1068, Y1086, Y1148, Y1173) to phenylalanine, as well as removal of these sites by truncation of the carboxyl-terminal 123 amino acid residues, resulted in reduced receptor phosphorylation of an in vivo specific substrate phospholipase C-gamma 1 to less than 50% compared to the wild-type receptor. The internalization rate constant Ke was also significantly lower in these mutants (0.15/min) compared to cells transfected with wild-type receptor (0.27/min). Additional mutation of tyrosine 992 to phenylalanine in the truncated receptor mutant (Dc-123F) further decreased the receptor internalization rate to a minimal level (ke = 0.07-0.10/min), equivalent to the ke measured for cells expressing kinase-negative receptor (A721). Moreover, tyrosine kinase activity of the Dc-123F receptor toward phospholipase C-gamma 1, compared to wild-type receptor, was reduced by 90%. Taken together, these results show that EGF receptor lacking five autophosphorylation sites functions similar to a kinase-negative receptor. Mutation of tyrosine residue Y992 alone in the context of full length EGF receptor, however, did not affect receptor internalization or kinase activity toward phospholipase C-gamma 1. These data indicate that tyrosine 992 is critical for substrate phosphorylation and internalization only in the context of the truncated receptor, and that minor autophosphorylation sites, such as Y992, may act as compensatory regulatory sties in the absence of the major EGF receptor autophosphorylation sites.     

Tyrosine 331 and phenylalanine 334 in Clostridium perfringens alpha-toxin are essential for cytotoxic activity

Differences in the biological properties of the Clostridium perfringens phospholipase C (alpha-toxin) and the C. bifermentans phospholipase C (Cbp) have been attributed to differences in their carboxy-terminal domains. Three residues in the carboxy-terminal domain of alpha-toxin, which have been proposed to play a role in membrane recognition (D269, Y331 and F334), are not conserved in Cbp (Y, L and I respectively). We have characterised D269Y, Y331L and F334I variant forms of alpha-toxin. Variant D269Y had reduced phospholipase C activity towards aggregated egg yolk phospholipid but increased haemolytic and cytotoxic activity. Variants Y331L and F334I showed a reduction in phospholipase C, haemolytic and cytotoxic activities indicating that these substitutions contribute to the reduced haemolytic and cytotoxic activity of Cbp.     

Recombinant glucocorticoid induced tumour necrosis factor receptor (rGITR) induced COX-2 activity in murine macrophage Raw 264.7 cells

Stimulation of murine macrophages with recombinant soluble glucocorticoid induced tumour necrosis factor receptor (GITR) induced cyclooxygenase (COX)-2 protein and generated significant amounts of PGE(2). Previous result demonstrated that macrophages express GITR and GITR ligand constitutively. Induction of COX-2 was synergistic with interferon (INF)-gamma. GITR/ligand system could deliver an activation signal to macrophages in inflammatory processes.     

Mechanisms of 4-hydroxytamoxifen anti-growth factor activity in breast cancer cells: alterations of growth factor receptor binding sites and tyrosine kinase activity

We previously demonstrated that antiestrogen 4-hydroxytamoxifen (OH-Tam) blocks the mitogenic activity of growth factors in breast cancer. We now investigate this mechanism by evaluating how OH-Tam affects growth factor binding and receptor tyrosine kinase activity. We show here that OH-Tam has an opposite effect on epidermal growth factor (EGF) and insulin-like growth factor-1 (IGF-1) binding in estrogen receptor (ER) positive cells. A decrease in IGF-1 binding sites may explain the reduced IGF-I mitogenic effect, whereas an increase in high affinity EGF binding associated with a decrease in in vitro receptor autophosphorylation rather favors the possibility of an alteration in EGF receptor tyrosine kinase activity. We conclude that OH-Tam may prevent growth factor action in ER+ cells both by modulating the concentration of growth factor binding sites and by altering growth factor receptor functionality.     

Role of amino acid residue 90 in bioactivity and receptor binding capacity of tumor necrosis factor mutants

We have previously produced two bioactive lysine-deficient mutants of TNF-alpha (mutTNF-K90R,-K90P) and found that these mutants have bioactivity superior to wild-type TNF (wtTNF). Because these mutants contained same amino acid except for amino acid 90, it is unclear which amino acid residue is optimal for showing bioactivity. We speculated that this amino acid position was exchangeable, and this amino acid substitution enabled the creation of lysine-deficient mutants with enhanced bioactivity. Therefore, we produced mutTNF-K90R variants (mutTNF-R90X), in which R90 was replaced with other amino acids, to assay their bioactivities and investigated the importance of amino acid position 90. As a result, mutTNF-R90X that replaced R90 with lysine, arginine and proline were bioactive, while other mutants were not bioactive. Moreover, these three mutants showed bioactivity as good as or better than wtTNF. R90 replaced with lysine or arginine had especially superior binding affinities. These results suggest that the amino acid position 90 in TNF-alpha is important for TNF-alpha bioactivity and could be altered to improve its bioactivity to generate a "super-agonist".     

Phe496 and Leu497 are essential for receptor binding and cytotoxic action of the murine interleukin-4 receptor targeted fusion toxin DAB389-mIL-4.

DAB389-mIL-4 is a murine interleukin-4 (mIL-4) diphtheria toxin-related fusion protein which has been shown to be selectively toxic to cells expressing the mIL-4 receptor. In this report, we have used site-directed and in-frame deletion mutagenesis to study the role of the putative C-terminal alpha-helix (helix E) of the mIL-4 component of DAB389-mIL-4 in the intoxication process. We demonstrate that deletion of the C-terminal 15 amino acids of the fusion toxin leads to loss of cytotoxicity. The substitution of Phe496 with either Pro, Ala or Tyr, results in a greater than 20-fold decrease in cytotoxic activity of the respective mutant fusion toxins. In addition, substitution of Leu497 with either Ala or Glu results in a similar loss of cytotoxic activity. All of these mutant forms of the mIL-4 fusion toxin demonstrate a significant decrease in binding affinity (Ki) to the mIL-4 receptor in a competitive radioligand binding assay. In marked contrast, however, the substitution of Asp495 with Asn results in a 4-fold increase in cytotoxic potency and binding affinity to mIL-4 receptor bearing cells in vitro.