Role of the C-terminus in the activity, conformation, and stability of interleukin-6.

Two murine interleukin-6 (mIL-6) variants were constructed using the polymerase chain reaction (PCR), one lacking the last five residues (183-187) at the C-terminus (pMC5) and another with the last five residues of mIL-6 substituted by the corresponding residues of human IL-6 (pMC5H). The growth stimulatory activity of pMC5 on the mouse hybridoma cell line 7TD1 was < 0.05% of mIL-6, whereas pMC5H and mIL-6 were equipotent. The loss of biological activity of pMC5 correlated with its negligible receptor binding affinity on 7TD1 cells, while the binding of pMC5H was comparable to that of mIL-6. Both pMC5 and pMC5H, like mIL-6, failed to interact with recombinant soluble human IL-6 receptor when assayed by surface plasmon resonance-based biosensor analysis. These studies suggest that the C-terminal seven amino acids of human IL-6, alone, do not define species specificity for receptor binding. A variety of biophysical techniques, as well as the binding of a conformational-specific monoclonal antibody, indicated that the global fold of the mIL-6 variants was similar to that of mIL-6, although small changes in the NMR spectra, particularly for pMC5, were observed. Some of these changes involved residues widely separated in the primary structure. For instance, interactions involving Tyr-22 were influenced by the C-terminal amino acids suggesting that the N- and C-termini of mIL-6 are in close proximity. Equilibrium unfolding experiments indicated that pMC5 was 0.8 kcal/mol less stable than mIL-6, whereas pMC5H was 1.4 kcal/mol more stable. These studies emphasize the structural importance of the C-terminal amino acids of IL-6 and suggest that truncation or mutation of this region could lead to small but significant alterations in other regions of the molecule.     

Purification and characterization of a recombinant murine interleukin-6. Isolation of N- and C-terminally truncated forms.

Murine interleukin-6 (IL-6), when expressed in Escherichia coli using the pUC9 vector, accumulated as insoluble aggregates or 'inclusion bodies'. After selective urea washing of the inclusion bodies, to remove extraneous proteins, murine IL-6 was solubilized with 8 M guanidine hydrochloride and then rapidly purified to homogeneity by gel-permeation chromatography followed by reversed-phase HPLC. It was demonstrated that complete disulfide bond formation in murine IL-6 occurred during the early urea washing/guanidine hydrochloride extraction steps, so no refolding step was required. When fully reduced murine IL-6 was dissolved in 8 M guanidine hydrochloride and allowed to air-oxidize, complete disulfide bond formation, monitored by analytical reversed-phase HPLC, was shown to occur within 13 h at 6 degrees C. About 25 mg pure protein was obtained from 37 g wet cells. This recombinant murine IL-6 had a specific activity in the hybridoma growth factor assay of 2 x 10(8) U/mg, which is equivalent to that of native murine IL-6. During the purification procedure, a number of variant forms of murine IL-6 were isolated and partially characterized. Two of these forms, T1 and T3, were C-terminal deletants of murine IL-6 lacking about 60 and 20 amino acids from the C-terminus, respectively, while the other form, T2, was an N-terminal deletant lacking 37 amino acids from the N-terminus. None of these variant forms of murine IL-6 bound to the murine IL-6 receptor and, consequently, all were inactive in the hybridoma growth factor assay.     

The cationic C-terminus of rat Muc2 facilitates dimer formation post translationally and is subsequently removed by furin.

Earlier immunolocalization experiments showed that the extreme cationic C-terminus of the rat intestinal mucin Muc2 (RMC) was present at the base of intestinal goblet cells in the vicinity of ER and golgi compartments, but was not found with the rest of the mucin in apical storage granules. This prompted us to investigate the possibility that an early proteolytic cleavage reaction occurs post-translationally. A plasmid pRMC, encoding the C-terminal 534 amino acids of the mucin, was expressed in COS-7 cells and was shown to undergo cleavage at an R-T-R-R sequence located within the C-terminal 14 amino acids. Cleavage did not occur with the construct RMCfH, a furin site-mutated (A-T-A-A) counterpart of pRMCH (poly His6 tagged RMC). Addition of a furin inhibitor to COS-7 cell incubations also prevented cleavage of RMC and RMCH products. 35S pulse-chase kinetic experiments revealed that a truncated mutant lacking the C-terminal 14 amino acids (pRMCDeltaCT) forms faulty (doublet) dimers in the ER. These were not secreted as efficiently as the normal dimer of wild-type (pRMC) constructs. Thus the cationic C-terminus of rMuc2 apppears to facilitate the correct formation of normal Muc2 domain dimers.     

Characterization of a recombinant murine interleukin-6: assignment of disulfide bonds

Murine interleukin 6 (mIL-6) has been synthesized as a fusion protein using a lac operon inducible plasmid in Escherichia coli. The first 8 amino acids are from the N-terminus of bacterial beta-galactosidase and the last 175 amino acids are from residue number 12 to the end of native mIL-6. This fusion protein is equipotent with the native molecule in the hybridoma growth factor assay and has comparable receptor binding characteristics. The two disulfide bridges in mIL-6 have been identified by Staphylococcus aureus V8 protease peptide mapping and Edman degradation of cystine-containing peptides. It has been shown that there are disulfide bonds between Cys46-Cys52 and Cys75-Cys85.     

Conserved amino acids at the C-terminus of rat phospholipase D1 are essential for enzymatic activity.

Rat brain phospholipase D1 (rPLD1) has two highly conserved motifs [H(X)K(X)4D, denoted HKD] located at the N-terminal and C-terminal halves, which are required for activity. Association of the two halves is essential for rPLD1 activity, which probably brings the two HKD domains together to form a catalytic center. In the present study, we find that an intact C-terminus is also essential for the catalytic activity of rPLD1. Serial deletion of the last four amino acids, EVWT, which are conserved in all mammalian PLD isoforms, abolished the catalytic activity of rPLD1. This loss of catalytic activity was not due to a lack of association of the N-terminal and C-terminal halves. Mutations of the last three amino acids showed that substitutions with charged or less hydrophobic amino acids all reduced PLD activity. For example, mutations of Thr1036 and Val1034 to Asp or Lys caused marked inactivation, whereas mutation to other amino acids had less effect. Mutation of Trp1035 to Leu, Ala, His or Tyr caused complete inactivation, whereas mutation of Glu1033 to Ala enhanced activity. The size of the amino acids at the C-terminus also affected the catalytic activity of PLD, reduced activity being observed with conservative mutations within the EVWT sequence (such as T/S, V/L or W/F). The enzyme was also inactivated by the addition of Ala or Val to the C-terminus of this sequence. Interestingly, the inactive C-terminal mutants could be complemented by cotransfection with a wild-type C-terminal half to restore PLD activity in vivo. These data demonstrate that the integrity of the C-terminus of rPLD1 is essential for its catalytic activity. Important features are the hydrophobicity, charge and size of the four conserved C-terminal amino acids. It is proposed that these play important roles in maintaining a functional catalytic structure by interacting with a specific domain within rPLD1.     

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.     

The role of the carboxyl-terminal 6 amino acid extension of human TSH beta subunit

The nucleotide sequence of human thyroid stimulating hormone (hTSH) gene can encode a protein of 138 amino acids. However, the mature polypeptide is lacking 6 amino acids of the carboxyl-terminus (C-terminus), suggesting posttranslational cleavage of these residues. To analyze a possible function of these 6 amino acids, we expressed two hTSH beta cDNAs with or without the 6 codons for C-terminal extension, together with alpha subunit cDNA in CHO cells, and determined the amino acid sequence of C-terminus of hTSH beta. hTSH beta propeptides without C-terminal extension were glycosylated, associated with alpha subunit and secreted, as normal propeptides were, and its heterodimer with alpha subunit showed normal TSH bioactivity in FRTL-5 bioassay. These data indicate that the 6 amino acid C-terminal extension is not necessary for the hTSH maturation in the process of the biosynthesis and for its bioactivity.     

C-terminal truncation and histidine-tagging of cytochrome c oxidase subunit II reveals the native processing site, shows involvement of the C-terminus in cytochrome c binding, and improves the assay for proton pumping

To enable metal affinity purification of cytochrome c oxidase reconstituted into phospholipid vesicles, a histidine-tag was engineered onto the C-terminal end of the Rhodobacter sphaeroides cytochrome c oxidase subunit II. Characterization of the natively processed wildtype oxidase and artificially processed forms (truncated with and without a his-tag) reveals Km values for cytochrome c that are 6-14-fold higher for the truncated and his-tagged forms than for the wildtype. This lowered ability to bind cytochrome c indicates a previously undetected role for the C-terminus in cytochrome c binding and is mimicked by reduced affinity for an FPLC anion exchange column. The elution profiles and kinetics indicate that the removal of 16 amino acids from the C-terminus, predicted from the known processing site of the Paracoccus denitrificans oxidase, does not produce the same enzyme as the native processing reaction. MALDI-TOF MS data show the true C-terminus of subunit II is at serine 290, three amino acids longer than expected. When the his-tagged form is reconstituted into lipid vesicles and further purified by metal affinity chromatography, significant improvement is observed in proton pumping analysis by the stopped-flow method. The improved kinetic results are attributed to a homogeneous, correctly oriented vesicle population with higher activity and less buffering from extraneous lipids.     

Functionally important conserved length of C-terminal regions of yeast and bovine ADP/ATP carriers, identified by deletion mutants studies, and water accessibility of the amino acids at the C-terminal region of the yeast carrier

Comparison of the amino acid sequence of yeast type 2 ADP/ATP carrier (yAAC2) with that of bovine type 1 AAC (bAAC1) revealed that the N- and C-terminus of yAAC2 are 15- and 6-amino acids longer, respectively, than those of bAAC1. In the present study, we focused on the difference in the C-terminal region between yAAC2 and bAAC1. Deletion of first six residues of C-terminus of yAAC did not markedly affect the function of yAAC2; however, further deletion of 1 amino acid (7th amino acid from the C-terminus) destroyed its function. On the contrary, deletion of the first amino acid residue of the C-terminus of bAAC1 caused failure of its functional expression in yeast mitochondria. Based on these results, we concluded that the 6-amino acid residue extension of the C-terminus of yAAC2 was not necessary for the function of this carrier and that the remainder of the C-terminal region of yAAC2, having a length conserved with that of bAAC1, is important for the transport function of AACs. We next prepared various single-Cys mutants in which each of 32 residues in the C-terminus of yAAC2 was replaced by a Cys residue. Since all mutants were successfully expressed in yeast mitochondria, we examined the reactivity of these cysteine residues with the membrane-impermeable sulfhydryl reagent eosin 5-maleimide (EMA). As a result, all cysteine residues that replaced the 9 continuous amino acids in Met310-Lys318 showed high reactivity with EMA regardless of the presence of carboxyatractyloside or bongkrekic acid; and so this region was concluded to be exposed to the water-accessible environment. Furthermore, based on the reactivities of cysteine residues that replaced amino acids in the sixth transmembrane segment, the probable structural features of the C-terminal region of this carrier in the presence of bongkrekic acid were discussed.     

The additional methionine residue at the N-terminus of bacterially expressed human interleukin-2 affects the interaction between the N- and C-termini

To gain insight into the origin of the difference in isoelectric point (pI) values for wild-type human interleukin-2 (IL-2) and IL-2 with an additional methionine residue at the N-terminus (Met-IL-2), conformational properties of the two molecular forms of IL-2 were compared by utilizing 1H NMR spectroscopy. Although overall conformations were conserved in the two forms, the presence of the additional methionine residue at the N-terminus induced chemical shift changes for residues Ala1 to Lys8 as well as for Thr133, which is located at the C-terminus. These observations indicate that the effect of the additional methionine residue is confined to the N- and C-terminal regions and unveil the existence of an interaction between the N- and C-terminal regions. The chemical shift change observed for Thr133 can be interpreted in terms of a change in pKa of the C-terminal carboxyl group, which interacts differently with the N-terminal amino group in the two forms of IL-2. It seems to be reasonable to conclude that the difference in pI values for the two forms of IL-2 is the consequence of the different interactions between the C- and N-terminal residues.     

C-terminal region regulates the functional expression of human noradrenaline transporter splice variants

The NET [noradrenaline (norepinephrine) transporter], an Na+/Cl--dependent neurotransmitter transporter, has several isoforms produced by alternative splicing in the C-terminal region, each differing in expression and function. We characterized the two major isoforms of human NET, hNET1, which has seven C-terminal amino acids encoded by exon 15, and hNET2, which has 18 amino acids encoded by exon 16, by site-directed mutagenesis in combination with NE (noradrenaline) uptake assays and cell surface biotinylation. Mutants lacking one third or more of the 24 amino acids encoded by exon 14 exhibited neither cell surface expression nor NE uptake activity, with the exception of the mutant lacking the last eight amino acids of hNET2, whose expression and uptake resembled that of the WT (wild-type). A triple alanine replacement of a candidate motif (ENE) in this region mimicked the influences of the truncation. Deletion of either the last three or another four amino acids of the C-terminus encoded by exon 15 in hNET1 reduced the cell surface expression and NE uptake, whereas deletion of all seven residues reduced the transport activity but did not affect the cell surface expression. Replacement of RRR, an endoplasmic reticulum retention motif, by alanine residues in the C-terminus of hNET2 resulted in a similar expression and function compared with the WT, while partly recovering the effects of the mutation of ENE. These findings suggest that in addition to the function of the C-terminus, the common proximal region encoded by exon 14 regulates the functional expression of splice variants, such as hNET1 and hNET2.     

Deletion of C-terminal 12 amino acids of GLUT1 protein does not abolish the transport activity.

We engineered the GLUT1 cDNA to delete C-terminal 12 amino acids of encoded GLUT1 protein. This mutated GLUT1 protein expressed in CHO cells by transfection of its cDNA was demonstrated to reside on the plasma membrane by cell surface labeling technique, and retain the transport activity, similar to that of the wild-type GLUT1. In addition, metabolic labeling of the intact cells with 35S indicated that the half-life of the mutated GLUT1 was not significantly different from that of the wild-type GLUT1. These results suggest that C-terminal 12 amino acids of GLUT1 are not important for the transport activity and the stability of the protein. Taken together with our previous results on the mutant without C-terminal 37 amino acids, the amino acids between the 37th and the 13th from the C-terminus appear to be essential for the transport activity.     

Escherichia coli methionyl-tRNA formyltransferase: role of amino acids conserved in the linker region and in the C-terminal domain on the specific recognition of the initiator tRNA

The formylation of initiator methionyl-tRNA by methionyl-tRNA formyltransferase (MTF) is important for the initiation of protein synthesis in eubacteria. We are studying the molecular mechanisms of recognition of the initiator tRNA by Escherichia coli MTF. MTF from eubacteria contains an approximately 100-amino acid C-terminal extension that is not found in the E. coli glycinamide ribonucleotide formyltransferase, which, like MTF, use N(10)-formyltetrahydrofolate as a formyl group donor. This C-terminal extension, which forms a distinct structural domain, is attached to the N-terminal domain through a linker region. Here, we describe the effect of (i) substitution mutations on some nineteen basic, aromatic and other conserved amino acids in the linker region and in the C-terminal domain of MTF and (ii) deletion mutations from the C-terminus on enzyme activity. We show that the positive charge on two of the lysine residues in the linker region leading to the C-terminal domain are important for enzyme activity. Mutation of some of the basic amino acids in the C-terminal domain to alanine has mostly small effects on the kinetic parameters, whereas mutation to glutamic acid has large effects. However, the deletion of 18, 20, or 80 amino acids from the C-terminus has very large effects on enzyme activity. Overall, our results support the notion that the basic amino acid residues in the C-terminal domain provide a positively charged channel that is used for the nonspecific binding of tRNA, whereas some of the amino acids in the linker region play an important role in activity of MTF.     

The C-terminus of eRF1 defines a functionally important domain for translation termination in Saccharomyces cerevisiae

Translation termination in eukaryotes is mediated by two release factors, eRF1 and eRF3, which interact to form a heterodimer that mediates termination at all three stop codons. By C-terminal deletion analysis of eRF1 from the yeast Saccharomyces cerevisiae, we show that the extreme C-terminus of this 437-amino-acid protein defines a functionally important domain for translation termination. A strain encoding eRF1 lacking the C-terminal 32 amino acids is not viable, whereas deletion of the C-terminal 19 amino acids is viable but shows a termination defect in vivo causing an enhancement of nonsense suppression. Using a combination of two-hybrid analysis and in vitro binding studies, we demonstrate that deletions encompassing the C-terminus of eRF1 cause a significant reduction in eRF3 binding to eRF1. All of the C-terminally truncated eRF1 still bind the ribosome, suggesting that the C-terminus does not constitute a ribosome-binding domain and eRF1 does not need to form a stable complex with eRF3 in order to bind the ribosome. These data, together with previously published data, suggest that the region between amino acids 411 and 418 of yeast eRF1 defines an essential functional domain that is part of the major site of interaction with eRF3. However, a stable eRF1:eRF3 complex does not have to be formed to maintain viability or efficient translation termination. Alignment of the seven known eukaryotic eRF1 sequences indicates that a highly conserved motif, GFGGIGG/A is present within the region of the C-terminus, although our deletion studies suggest that it is sequences C-terminal to this region that are functionally important.     

Synthesis, activity, and preliminary structure of the fourth EGF-like domain of thrombomodulin.

The fourth EGF-like domain of thrombomodulin (TM4), residues E346-F389 in the TM sequence, has been synthesized. Refolding of the synthetic product under redox conditions gave a single major product. The disulfide bonding pattern of the folded, oxidized domain was (1-3, 2-4, 5-6), which is the same as that found in EGF protein. TM4 was tested for TM anticoagulant activity because deletion and substitution mutagenesis experiments have shown that the fourth EGF-like domain of TM is essential for TM cofactor activity. TM4 showed no TM-like activity in two assay systems, both for inhibition of fibrin clot formation, and for cofactor activity in thrombin activation of protein C. A preliminary structure of TM4 was determined by 2D 1H NMR from 519 NOE-derived distance constraints. Distance geometry calculations yielded a single convergent structure. The structure resembles the structure of EGF and other known EGF-like domains but has some key differences. The central two-stranded beta-sheet is conserved despite the differences in the number of amino acids in the loops. The C-terminal loop formed by the disulfide bond between C372 and C386 in TM4 is five amino acids longer than the analogous loop between C33 and C42 of EGF protein. This loop appears to have a different fold in TM4 than in EGF protein. The loop forms the two outside strands of a broken, irregular tri-stranded beta-sheet, and amino acids H384-F389 lie between the two strands forming the middle strand of the sheet. Thus, although the C-terminus of EGF protein forms one of the outside strands of a tri-stranded antiparallel sheet, the C-terminus of TM4 forms the inside strand of an irregular tri-stranded parallel-anti-parallel sheet. The residues D349, E357, and E374, which were shown to be critical for cofactor activity by alanine scanning mutagenesis, all lie in a patch near the C-terminal loop, and are solvent accessible. The other critical residues, Y358 and F376, are largely buried and appear to play essential structural rather than functional roles.     

RAS2 protein of Saccharomyces cerevisiae undergoes removal of methionine at N terminus and removal of three amino acids at C terminus

RAS2 protein of Saccharomyces cerevisiae undergoes post-translational modifications involving methyl esterification and palmitic acid addition, resulting in their association with the plasma membrane. In this paper, we provide evidence that two kinds of proteolytic events accompany the biosynthesis. This is shown by separating and characterizing three intracellular forms of RAS2 protein: precursor, intermediate, and mature (fatty acid-acylated) forms. N-Terminal sequencing has revealed that all three forms start with proline, which is the second amino acid expected from the RAS2 gene sequence. Thus, the first methionine is removed very early during the biosynthesis. Isolation and sequencing of C-terminal peptides indicate that three C-terminal amino acids present in the precursor form are removed in the intermediate and in the fatty acid acylated forms. C-Terminal proteolysis appears to accompany methyl esterification, since the methylation occurs with the intermediate and the fatty acid-acylated forms, but not with the precursor. Palmitic acid is identified as the major fatty acid attached to the fatty acid-acylated form.