Role of disulfide bonds in biologic activity of human interleukin-6.

We have examined the functional importance of the two disulfide bonds formed by the four conserved cysteines of human interleukin (IL-6). Using a bacterial expression system, we have synthesized a series of recombinant IL-6 mutants in which the constituent cysteines of the first (Cys45-Cys51), second (Cys74-Cys84), or both disulfide bonds of recombinant human interleukin-6 were replaced by other amino acids. Each mutant was partially purified and tested in four representative bioassays. While mutants lacking Cys45 and Cys51 retained activity similar to nonmutated recombinant IL-6, the activity of mutants lacking Cys74 and Cys84 was significantly reduced, especially in assays involving human cell lines. These results indicate that the first disulfide bond of human interleukin-6 is not required for maintenance of normal biologic activity. However, the fact that mutants lacking Cys45 and Cys51 were more active than corresponding cysteine-free mutants indicates that the disulfide bond formed by these residues contributes to biologic activity in the absence of the second disulfide bond. Competition binding studies with representative mutants indicate that their affinity for the human IL-6 receptor parallels their biologic activities on human cells.     

Effects of site-specific mutations on biologic activities of recombinant human IL-6

To examine structure-activity relationships of human IL-6, we have determined the effects of specific mutations on the biologic activity of a human rIL-6 expressed in bacteria. Three types of mutants were examined: 1) a variant that contains serines in place of the four naturally occurring cysteines; 2) a series of cysteine-containing deletion mutants, each having a single internal 20 amino acid deletion; and 3) a cysteine-free variant containing a single 20 amino acid deletion. The mutants of the second type constitute a set of nonoverlapping, adjacent deletions spanning amino acids 4 through 183 of the 184 amino acids in natural human IL-6. All of the mutants were expressed, along with the full length, cysteine-containing analogue, in Escherichia coli as fusion proteins, joined to beta-galactosidase through a collagen linker. This system allows microgram quantities of the rIL-6 variants to be partially purified from small bacterial cultures without chromatographic or refolding steps. Each of the rIL-6 variants was released from the beta-galactosidase fusion protein with collagenase, and the recovered rIL-6 was quantitated by laser densitometry of Coomassie-stained, SDS polyacrylamide gels. The sp. ac. of each of the rIL-6 variants was determined using four assays: induction of IgM secretion from an EBV transformed human B cell line, induction of fibrinogen secretion from a human hepatoma cell line, induction of fibrinogen secretion from a rat hepatoma cell line, and induction of proliferation of a murine hybridoma cell line. Replacement of cysteines with serines reduced activity relative to cysteine-containing rIL-6 to about 20% in the rat hepatoma assay and about 3% in the mouse hybridoma assay, whereas activity in both of the human cell lines was reduced to less than 0.1%. These data suggest that the murine and rat cell lines are less selective than the human cell lines in their requirements for recognition of biologically active IL-6. Each of the deletions, except that of amino acids 4 through 23, resulted in loss of activity in all four assays. These results suggest that the information necessary for activity is not contained within any one portion of the IL-6 molecule, but rather that multiple segments of the protein are required for each of the biologic activities that we tested.     

Two observed regions in B lymphocyte stimulator important for its biological activity

B lymphocyte stimulator (BLyS),a member of the tumor necrosis factor superfamily ofligands,is a crucial survival factor for B cells.We successfully constructed seven mutants of the functionalsoluble fragment of human BLyS (named cBLyS,amino acid 134-285),including three deletion mutants andfour site-directed mutants.All the mutant proteins were expressed in Escherichia coli and purified by Ni-NTA affinity chromatography.The biological activities of these mutants were assessed by the ligand-recep-tor binding assay,B cell proliferation assay and immune effect response in vivo.Our results indicated thatfour residues,H~(218),F~(220),T~(228) and L~(229),are indispensable for the biological activity of cBLyS,whereas tworegions,amino acid 134-148 and amino acid 271-285,are related to the biological activity of BLyS.Theprotein of deletion of amino acid 134-148 leads to a complete defection in raising the antigen-specific IgMtiter.The deletion of amino acid 271-285 reduces the effectiveness compared with the native cBLyS.Thisindicates that the region of amino acid 134-148 is indispensable for cBLyS to function normally.     

Definition of regions in human c-myc that are involved in transformation and nuclear localization.

To study the relationship between the primary structure of the c-myc protein and some of its functional properties, we made in-frame insertion and deletion mutants of the normal human c-myc coding domain that was expressed from a retroviral promoter-enhancer. We assessed the effects of these mutations on the ability of c-myc protein to cotransform normal rat embryo cells with a mutant ras gene, induce foci in a Rat-1-derived cell line (Rat-1a), and localize in nuclei. Using the cotransformation assay, we found two regions of the protein (amino acids 105 to 143 and 321 to 439) where integrity was critical: one region (amino acids 1 to 104) that tolerated insertion and small deletion mutations, but not large deletions, and another region (amino acids 144) to 320) that was largely dispensable. Comparison with regions that were important for transformation of Rat-1a cells revealed that some are essential for both activities, but others are important for only one or the other, suggesting that the two assays require different properties of the c-myc protein. Deletion of each of three regions of the c-myc protein (amino acids 106 to 143, 320 to 368, and 370 to 412) resulted in partial cytoplasmic localization, as determined by immunofluorescence or immunoprecipitation following subcellular fractionation. Some abnormally located proteins retained transforming activity; most proteins lacking transforming activity appeared to be normally located.     

Site-specific mutagenesis of the human interleukin-1 beta gene: the role of arginine residue at the N-terminal region

Using the expression system for site-specific mutagenesis in Escherichia coli, we have made deletion mutants at the N-terminal or C-terminal region of human interleukin-1 beta (IL-1 beta) consisting of 153 amino acids. The truncated mutants showed that at least 147 amino acids (numbers 4-150) in IL-1 beta are necessary for the exertion of biological activity. When we changed the arginine at the 4th position (Arg4) in IL-1 beta to other specific amino acids, there was a marked difference in the relative extent of biological and receptor binding activities among the mutants. The order of the mutants was Arg4 = Lys4 greater than Gln4 greater than Gly4 = des-Arg4 greater than Asp4. Our results demonstrate that the arginine residue at the 4th position in IL-1 beta is important, but not essential, for IL-1 beta to exhibit its biological and receptor binding activities, and the positive charge at this site plays a key role for IL-1 beta to exert the activities.     

Analysis of human IL-6 mutants expressed in Escherichia coli. Biologic activities are not affected by deletion of amino acids 1-28

We have constructed and analyzed amino terminally deleted analogs of IL-6. Progressively shortened variants of mature IL-6 were constructed at the cDNA level and expressed in Escherichia coli. Mutant proteins were recovered from refractile bodies by solubilizing in 6 M guanidine-HCl. The mutant protein concentration in these preparations was estimated by Western blotting by using an IL-6-specific mAb and the biologic activity was measured in the B9 (hybridoma growth factor) assay. The first 28 amino acids of mature IL-6 could be removed without significantly affecting biologic activity. A further removal of amino acids 29 and 30 resulted in an approximately 50-fold decrease, whereas removal of amino acids 31 to 34 virtually abolished the activity. The mutants showed the same reaction pattern in three other IL-6 assays: induction of murine thymocyte proliferation, induction of fibrinogen synthesis by a human hepatoma cell line (HepG2), and the induction of IgM synthesis by an EBV-transformed B cell line. This suggests that a single functional domain might be responsible for all four activities of IL-6.     

Localization of the catalytic activity in restrictocin molecule by deletion mutagenesis.

Restrictocin, produced by the fungus Aspergillus restrictus, is a highly specific ribonucleolytic toxin which cleaves a single phosphodiester bond between G4325 and A4326 in the 28S rRNA. It is a nonglycosylated, single-chain, basic protein of 149 amino acids. The putative catalytic site of restrictocin includes Tyr47, His49, Glu95, Arg120 and His136. To map the catalytic activity in the restrictocin molecule, and to study the role of N- and C-terminus in its activity, we have systematically deleted amino-acid residues from both the termini. Three N-terminal deletions removing 8, 15 and 30 amino acids, and three C-terminal deletions lacking 4, 6, and 11 amino acids were constructed. The deletion mutants were expressed in Escherichia coli, purified to homogeneity and functionally characterized. Removal of eight N-terminal or four C-terminal amino acids rendered restrictocin partially inactive, whereas any further deletions from either end resulted in the complete inactivation of the toxin. The study demonstrates that intact N- and C-termini are required for the optimum functional activity of restrictocin.     

Structure-function analysis of human IL-6. Epitope mapping of neutralizing monoclonal antibodies with amino- and carboxyl-terminal deletion mutants.

To study the active site(s) of IL-6 we combined mutagenesis of IL-6 with epitope mapping of IL-6 specific mAb. In addition to amino-terminal deletion mutants we described previously, carboxyl-terminal deletion mutants were prepared. Functional analysis showed that deletion of only five carboxyl-terminal amino acids already reduced the bioactivity 1000-fold. A panel of mAb to IL-6 was subsequently analyzed by antibody competition experiments and binding to the amino- and carboxyl-terminal deletion mutants. On the basis of the competition experiments the six neutralizing mAb were divided in two groups (I and II). The binding pattern with the deletion mutants suggested that the region recognized by the four mAb in group I is composed of residues of amino- and carboxyl-terminus: binding of two mAb was abolished after deletion of amino acid Ala I-Ile26, of the third mAb after deletion of the four carboxyl-terminal amino acids whereas the fourth mAb did not bind to either mutant. Group II mAb retained binding to these mutants. Taken together these data suggest that in the native IL-6 molecule amino acid residues of amino and carboxyl terminus are in close proximity and that together they constitute an active site. Furthermore our data suggest that the part of the molecule recognized by group II antibodies is a second site involved in biologic activity.     

Interference with viral infection by RNA replicase deleted at the carboxy-terminal region

Escherichia coli cells harboring an altered Q beta RNA replicase which has amino acid substitutions of the glycine residue at position 357 in the conserved sequence Tyr356-Gly357-Asp358-Asp359 of the beta-subunit protein lost the replicase activity but interfered with proliferation of Q beta phage [Inokuchi and Hirashima (1987) J. Virol. 61, 3946-3949]. To examine the mechanism of the interference, we further analyzed various mutants lacking the carboxy-terminal region of the beta-subunit protein. The cells expressing the beta-subunit gene with up to 17% deletion from the carboxy-terminus of the protein prevented the proliferation of Q beta phage. However, in the case that the deletion extended beyond 25% from the carboxy-terminus, the cells showed no interference. In addition, when the interference took place, the phage coat protein synthesis was inhibited. These results indicate that the region between amino acids 440 and 487 of the beta-subunit protein is involved in the interference and suggest that the defective replicase inhibits the phage coat protein synthesis by competing with the ribosomes at the initiation site of the coat gene.     

Regions of the mouse CD14 molecule required for toll-like receptor 2- and 4-mediated activation of NF-kappa B

Regions of mouse CD14 required for Toll-like receptor 2 (TLR2)- and TLR4-mediated activation of NF-kappaB were studied in transiently transfected 293 cells. Wild-type CD14 enhanced lipopolysaccharide (LPS)-induced NF-kappaB-dependent reporter activity in cells expressing TLR4/MD-2, and deletion of amino acid regions 35-44, 144-153, 235-243, and 270-275 impaired the TLR4-mediated activation. Unlike human CD14, mouse CD14 truncated at amino acid 151 lost the activity. Deletion of amino acids 35-44 or 235-243 also abrogated TLR2-mediated activation of NF-kappaB, whereas mutants lacking 144-153 and 270-275 retained the activity. Deletion and alanine substitution experiments revealed that amino acids 151-153 and 273-275 were required for the TLR4-mediated activation. Both deletion mutants lacking amino acids 35-44 and 235-243 and alanine substitution mutants in regions 151-153 and 273-275 were expressed on the cell surface and retained the ability to associate with TLR4. A cross-linking study with photoreactive LPS showed that the labeling intensities to CD14 mutants/TLR4/MD-2 were paralleled by the ability of CD14 mutants to increase TLR4-mediated activation. These results indicate that different regions of mouse CD14 are required for TLR4- and TLR2-mediated activation of NF-kappaB and suggest that amino acids 35-44, 151-153, 235-243, and 273-275 of mouse CD14 play an important role in LPS binding and its transfer to TLR4/MD-2.     

DISC1-kendrin interaction is involved in centrosomal microtubule network formation

Disrupted-In-Schizophrenia 1 (DISC1) was identified as a novel gene disrupted by a (1;11)(q42.1;q14.3) translocation segregating with schizophrenia, bipolar disorder and other major mental illnesses in a Scottish family. We previously identified 446-533 amino acids of DISC1 as the kendrin-binding region by means of a directed yeast two-hybrid interaction assay and showed that the DISC1-kendrin interaction is indispensable for the centrosomal localization of DISC1. In this study, to confirm the DISC1-kendrin interaction, we examined the interaction between deletion mutants of DISC1 and kendrin. Then, we demonstrated that the carboxy-terminus of DISC1 is indispensable for the interaction with kendrin. Furthermore, the immunocytochemistry revealed that the carboxy-terminus of DISC1 is also required for the centrosomal targeting of DISC1. Overexpression of the DISC1-binding region of kendrin or the DISC1 deletion mutant lacking the kendrin-binding region impairs the microtubule organization. These findings suggest that the DISC1-kendrin interaction plays a key role in the microtubule dynamics.     

Definition of the human raf amino-terminal regulatory region by deletion mutagenesis.

Activation of transforming potential of the cellular raf gene has uniformly been associated with the deletion of amino-terminal coding sequences. In order to determine whether 5' truncation alone could activate cellular raf, we constructed 21 human c-raf-1 cDNAs with variable BAL 31-generated deletions distal to a Moloney murine sarcoma virus long terminal repeat and a consensus translation initiation sequence. The deletions ranged from 136 to 1,399 nucleotides of coding sequence and shortened the 648-amino-acid raf protein by 44 to 465 amino acids. The full-length c-raf-1 cDNA was nontransforming upon transfection of NIH 3T3 cells, as were four mutants with deletions of 142 or fewer amino acids. Seven of nine mutants with deletions of 154 to 273 amino acids induced transformation with efficiencies ranging from 0.25 to 70 foci per micrograms of DNA. Mutants with deletions of 303 to 324 amino acids displayed high transforming activities (comparable with that of v-raf), with a peak activity of 2,400 foci per microgram of DNA when 305 amino acids were deleted. Deletions of greater than 383 amino acids, extending into the raf kinase domain, lacked transforming activity. Northern (RNA) blotting and immunoprecipitation assays indicated that transfected NIH cells expressed raf RNAs and proteins of the expected sizes. Thus, 5' truncation alone can activate raf transforming potential, with a sharp peak of activation around amino acid 300. Analysis of three raf genes previously detected by transfection of tumor DNAs indicated that these genes were activated by recombination in raf intron 7 and encoded fusion proteins containing amino-terminal non-raf sequences. The extend of deletion of raf sequences in these recombinant genes corresponded to BAL 31 mutants which did not display high transforming activity, suggesting that the fused non-raf coding sequences may also contribute to biological activity.     

A mutational analysis of phosphatidylinositol-3-kinase activation by human colony-stimulating factor-1 receptor.

Colony-stimulating factor-1 (CSF1) is a cell lineage-specific hemopoietin required for the growth, differentiation, and survival of macrophages and their precursors. The human CSF1 receptor (CSF1R) is a 150-kDa transmembrane glycoprotein whose cytoplasmic tyrosine kinase domain is split by a kinase insert (KI) region of approximately 70 amino acids. We tested the ability of CSF1R KI domain deletion mutants to stimulate phosphatidylinositol-3-kinase (PtdIns-3-kinase), an enzyme whose activity is augmented by tyrosine kinase oncogenes and receptor tyrosine kinases, and to support mitogenesis in transfected cells. Receptor immunoprecipitates from CSF1-stimulated cells contained greater than 5-fold more PtdIns-3-kinase activity compared to nonstimulated cells. High performance liquid chromatography analysis of the PtdIns-3-kinase product scraped from thin layer chromatography plates indicated that PtdIns-3-P was produced. CSF1R KI domain deletion mutants retained tyrosine kinase activity in vitro. Receptor immunoprecipitates of two partially overlapping 28 and 30 amino acid KI deletion mutants of CSF1R retained some PtdIns-3-kinase activity, in contrast to immunoprecipitates of CSF1R lacking 67 amino acids of the KI domain. Each deletion mutant stimulated CSF1-dependent DNA synthesis in transfected cells at much reduced levels compared to wild-type receptor expressing cells. These data suggest a role for the CSF1R KI domain in PtdIns-3-kinase association and for CSF1-induced thymidine incorporation into DNA.     

Truncation of NH2-terminal amino acid residues increases agonistic potency of leukotactin-1 on CC chemokine receptors 1 and 3

Leukotactin-1 (Lkn-1) is a human CC chemokine that binds to both CC chemokine receptor 1 (CCR1) and CCR3. Structurally, Lkn-1 is distinct from other human CC chemokines in that it has long amino acid residues preceding the first cysteine at the NH(2) terminus, and contains two extra cysteines. NH(2)-terminal amino acids of Lkn-1 were deleted serially, and the effects of each deletion were investigated. In CCR1-expressing cells, serial deletion up to 20 amino acids (Delta20) did not change the calcium flux-inducing activity significantly. Deletion of 24 amino acids (Delta24), however, increased the agonistic potency approximately 100-fold. Deletion of 27 or 28 amino acids also increased the agonistic potency to the same level shown by Delta24. Deletion of 29 amino acids, however, abolished the agonistic activity almost completely showing that at least 3 amino acid residues preceding the first cysteine at the NH(2) terminus are essential for the biological activity of Lkn-1. Loss of agonistic activity was due to impaired binding to CCR1. In CCR3-expressing cells, Delta24 was the only form of Lkn-1 mutants that revealed increased agonistic potency. Our results indicate that posttranslational modification is a potential mechanism for the regulation of biological activity of Lkn-1.     

Functional characterization of the placental fusogenic membrane protein syncytin

Syncytin is an envelope protein of the human endogenous retrovirus family W (HERV-W). Syncytin is specifically expressed in the human placenta and mediates trophoblast cell fusion into the multinucleated syncytiotrophoblast layer. It is a polypeptide of 538 amino acids and is predicted to be posttranslationally cleaved into a surface (SU) subunit and a transmembrane (TM) subunit. Functional characterization of syncytin protein can aid understanding of the molecular mechanism underlying syncytin-mediated cell fusion. In this report, we studied the structure-function relationship of syncytin in 293T and HeLa cells transiently expressing wild-type syncytin or syncytin mutants generated by linker scanning and deletion mutagenesis. Of the 22 linker-inserted mutants, mutants InS51, InV139, InE156, InS493, InA506, and InL529 were fusogenic, suggesting that regions around amino acids S51, V139, and E156 in the SU subunit and S493, A506, and L529 in the cytoplasmic domain (CTM) of syncytin are flexible in conformation. Of the 17 deletion mutants, nine mutants with deletions in the region from amino acids 479 to 538 were fusogenic. The deletion mutant DelI480, containing only the first four amino acid residues in the cytoplasmic domain, had enhanced fusogenic activity in comparison with the wild-type. In addition, two heptad repeat regions (HRA and B) were defined in the TM subunit of syncytin. A peptide inhibitor derived from the C-terminal heptad repeat region (HRB) was shown to potently inhibit syncytin-mediated cell fusion. Our results suggest that the cytoplasmic domain of syncytin is not essential for syncytin-mediated fusion but may play a regulatory role, and an intramolecular interaction between HRA and B is involved in the fusion process.     

Effect of semi-random mutagenesis at the C-terminal 4 amino acids of human interleukin-6 on its biological activity.

The carboxyl(C)-terminus of human interleukin-6 (hIL-6) has a critical role in the expression of the biological activity of this cytokine. To define the structure-function relationships of this region, semi-random mutagenesis of the C-terminal Leu181-Arg182-Qln183-Met184 sequence of hIL-6 was performed. The mutants were produced in Escherichia coli, renatured, and purified. Alterations of the C-terminal 4 amino acids caused a significant reduction of the proliferative effect of the mutants on MH60.BSF2 and KT-3 cells, and also led to a drastic decrease in receptor binding affinity. These results suggest the importance of a positively charged residue at position 182 or 183 and an alpha-helix at position 181 for the biological activity of hIL-6.     

Carboxy-terminal truncations of epidermal growth factor (EGF) receptor affect diverse EGF-induced cellular responses.

The binding of epidermal growth factor (EGF) to its receptor induces tyrosine phosphorylation of phospholipase C gamma (PLC gamma), which appears to be necessary for its activation leading to phosphatidyl inositol (PI) hydrolysis. Moreover, EGF-receptor (EGF-R) activation and autophosphorylation results in binding of PLC gamma to the tyrosine phosphorylated carboxy-terminus of the receptor. To gain further insights into the mechanisms and interactions regulating these processes, we have analyzed transfected NIH-3T3 cells expressing two EGF-R carboxy-terminal deletion mutants (CD63 and CD126) with reduced capacity to stimulate PI hydrolysis, Ca2+ rises, and DNA synthesis. In fact, the CD126 mutant lacking 126 carboxy-terminal amino acids, including four tyrosine autophosphorylation sites, was unable to stimulate PI hydrolysis or Ca2+ rise in response to EGF. Surprisingly, EGF binding to the cell lines expressing CD63 or CD126 mutants was followed by similar stimulation of tyrosine phosphorylation of PLC gamma. Our results suggest that although necessary, tyrosine phosphorylation of PLC gamma may not be sufficient for stimulation and PI hydrolysis. It is clear, however, that the carboxy-terminal region of EGF-R is involved in regulation of interactions with cellular targets and therefore plays a crucial role in postreceptor signaling pathways.