The structural basis of Trp192 and the C-terminal region in trichosanthin for activity and conformational stability

Trichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) possessing N-glycosidase activity. TCS has various pharmacological properties, including immunomodulatory, anti-tumor and anti-HIV activities. Up to seven C-terminal residues of TCS (TCS-C7) can be deleted resulting in lower antigenicity with minimal effects on its activity. However, an additional problem is that the minimal effects on activity are higher than the reduction in antigenicity. In the present work, the crystal structure of TCS-C7 was determined. It shows the details of the C-terminal residues of TCS-C7, and in particular the hydrogen bonds between P35 and L240, S196 and L240, and W192 and L239, which play an important role in maintaining the structure of TCS-C7. Further analysis shows that the hydrogen bonds related to Leu240 are key in maintaining the relationship between N- and C-terminal domains. The major role of the C-terminal tail appears to stabilize the structure of TCS. The conformation between helix H7 at the N-terminal domain and the C-terminal tail at the C-terminal domain is also revealed. Two mutants, TCS-W192F and TCS-C7-W192F, were prepared and crystal structures were determined. These variants have greatly reduced ribosome-inactivating activities compared with TCS and TCS-C7, respectively, and TCS-W192F and TCS-C7-W192F have a similar stability in guanidine hydrochloride compared with TCS-C7. This suggests that Trp192 can affect the ribosome-inactivating activity of TCS.     

Antigenicity of UV radiation-induced murine tumors correlates positively with the level of adenosine deaminase activity

Summary The specific activities of adenosine deaminase (ADA) in 16 murine tumor cell lines derived from seven UV light-induced neoplasms (melanoma and fibrosarcoma) were determined. In each case, the specific activity of ADA correlated positively with the antigenicity of the tumor cells. Highly antigenic cell lines that regress upon introduction into syngeneic hosts had on average 4- to 6-fold higher ADA specific activities than cell lines of low antigenicity that grow progressively in syngeneic hosts. The antigenic differences are probably not related to intracellular cAMP levels, as the level of cAMP differed only 2-fold between the two groups of cell lines.     

The highly processive DNA polymerase of bacteriophage T5. Role of the unique N and C termini

The DNA polymerase encoded by bacteriophage T5 has been reported previously to be processive and to catalyze extensive strand displacement synthesis. The enzyme, purified from phage-infected cells, did not require accessory proteins for these activities. Although T5 DNA polymerase shares extensive sequence homology with Escherichia coli DNA polymerase I and T7 DNA polymerase, it contains unique regions of 130 and 71 residues at its N and C termini, respectively. We cloned the gene encoding wild-type T5 DNA polymerase and characterized the overproduced protein. We also examined the effect of N- and C-terminal deletions on processivity and strand displacement synthesis. T5 DNA polymerase lacking its N-terminal 30 residues resembled the wild-type enzyme albeit with a 2-fold reduction in polymerase activity. Deletion of 24 residues at the C terminus resulted in a 30-fold reduction in polymerase activity on primed circular DNA, had dramatically reduced processivity, and was unable to carry out strand displacement synthesis. Deletion of 63 residues at the C terminus resulted in a 20,000-fold reduction in polymerase activity. The 3' to 5' double-stranded DNA exonuclease activity associated with T5 DNA polymerase was reduced by a factor of 5 in the polymerase truncated at the N terminus but was stimulated by a factor of 7 in the polymerase truncated at the C terminus. We propose a model in which the C terminus increases the affinity of the DNA for the polymerase active site, thus increasing processivity and decreasing the accessibility of the DNA to the exonuclease active site.     

The C-terminal domain of 4-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii is an autoinhibitory domain

p-Hydroxyphenylacetate (HPA) 3-hydroxylase from Acinetobacter baumannii consists of a reductase component (C(1)) and an oxygenase component (C(2)). C(1) catalyzes the reduction of FMN by NADH to provide FMNH(-) as a substrate for C(2). The rate of reduction of flavin is enhanced ～20-fold by binding HPA. The N-terminal domain of C(1) is homologous to other flavin reductases, whereas the C-terminal domain (residues 192-315) is similar to MarR, a repressor protein involved in bacterial antibiotic resistance. In this study, three forms of truncated C(1) variants and single site mutation variants of residues Arg-21, Phe-216, Arg-217, Ile-246, and Arg-247 were constructed to investigate the role of the C-terminal domain in regulating C(1). In the absence of HPA, the C(1) variant in which residues 179-315 were removed (t178C(1)) was reduced by NADH and released FMNH(-) at the same rates as wild-type enzyme carries out these functions in the presence of HPA. In contrast, variants with residues 231-315 removed behaved similarly to the wild-type enzyme. Thus, residues 179-230 are involved in repressing the production of FMNH(-) in the absence of HPA. These results are consistent with the C-terminal domain in the wild-type enzyme being an autoinhibitory domain that upon binding the effector HPA undergoes conformational changes to allow faster flavin reduction and release. Most of the single site variants investigated had catalytic properties similar to those of the wild-type enzyme except for the F216A variant, which had a rate of reduction that was not stimulated by HPA. F216A could be involved with HPA binding or in the required conformational change for stimulation of flavin reduction by HPA.     

C-terminal amino acid residues are required for the folding and cholesterol binding property of perfringolysin O, a pore-forming cytolysin.

Perfringolysin O (theta-toxin) is a pore-forming cytolysin whose activity is triggered by binding to cholesterol in the plasma membrane. The cholesterol binding activity is predominantly localized in the beta-sheet-rich C-terminal half. In order to determine the roles of the C-terminal amino acids in theta-toxin conformation and activity, mutants were constructed by truncation of the C terminus. While the mutant with a two-amino acid C-terminal truncation retains full activity and has similar structural features to native theta-toxin, truncation of three amino acids causes a 40% decrease in hemolytic activity due to the reduction in cholesterol binding activity with a slight change in its higher order structure. Furthermore, both mutants were found to be poor at in vitro refolding after denaturation in 6 M guanidine hydrochloride, resulting in a dramatic reduction in cholesterol binding and hemolytic activities. These activity losses were accompanied by a slight decrease in beta-sheet content. A mutant toxin with a five-amino acid truncation expressed in Escherichia coli is recovered as a further truncated form lacking the C-terminal 21 amino residues. The product retains neither cholesterol binding nor hemolytic activities and shows a highly disordered structure as detected by alterations in the circular dichroism and tryptophan fluorescence spectra. These results show that the C-terminal region of theta-toxin has two distinct roles; the last 21 amino acids are involved to maintain an ordered overall structure, and in addition, the last two amino acids at the C-terminal end are needed for protein folding in vitro, in order to produce the necessary conformation for optimal cholesterol binding and hemolytic activities.     

Regulation of ribosomal protein S6 phosphorylation by casein kinase 1 and protein phosphatase 1

Ribosomal protein S6 (rpS6) is a critical component of the 40 S ribosomal subunit that mediates translation initiation at the 5'-m(7)GpppG cap of mRNA. In response to mitogenic stimuli, rpS6 undergoes ordered C-terminal phosphorylation by p70 S6 kinases and p90 ribosomal S6 kinases on four conserved Ser residues (Ser-235, Ser-236, Ser-240, and Ser-244) whose modification potentiates rpS6 cap binding activity. A fifth site, Ser-247, is also known to be phosphorylated, but its function and regulation are not well characterized. In this study, we employed phospho-specific antibodies to show that Ser-247 is a target of the casein kinase 1 (CK1) family of protein kinases. CK1-dependent phosphorylation of Ser-247 was induced by mitogenic stimuli and required prior phosphorylation of upstream S6 kinase/ribosomal S6 kinase residues. CK1-mediated phosphorylation of Ser-247 also enhanced the phosphorylation of upstream sites, which implies that bidirectional synergy between C-terminal phospho-residues is required to sustain rpS6 phosphorylation. Consistent with this idea, CK1-dependent phosphorylation of rpS6 promotes its association with the mRNA cap-binding complex in vitro. Additionally, we show that protein phosphatase 1 (PP1) antagonizes rpS6 C terminus phosphorylation and cap binding in intact cells. These findings further our understanding of rpS6 phospho-regulation and define a direct link between CK1 and translation initiation.     

Site-directed PEGylation of trichosanthin retained its anti-HIV activity with reduced potency in vitro

Trichosanthin (TCS) was the first ribosome inactivating protein found to possess anti-HIV-1 activity. Phase I/II clinical trial of this compound had been done. Antigenicity and short plasma half-life were the major side effects preventing further clinical trial. Modification of TCS is therefore necessary to revive the interest to develop this compound as an anti-HIV agent. Three potential antigenic sites (Ser-7, Lys-173, and Gln-219) were identified by computer modeling. Through site-directed mutagenesis, these three antigenic amino acids were mutated to a cysteine residue resulting in 3 TCS mutants, namely S7C, K173C, and Q219C. These mutants were further coupled to polyethylene glycol with a molecular size of 20 kDa (PEG) via the cysteine residue. This produced another three TCS derivatives, namely PEG_20k-S7C, PEG_20k-K173C, and PEG_20k-Q219C. PEGylation had been widely used recently to decrease immunogenicity by masking the antigenic sites and prolong plasma half-life by expanding the molecular size. The in vitro anti-HIV-1 activity of these mutants and derivatives was tested. Results showed that the anti-HIV-1 activity of S7C, K173C, and Q219C was decreased by about 1.5- to 5.5-fold with slightly lower cytotoxicity. On the other hand, PEGylation produced larger decrease (20- to 30-fold) in anti-HIV activity. Cytotoxicity was, however, weakened only slightly by about 3-fold. The in vitro study showed that the anti-HIV activity of PEGylated TCS was retained with reduced potency. The in vivo activity is expected to have only slightly changed due to other beneficial effects like prolonged half-life.     

Identification of Renibacterium salmoninarum surface proteins by radioiodination

Abstract Treatment of Clostridium perfringens alpha toxin with aminopeptidase resulted in no effect on various activities of the toxin. Aminopeptidase did not hydrolyze the native toxin or the toxin treated with urea in the presence of EDTA. Treatment with carboxypeptidase for 30 min resulted in a 75% decrease in these activities. Incubation of the native toxin with carboxypeptidase for 30 min released approximately 15 amino acids from the C-terminus of the toxin. The biological activities of a mutant toxin lacking 20 C-terminal residues of the toxin (AT_1–350) showed about 59–87% of the activity of native toxin. The mutant toxin showed partial antigenic identity with the native toxin. These data suggest that the C-terminal domain contributes to maintaining the active form of the toxin.     

Design of a highly potent anti-hypertensive peptide based on ovokinin(2-7)

Ovokinin(2-7) (RADHPF), an orally active antihypertensive peptide derived from ovalbumin, lowers blood pressure in SHRs at a dose of 10 mg/kg. Attempts were made to potentiate its anti-hypertensive activity by replacing the amino acid residues in [Pro2, Phe3]-ovokinin(2-7), which was previously reported to have 33-fold stronger activity than ovokinin(2-7). The anti-hypertensive activity of [Pro2, Phe3]-ovokinin(2-7) was improved by replacement of the C-terminal Phe residue with Trp. Then, the best amino acid residues at other positions for the anti-hypertensive effect were selected. RPLKPW, the most potent derivative obtained, showed significant anti-hypertensive activities at a dose of 0.1 mg/kg after oral administration in spontaneously hypertensive rats (SHRs). Thus, RPLKPW showed 100-fold more potent anti-hypertensive activity than ovokinin(2-7).     

Identification of residues critical for toxicity in Clostridium perfringens phospholipase C, the key toxin in gas gangrene.

Clostridium perfringens phospholipase C (PLC), also called alpha-toxin, is the major virulence factor in the pathogenesis of gas gangrene. The toxic activities of genetically engineered alpha-toxin variants harboring single amino-acid substitutions in three loops of its C-terminal domain were studied. The substitutions were made in aspartic acid residues which bind calcium, and tyrosine residues of the putative membrane-interacting region. The variants D269N and D336N had less than 20% of the hemolytic activity and displayed a cytotoxic potency 103-fold lower than that of the wild-type toxin. The variants in which Tyr275, Tyr307, and Tyr331 were substituted by Asn, Phe, or Leu had 11-73% of the hemolytic activity and exhibited a cytotoxic potency 102- to 105-fold lower than that of the wild-type toxin. The results demonstrated that the sphingomyelinase activity and the C-terminal domain are required for myotoxicity in vivo and that the variants D269N, D336N, Y275N, Y307F, and Y331L had less than 12% of the myotoxic activity displayed by the wild-type toxin. This work therefore identifies residues critical for the toxic activities of C. perfringens PLC and provides new insights toward understanding the mechanism of action of this toxin at a molecular level.     

The domain organization of streptokinase: nuclear magnetic resonance, circular dichroism, and functional characterization of proteolytic fragments.

Streptococcus equisimilis streptokinase (SK) is a bacterial protein of unknown tertiary structure and domain organization that is used extensively to treat acute myocardial infarction following coronary thrombosis. Six fragments of SK were generated by limited proteolysis with chymotrypsin and purified. NMR and CD experiments have shown that the secondary and tertiary structure present in the native molecule is preserved within all fragments, except the N-terminal fragment SK7. NMR spectra demonstrate the presence in SK of three structurally autonomous domains and a less structured C-terminal "tail." Cleavage within the N-terminal domain generates an N-terminal fragment, SK7, which remains noncovalently associated with the remainder of the molecule; in isolation, SK7 adopts an unfolded conformation. The abilities of these fragments to induce active site formation within human plasminogen upon formation of their heterodimeric complex were assayed. The lowest mass SK fragment exhibiting Plg-dependent activator activity was shown to be SK27 (mass 27,000, residues 147-380), which contains both central and C-terminal domains, although this activity was reduced approximately 6,000-fold relative to that of full-length SK. The activity of a 36,000 mass fragment, SK36 (residues 64-380), which differs from SK27 in possessing a portion of the N-terminal domain, was reduced to 0.1-1.0% of that of SK. Other fragments (masses 7,000, 11,000, 16,000, 17,000, 25,000, and 26,000), representing either single domains or single domains extended by portions of other domains, were inactive. However, SK7 (residues 1-63), at a 100-fold molar excess concentration, greatly potentiated the activities of SK27 and SK36, by up to 50- and > 130-fold, respectively. These findings demonstrate that all of SK's three domains are essential for native-like SK activity. The central and C-terminal domains mediate plasminogen-binding and active site-generating functions, whereas the N-terminal domain mediates an activity-potentiating function.     

Crystallization and preliminary x-ray crystallographic studies of trichosanthin delta C7

Trichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) which possesses rRNA N-glycosidase activity. TCS has various pharmacological properties. It is possible to reduce the antigenicity of TCS by deleting up to seven C-terminal residues of TCS (TCS-C7) with minimal effect on its activity. TCS-C7 has been crystallized and the crystal diffracted to 1.8 A. It belongs to space group P2(1), with unit-cell parameters a=71.6A, b=74.4A, c=87.6A, beta=97.0 degrees. It is given that there are four molecules per asymmetric unit.     

Site-directed polyethylene glycol modification of trichosanthin: effects on its biological activities, pharmacokinetics, and antigenicity

Trichosanthin (TCS), a type I ribosome-inactivating protein (RIP), was modified with polyethylene glycol (PEG) in order to reduce its antigenicity and prolong its half-life. Computer modeling identified three potential antigenic sites namely Q219, K173 and S7. By site-directed mutagenesis, these sites were changed into cysteine through which PEG can be covalently attached. The resulting TCS had a PEG coupled directly above one of its potential antigenic determinants, hence masking the antigenic region and prevent binding of antibodies specific to this site. In general, mutation did not bring about significant changes in ribosome-inactivating activity, cytotoxicity, and abortifacient activity of TCS. However, the in vitro activities of PEG modified (PEGylated) TCS muteins were 3-20 folds lower and the in vivo activity 50% less than that of nTCS. Pharmacokinetics study indicated that all three PEGylated TCS muteins showed 6-fold increase in mean residence time as compared to unmodified muteins. The binding affinity of an IgE monoclonal antibody (TE1) to TCS was greatly reduced after PEG modification (PEGylation) at position Q219, suggesting that TE1 recognized an epitope very near to residue Q219. PEGylated TCS muteins induced similar IgG response but 4-16 fold lower IgE response in mice compared with nTCS.     

Antigenic Reactivities of Chemically Modified β-Lactoglobulins with Antiserum to Bovine β-Lactoglobulin

Analysis of the quantitative precipitation of bovine β-lactoglobulin (β-Lg) with rabbit antiserum to β-Lg indicated that there were at least four antigenic sites on β-Lg. To study the antigenic property of bovine β-Lg, we examined the antigenic reactivity of anti β-Lg serum with β-Lg specifically modified with chemical reagents by immunodiffusion analysis, a quantitative precipitin test, and an enzyme-linked immunosorbent assay.

Modification of arginine residues, tryptophan residues, or sulfhydryl groups had little effect on the antigenic reactivity. A significant decrease in the reactivity was observed when β-Lg was acetylated, succinylated, modified with diethylpyrocarbonate or coupled with glycine amide.

These results suggest that 1.1 of three arginine residues, two tryptophan residues, and one sulfhydryl group are out of the antigenic sites, but there is a possibility that the amino group, histidine residue and carboxyl group may play an important role in the antigenicity of bovine β-Lg.     

Primary structure and functional characterization of rat C5a: an anaphylatoxin with unusually high potency.

The anaphylatoxin C5a is a pro-inflammatory factor generated from C5 during complement activation. C5a derived from rat C5 exhibits significantly greater potency compared to C5a from other species. Rat C5a was 25-fold more potent than human C5a for eliciting spasmogenic contraction of guinea pig ileum. Proteolytic removal of the C-terminal arginine of C5a (C5adesArg) reduced spasmogenic potency of rat C5a by only 4-fold compared to a 3,000-fold reduction for human C5adesArg. In addition, rat C5adesArg was 50-fold more potent than human C5adesArg in a guinea pig vascular permeability (in vivo) assay and as a chemotactic factor for human neutrophils. C5a and C5adesArg were purified from zymosan-activated rat serum. Rat C5a, like human C5a, is glycosylated but contains 77 amino acid residues instead of the 74 residues of human C5a. Comparison of the primary structures of rat and human C5a indicated differences at 30 positions including an insert of 3 residues (LLH) in the rat molecule between residue positions 3 and 4 in human C5a. Insertion of residues LLH between Gln-3 and Lys-4 in a recombinant human C5a molecule using site-directed mutagenesis failed to enhance potency. Synthetic C-terminal analogues of rat C5a proved to be measurably more potent than the corresponding human C5a analogues (Ember JA et al., 1993, Protein Sci 2(Suppl 1):159 [Abstr]). We conclude that multiple sequence differences in the C-terminal effector portion and/or elsewhere in rat C5a, but not the LLH insert, account for the significant enhancement in potency of rat C5a over C5a from other species.     

Investigation of the C-terminal redox center of high-Mr thioredoxin reductase by protein engineering and semisynthesis

High-molecular weight thioredoxin reductases (TRs) catalyze the reduction of the redox-active disulfide bond of thioredoxin, but an important difference in the TR family is the sequence of the C-terminal redox-active tetrapeptide that interacts directly with thioredoxin, especially the presence or absence of a selenocysteine (Sec) residue in this tetrapeptide. In this study, we have employed protein engineering techniques to investigate the C-terminal redox-active tetrapeptides of three different TRs: mouse mitochondrial TR (mTR3), Drosophila melanogaster TR (DmTR), and the mitochondrial TR from Caenorhabditis elegans (CeTR2), which have C-terminal tetrapeptide sequences of Gly-Cys-Sec-Gly, Ser-Cys-Cys-Ser, and Gly-Cys-Cys-Gly, respectively. Three different types of mutations and chemical modifications were performed in this study: insertion of alanine residues between the cysteine residues of the Cys-Cys or Cys-Sec dyads, modification of the charge at the C-terminus, and altering the position of the Sec residue in the mammalian enzyme. The results show that mTR3 is quite accommodating to insertion of alanine residues into the Cys-Sec dyad, with only a 4-6-fold drop in catalytic activity. In contrast, the activity of both DmTR and CeTR2 was reduced 100-300-fold when alanine residues were inserted into the Cys-Cys dyad. We have tested the importance of a salt bridge between the C-terminus and a basic residue that was proposed for orienting the Cys-Sec dyad of mTR3 for proper catalytic position by changing the C-terminal carboxylate to a carboxamide. The result is an enzyme with twice the activity as the wild-type mammalian enzyme. A similar result was achieved when the C-terminal carboxylate of DmTR was converted to a hydroxamic acid or a thiocarboxylate. Last, reversing the positions of the Cys and Sec residues in the catalytic dyad resulted in a 100-fold loss of catalytic activity. Taken together, the results support our previous model of Sec as the leaving group during reduction of the C-terminus during the catalytic cycle.     

Site-directed mutagenesis of yeast C1-tetrahydrofolate synthase: analysis of an overlapping active site in a multifunctional enzyme

C1-tetrahydrofolate (THF) synthase is a trifunctional protein possessing the activities 10-formyl-THF synthetase, 5,10-methenyl-THF cyclohydrolase, and 5,10-methylene-THF dehydrogenase. The current model divides this protein into two functionally independent domains with dehydrogenase/cyclohydrolase activities sharing an overlapping active site on the N-terminal domain and synthetase activity associated with the C-terminal domain. Previous chemical modification studies on C1-THF synthase from the yeast Saccharomyces cerevisiae indicated at least two cysteinyl residues involved in the dehydrogenase/cyclohydrolase reactions [Appling, D. R., & Rabinowitz, J. C. (1985) Biochemistry 24, 3540-3547]. In the present work, site-directed mutagenesis of the S. cerevisiae ADE3 gene, which encodes C1-THF synthase, was used to individually change each cysteine contained within the dehydrogenase/cyclohydrolase domain (Cys-11, Cys-144, and Cys-257) to serine. The resulting proteins were overexpressed in yeast and purified for kinetic analysis. Site-specific mutations in the dehydrogenase/cyclohydrolase domain did not affect synthetase activity, consistent with the proposed domain structure. The C144S and C257S mutations result in 7- and 2-fold increases, respectively, in the dehydrogenase Km for NADP+. C144S lowers the dehydrogenase maximal velocity roughly 50% while C257S has a maximal velocity similar to that of the wild type. Cyclohydrolase catalytic activity is reduced 20-fold by the C144S mutation but increased 2-fold by the C257S mutation. Conversion of Cys-11 to serine has a negligible effect on dehydrogenase/cyclohydrolase activity. A double mutant, C144S/C257S, results in catalytic properties roughly multiplicative of the individual mutations.(ABSTRACT TRUNCATED AT 250 WORDS)