肠毒素C2蛋白C,N末端对其活性的影响

目的:分析金黄色葡萄球菌肠毒素C2(SEC2)中N,C末端对其超抗原活性和可溶性表达能力的影响。方法:应用基因工程技术对SEC2的N,C末端进行部分删除,获得三种突变蛋白,并对其进行体外超抗原活性和可溶性表达能力的比较。结果:对SEC2的N,C末端的删除都在一定程度上影响其超抗原活性和可溶性表达能力,其中,N末端的两个删除突变体的超抗原活性分别降低40%和48%,而删除C末端则使其可溶性表达水平下降到野生型的20%左右。结论:SEC2蛋白分子的N末端对其超抗原活性起主要作用,C末端对其可溶性表达具有显著影响,而完整的SEC2分子对于其发挥最大生物学活性是必要的。     

Functional analysis of the disulphide loop mutant of staphylococcal enterotoxin C2

The superantigen staphylococcal enterotoxin C2 (SEC2) tremendously activate T lymphocytes bearing certain T-cell receptor Vβ domains when binding to MHC II molecules, which launches a powerful response of tumour inhibition in vitro as well as in vivo. However, the toxicity of SEC2 performed in clinic limited its broad application for immunotherapy. The previous studies suggested that the disulphide loop may be important for the toxicity of some SEs, which prompted us to investigate the potential roles of the disulphide loop in biological activity of SEC2. Site-directed mutagenesis was used to disturb the formation of the disulphide bond by substituting Ala or Ser for Cys-93 and Cys-110. The expressed mutants in Escherichia coli were used to determine their superantigen activity and toxicity. Results showed that all of the mutated proteins exhibited reduced abilities to induce T-cell proliferation and cytotoxic effects on tumour cells L929 and Hepa1-6, suggesting that the disulphide loop plays functional role in maintaining the maximal superantigen activity of SEC2. Furthermore, the toxicity assays in vivo showed that all of the mutants induced a reduced emetic and pyrogenic responses compared with native SEC2, which might be important for further construction of lowly toxic superantigen agent.     

An engineered superantigen SEC2 exhibits promising antitumor activity and low toxicity

Recent studies suggested that the histidine residues at 118 and 122 play an important role for the toxicity of staphylococcal enterotoxin C subtype 2 (SEC2), and the substitutions of both histidines with alanine can severely impair the fever activity of SEC2. We hypothesized that promising SEC2 antitumor agent with low toxicity and enhanced superantigen activity can be constructed by introducing related mutations at protein functional sites of SEC2. We showed that the SEC2 mutants H122A and H118A/H122A exhibited improved superantigen activity after introducing the point mutations at Thr20 and Gly22. A resultant mutant, named as SAM-3, has considerable abilities to inhibit the growth of H22 and Hepa1-6 tumor cells in vitro and colon 26 solid tumor in vivo. Furthermore, SAM-3 also exhibits significantly reduced toxicity compared with native SEC2. The study provides a novel strategy for designing promising superantigen immunotherapeutic agent. The constructed SEC2 mutant SAM-3 can be used as a powerful candidate for cancer immunotherapy and could compensate the deficiency caused by toxicity of native SEC2 in clinic.     

Enhancement of superantigen activity and antitumor response of staphylococcal enterotoxin C2 by site-directed mutagenesis

Bacterial superantigen staphylococcal enterotoxins (SEs) tremendously stimulate polyclonal T cells bearing particular TCR Vβ domains when binding to MHC II molecules, suggesting that they could be a candidate of new antitumor agent. SEC2, an important member of superantigen family, has been used in clinical trial as an immuntherapy agent for cancer treatment in China, and obtained some encouraging effects. However, the presence of immunosuppression and endotoxic activity limits the therapeutic dosage of SEC2, and influences its antitumor effect in clinic. Therefore, the enhancement of superantigen activity and antitumor effect of SEC2 could effectively make compensation for the disadvantages mentioned above. In this study, a superantigen SEC2(T20L/G22E) mutant was generated by site-directed mutagenesis, and efficiently expressed in E. coli BL21(DE3). The results showed that SEC2(T20L/G22E) mutant exhibited a significantly enhanced superantigen activity and antitumor response, compared with native SEC2 in vitro. Further toxicity assay in vivo indicated that SEC2(T20L/G22E) mutant had no significant increase in emetic and pyrogenic activity compared with SEC2, which suggested that the mutant SEC2(T20L/G22E) could be used as a potentially powerful candidate for cancer immunotherapy, and could make compensation for the deficiency of native SEC2 in clinic.     

Construction of novel bifunctional chimeric proteins possessing antitumor and thrombolytic activities

Based on their respective antitumor and thrombolytic activities, the superantigen staphylococcal enterotoxin C2 (SEC2) and staphylokinase (Sak) were chosen for the construction of the novel chimeric proteins Sak-linker- SEC2 and SEC2-linker-Sak using a linker composed of nine Ala residues. Both chimeric proteins possessed nearly the same PBMC proliferation stimulating activity and antitumor activity as SEC2 and thrombolytic activity as Sak. Neither the SEC2 or Sak component of each chimeric protein affected the activity of the other component. The results presented in this study provide a possible strategy to prevent and cure tumor thrombus.     

Dissection of the enzymatic and immunologic functions of macrophage migration inhibitory factor. Full immunologic activity of N-terminally truncated mutants.

Macrophage migration inhibitory factor (MIF) is a cytokine with broad regulatory functions in innate immunity. MIF belongs to the few cytokines displaying catalytic activities, i.e. MIF has a Pro2-dependent tautomerase and a Cys-Ala-Leu-Cys (CALC) cysteine-based thiol-protein oxidoreductase activity. Previous studies have addressed the roles of the catalytic site residues and the C-terminus. The two activities have not been directly compared. Here we report on the N-terminal mutational analysis and minimization of MIF and on a dissection of the two catalytic activities by comparing mutants P2AMIF, Delta4MIF, Delta5MIF, Delta6MIF, Delta7MIF, Delta8MIF, and Delta10MIF with the cysteine mutants of MIF. As N-terminal deletion was predicted to interfere with protein structure due to disruption of the central beta sheet, it was surprising that deletion of up to six N-terminal residues resulted in normally expressed proteins with wild-type conformation. Strikingly, such mutants exhibited full MIF-specific immunologic activity. While mutation of Pro2 eliminated tautomerase activity, the CALC cysteine residues had no influence on this activity. However, mutant C81SMIF, which otherwise has full biologic activity, only had 32% tautomerase activity. Deletion of four N-terminal residues did not interfere with insulin reduction by MIF. By contrast, reduction of 2-hydroxyethyldisulfide (HED) was markedly affected by N-terminal manipulation, with P2AMIF and Delta2MIF exhibiting 40% activity, and Delta4MIF completely failing to reduce HED. This study constitutes the first comparison of the two catalytic activities of MIF and should assist in understanding the molecular links between the catalytic and immunologic activities of this cytokine and in providing guidelines for N-terminal protein minimization.     

The Gly-Gly linker region of the insect cytokine growth-blocking peptide is essential for activity

Growth-blocking peptide (GBP) is a 25-amino acid cytokine isolated from the lepidopteran insect Pseudaletia separata. GBP exhibits various biological activities such as regulation of larval growth of insects, proliferation of a few kinds of cultured cells, and stimulation of a class of insect immune cells called plasmatocytes. The tertiary structure of GBP consists of a well structured core domain and disordered N and C termini. Our previous studies revealed that, in addition to the structured core, specific residues in the unstructured N-terminal region (Glu1 and Phe3) are also essential for the plasmatocyte-stimulating activity. In this study, a number of deletion, insertion, and site-directed mutants targeting the unstructured N-terminal residues of GBP were constructed to gain more detailed insight into the mode of interaction between the N-terminal region and GBP receptor. Alteration of the backbone length of the linker region between the core structure and N-terminal domain reduced plasmatocyte-stimulating activity. The substitutions of Gly5 or Gly6 in this linker region with more bulky residues, such as Phe and Pro, also remarkably reduced this activity. We conclude that the interaction of GBP with its receptor depends on the relative position of the N-terminal domain to the core structure, and therefore the backbone flexibility of Gly residues in the linker region is necessary for adoption of a proper conformation suited to receptor binding. Additionally, antagonistic experiments using deletion mutants confirmed that not only the core domain but also the N-terminal region of GBP are required for "receptor-binding," and furthermore Phe3 is a binding determinant of the N-terminal domain.     

Participation of two N-terminal residues in LPS-neutralizing activity of sarcotoxin IA

Sarcotoxin IA is a cecropin-type antibacterial peptide of flesh fly. Using a mutant sarcotoxin IA lacking two N-terminal residues, we demonstrated that these residues are indispensable for its antibacterial activity against Escherichia coli and LPS-binding. Contrary to the native sarcotoxin IA, the mutant sarcotoxin IA could not neutralize various biological activities of LPS. It was suggested that sarcotoxin IA firmly binds to the lipid A core of LPS via these two N-terminal residues and forms a stable binding complex that exhibits no appreciable biological activity like native LPS.     

Zinc-mediated dimerization and its effect on activity and conformation of staphylococcal enterotoxin type C

Staphylococcal enterotoxins are superantigen exotoxins that mediate food poisoning and toxic shock syndrome in humans. Despite their structural and functional similarities, superantigens display subtle differences in biological properties and modes of receptor binding as a result of zinc atoms bound differently in their crystal structures. For example, the crystal structures of the staphylococcal enterotoxins in the type C serogroup (SECs) contain a zinc atom coordinated by one aspartate and two histidine residues from one molecule and another aspartate residue from the next molecule, thus forming a dimer. This type of zinc ligation and zinc-mediated dimerization occurs in several SECs, but not in most other staphylococcal enterotoxin serogroups. This prompted us to investigate the potential importance of zinc in SEC-mediated pathogenesis. Site-directed mutagenesis was used to replace SEC zinc binding ligands with alanine. SEC mutants unable to bind zinc did not have major conformational alterations although they failed to form dimers. Zinc binding was not essential for T cell stimulation, emesis, or lethality although in general the mutants were less pyrogenic. Thus the zinc atom in SECs might represent a non-functional heavy atom in an exotoxin group that has diverged from related bacterial toxins containing crucial zinc atoms.     

N-terminal domain of eotaxin-3 is important for activation of CC chemokine receptor 3

Eotaxin-3 belongs to the CC chemokine family, and specifically recognizes CC chemokine receptor (CCR) 3 that is expressed on eosinophils, basophils and helper T type 2 cells. The three-dimensional structure of eotaxin-3 determined by nuclear magnetic resonance has revealed that the N-terminal nine residues preceding the first cysteine comprise an unstructured domain, which is also observed in other chemokine molecules. In order to determine the function of the N-terminal domain of eotaxin-3, we constructed various N-terminal-deletion mutants, and then examined their binding and chemotactic activities toward eosinophils in vitro. Competitive binding studies showed that the binding affinity of truncated mutant toward CCR3 was almost the same as that of wild-type eotaxin-3 even though the N-terminal truncation involved the first through to the ninth residues. In contrast, the chemotactic activity gradually decreased with extension of the N-terminal deletion, and when the deletion extended to the eighth residue, the activity was not detected at all. Thus, the N-terminal nine residues are not critical for binding but the N-terminal eight residues are essential for activation of CCR3. The truncated eotaxin-3 proteins lacking the N-terminal eight or nine residues inhibited the chemotactic activity of chemokines that recognize CCR3. The truncated mutants can possibly be used for anti-allergic and anti-HIV-1 therapy.     

人白介素15及突变体的分子设计

以人白介素－２晶体结构为模板同源模型建人白介素１５及其两株突变体（Ｎ端缺失４个氨基酸、Ｃ端缺失３个氨基酸）的空间构象。在ＣＶＦＦ力场下,经过分子力学优化、常温分子动力学模拟获得稳定立体结构模型。借助空间构象残基的亲疏水性分析,利用Ｄｅｌｐｈｉ一性分析蛋白表达静电分布,进行从理论上预测人白介素１５及两株突变体生物学功能的性、差异性。结合分子生物实验,在ＰＢＶ２２０载体中克隆表达获得人白介素１５及两载     

Deletion analysis of yeast Sec65p reveals a central domain that is sufficient for function in vivo

The Saccharomyces cerevisiae SEC65 gene encodes a 32 kDa subunit of yeast signal recognition particle that is homologous to human SRP19. Sequence comparisons suggest that the yeast protein comprises three distinct domains. The central domain (residues 98–171) exhibits substantial sequence similarity to the 144 residue SRP19. In contrast, the N-terminal and C-terminal domains (residues 1–97 and 172–273 respectively) share no similarity to SRP19, with the exception of a cluster of positively charged residues at the extreme C-terminus of both proteins. Here, we report the cloning of a Sec65p homologue from the yeast Candida albicans that shares the same extended domain structure as its S. cerevisiae counterpart. This conservation of sequence is reflected at the functional level, as the C. albicans gene can complement the conditional lethal sec65-1 mutation in S. cerevisiae . In order to examine the role of the N- and C- terminal domains in Sec65p function, we have engineered truncation mutants of S. cerevisiae SEC65 and tested these for complementing activity in vivo and for SRP integrity in vitro . These studies indicate that a minimal Sec65p comprising residues 76–209, which includes the entire central SRP19-like domain, is sufficient for SRP function in yeast.     

trans-sialidase of Trypanosoma cruzi: location of galactose-binding site(s).

Trypanosoma cruzi expresses a trans-sialidase on its surface, which catalyzes the transfer of sialic acid from mammalian host glycans to its own surface glycoproteins. It has been proposed that the enzyme consists of three domains prior to a long C-terminal repeating sequence that is not required for enzyme activity. The first of these domains shares significant sequence identity with bacterial sialidases which catalyse the hydrolysis of sialic acid. Here we report the sequence of the N-terminal domains of the TS19y trans-sialidase gene, which was expressed in bacteria with the same specific activity as natural enzyme of T. cruzi. Various deletion mutants of TS19y, without the C-terminal tandem repeat, have been cloned and expressed and their trans-sialidase and sialidase activities measured. These experiments show that all three N-terminal domains are required for full trans-sialidase activity, though only the first is necessary for sialidase activity. Some transferase activity is observed, however, even with the shortest construct comprising the first N-terminal domain. Deletion mutants to probe the role of the N-terminal residues of the first domain suggest that the first 33 residues are also required for trans-sialidase activity, but not for sialidase activity. Molecular modelling of the first N-terminal domain of TS19y based on our structures of bacterial sialidases and site-directed mutations suggests the location of a galactose-binding site within this domain.     

Protection of GroEL by its methionine residues against oxidation by hydrogen peroxide

GroEL undergoes an important functional and structural transition when oxidized with hydrogen peroxide (H2O2) concentrations between 15 and 20mM. When GroEL was incubated for 3h with 15 mM H2O2, it retained its quaternary structure, chaperone and ATPase activities. Under these conditions, GroEL's cysteine and tyrosine residues remained intact. However, all the methionine residues of the molecular chaperone were oxidized to the corresponding methionine-sulfoxides under these conditions. The oxidation of the methionine residues was verified by the inability of cyanogen bromide to cleave at the carboxyl side of the modified methionine residues. The role for the proportionately large number (23) of methionine residues in GroEL has not been identified. Methionine residues have been reported to have an antioxidant activity in proteins against a variety of oxidants produced in biological systems including H2O2. The carboxyl-terminal domain of GroEL is rich in methionine residues and we hypothesized that these residues are involved in the protection of GroEL's functional structure by scavenging H2O2. When GroEL was further incubated for the same time, but with increasing concentrations of H2O2 (>15 mM), the oxidation of GroEL's cysteine residues and a significant decrease of the tyrosine fluorescence due to the formation of dityrosines were observed. Also, at these higher concentrations of H2O2, the inability of GroEL to hydrolyze ATP and to assist the refolding of urea-unfolded rhodanese was observed.     

The serine protease and RNA-stimulated nucleoside triphosphatase and RNA helicase functional domains of dengue virus type 2 NS3 converge within a region of 20 amino acids

NS3 protein of dengue virus type 2 has a serine protease domain within the N-terminal 180 residues. NS2B is required for NS3 to form an active protease involved in processing of the viral polyprotein precursor. The region carboxy terminal to the protease domain has conserved motifs present in several viral RNA-stimulated nucleoside triphosphatase (NTPase)/RNA helicases. To define the functional domains of protease and NTPase/RNA helicase activities of NS3, full-length and amino-terminal deletion mutants of NS3 were expressed in Escherichia coli and purified. Deletion of 160 N-terminal residues of NS3 (as in NS3del.2) had no detrimental effect on the basal and RNA-stimulated NTPase as well as RNA helicase activities. However, mutagenesis of the conserved P-loop motif of the RNA helicase domain (K199E) resulted in loss of ATPase activity. The RNA-stimulated NTPase activity was significantly affected by deletion of 20 amino acid residues from the N terminus or by substitutions of the cluster of basic residues, 184RKRK-->QNGN, of NS3del.2, although both mutant proteins retained the conserved RNA helicase motifs. Furthermore, the minimal NS3 protease domain, required for cleavage of the 2B-3 site, was precisely defined to be 167 residues, using the in vitro processing of NS2B-NS3 precursors. Our results reveal that the functional domains required for serine protease and RNA-stimulated NTPase activities map within the region between amino acid residues 160 and 180 of NS3 protein and that a novel motif, the cluster of basic residues 184RKRK, plays an important role for the RNA-stimulated NTPase activity.     

Identification of amino acids important for the biochemical activity of Methanothermobacter thermautotrophicus MCM

Methanothermobacter thermautotrophicus minichromosomal maintenance protein (mtMCM) is a 75 kDa protein that self-assembles into a double hexamer structure. The double hexamer formed by the N-terminal region of mtMCM has a highly charged (overwhelmingly net positive) inner channel. Here we investigate the effects of point mutations of some of these charged residues on the biological activities of mtMCM. Although all of the mutants were similar to the wild type in protein folding and complex assembly, we found that mutations impaired helicase activity. The study of the DNA binding and ATPase activities of these mutants revealed that the impairment of the helicase activity was highly correlated with a decrease in DNA binding, providing evidence consistent with the role of these charged residues of the inner channel in interactions with DNA.     

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.     

Role of unique basic residues in cytotoxic, antibacterial and antiparasitic activities of human eosinophil cationic protein

Eosinophil granule proteins, eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin are members of the RNase A superfamily, which play a crucial role in host defense against various pathogens as they are endowed with several biological activities. Some of the biological activities possessed by ECP have been attributed to its strong basic character. In the current study, we have investigated the role of five unique basic residues, Arg22, Arg34, Arg61, Arg77 and His64 of ECP in its catalytic, cytotoxic, antibacterial and antiparasitic activities. These residues were changed to alanine to generate single and double mutants. None of the selected residues was found to be involved in the RNase activity of ECP. The substitution of all five residues individually was detrimental for the cytotoxic, antibacterial and antiparasitic activities of ECP; however, mutation of Arg22 and Arg34 resulted in the most significant effects. The double mutants also had reduced biological activities. All ECP mutants that had significantly reduced toxicity also had reduced membrane destabilization activity. Our study demonstrates that Arg22, Arg34, Arg61, Arg77 and His64 of ECP are crucial for its membrane destabilization activity, which appears to be the underlying mechanism of its cytotoxic, antibacterial and antiparasitic activities.     

Mutational analysis of autonomously functioning trans-activating peptides encoded by adenovirus E1A: evidence for two subdomains.

We have shown previously that a chemically synthesized adenovirus E1A region 3 peptide of 49 amino acids, protein domain 3 (PD3; residues 140 to 188 of the 289-amino-acid protein), trans activates viral genes in vitro and in vivo. To study structure-function relationships, we synthesized N-terminal deletion and cysteine substitution mutant peptides and tested their activities in a cell microinjection assay. Peptides lacking 1 to 12 N-terminal residues exhibited 5- to 50-fold-reduced molar specific activities, whereas those lacking 16 or 18 residues were inactive. Substitution of each of five PD3 cysteine residues with alanine resulted in substantial losses of activity: mutants in the PD3 N-terminal portion showed 40 to 55% of wild-type activity but required a 20-fold-higher concentration than PD3, whereas those in the C-terminal half were as much less active. These peptide mutant studies suggest the existence of two PD3 functional regions: one, localized in the C-terminal 70 to 75% of the molecule, is essential for trans activation; the other, localized in the N-terminal 25 to 30%, can be overridden to a significant extent at high peptide concentrations.     

Several acidic amino acids in the N-domain of insulin-like growth factor-binding protein-5 are important for its transactivation activity

Insulin-like growth factor-binding protein (IGFBP)-5 is a secreted protein that binds to IGFs and modulates IGF actions. IGFBP-5 is also found in the nuclei of cultured cells and has transactivation activity. Here we report the nuclear localization of endogenous IGFBP-5 in mouse embryonic skeletal cells. Chromatin immunoprecipitation experiments indicated that IGFBP-5 interacts with the nuclear histone-DNA complex. Using a series of deletion mutants, the transactivation domain of IGFBP-5 was mapped to its N-terminal region. Intriguingly, the transactivation activity of IGFBP-5 is masked by negative regulatory elements located in the L- and C-domains. Among the other IGFBPs, the N-domains of IGFBP-2 and -3 also had strong transactivation activity, whereas those of IGFBP-1 and -6 had no activity. The IGFBP-4 N-domain had modest activity. Sequence analysis revealed several amino acids in the IGFBP-5 N-domain that are not present in IGFBP-1. The activities of mutants in which these residues were changed to the corresponding IGFBP-1 sequence were determined. Mutations that changed acidic residues to neutral residues (e.g. E8A, D11S, E12A, E30S/P31A, E43L, and E52A) or a polar to a basic residue (e.g. Q56R) significantly reduced transactivation activity. The E8A/D11S/E12A triple mutant and E52A/Q56R double mutants showed further reduced activity. The combinatory mutants had essentially no transactivation activity. Taken together, our results indicate that there are several conserved residues in the IGFBP-5 N-terminal region that are critical for transactivation and that IGFBP-2 and -3 also have strong transactivation activity in their N-domains.