共查询到20条相似文献,搜索用时 31 毫秒
1.
Luke C Schick C Tsu C Whisstock JC Irving JA Brömme D Juliano L Shi GP Chapman HA Silverman GA 《Biochemistry》2000,39(24):7081-7091
The human squamous cell carcinoma antigens (SCCA) 1 and 2 are members of the serpin family that are 92% identical in their amino acid sequence. Despite this similarity, they inhibit distinct classes of proteinases. SCCA1 neutralizes the papain-like cysteine proteinases, cathepsins (cat) S, L, and K; and SCCA2 inhibits the chymotrypsin-like serine proteinases, catG and human mast cell chymase. SCCA2 also can inhibit catS, as well as other papain-like cysteine proteinases, albeit at a rate 50-fold less than that of SCCA1. Analysis of the mechanism of inhibition by SCCA1 revealed that the reactive site loop (RSL) is important for cysteine proteinase inhibition. The inhibition of catS by a mutant SCCA2 containing the RSL of SCCA1 is comparable to that of wild-type SCCA1. This finding suggested that there were no motifs outside and only eight residues within the RSL that were directing catS-specific inhibition. The purpose of this study was to determine which of these residues might account for the marked difference in the ability of SCCA1 and SCCA2 to inhibit papain-like cysteine proteinases. SCCA2 molecules containing different RSL mutations showed that no single amino acid substitution could convert SCCA2 into a more potent cysteine proteinase inhibitor. Rather, different combinations of mutations led to incremental increases in catS inhibitory activity with residues in four positions (P1, P3', P4', and P11') accounting for 80% of the difference in activity between SCCA1 and SCCA2. Interestingly, the RSL cleavage site differed between wild-type SCCA2 and this mutant. Moreover, these data established the importance of a Pro residue in the P3' position for efficient inhibition of catS by both wild-type SCCA1 and mutated SCCA2. Molecular modeling studies suggested that this residue might facilitate positioning of the RSL within the active site of the cysteine proteinase. 相似文献
2.
Galectins are a family of animal lectins defined by their β-galactoside-binding specificity and a consensus sequence in their
carbohydrate-recognition domain. Galectin-1 (Gal-1) is expressed as a non-covalently linked homodimer present in a variety
of tissues. Here we describe its isolation from human platelets by a procedure involving ionic exchange chromatography and
affinity chromatography on lactose-agarose. Platelet Gal-1 co-purifies with actin, forming an actin-Gal-1 complex which does
no dissociate even after treatment with sodium dodecyl sulfate. The presence of both proteins was confirmed by Western blot
and by trypsin digestion followed by mass spectrometry identification. By hemagglutination assays we studied the response
of recombinant Gal-1/actin, mixed and pre-incubated in different proportions, and then tested against neuraminidase treated
rabbit red blood cells. The complex formation was confirmed by confocal microscopy, showing that both proteins co-localised
in resting platelets as well as in thrombin-activated ones. These results suggest that endogenous Gal-1 forms an intracellular
complex with monomeric actin and that, after platelet activation, Gal-1 could play a role in the polymerization-depolymerization
process of actin, which concludes in platelet aggregation. 相似文献
3.
We have constructed a mutant Na,K-ATPase alpha1-subunit with all native cysteine residues replaced. Using the baculovirus system, this cysteine-less alpha1-subunit and wild-type beta1-subunit were expressed in High Five cells. After 3 days of infection, cells were fractionated, and endoplasmic reticulum, Golgi apparatus, and plasma membranes were isolated. The molecular activity of the cysteine-less mutant in the plasma membranes was close to the wild-type protein (8223 min(-)(1) versus 6655 min(-)(1)). Cation and ATP activation of Na,K-ATPase activities revealed that replacing all 23 cysteines resulted in only a 50% reduction of K(m) for Na(+), a 2-fold increase in K(m) for K(+), and no changes in K(m) for ATP. The distribution of alpha-subunits among the membranes showed a high percentage of cysteine-less protein in the endoplasmic reticulum and Golgi apparatus compared with the wild-type protein. Furthermore, the cellular stability of the alphabeta assembly appeared reduced in the cysteine-less mutant. Cells harvested after more than 3 days of infection showed extensive degradation of the cysteine-less alpha-subunit, which is not observed with the wild-type enzyme. Thus the Na,K-ATPase contains no cysteine residues that are critical for function, but the folding and/or assembly pathway of this enzyme is affected by total cysteine substitution. 相似文献
4.
Kim BM Schultz LW Raines RT 《Protein science : a publication of the Protein Society》1999,8(2):430-434
Human ribonuclease inhibitor (hRI) is a cytosolic protein that protects cells from the adventitious invasion of pancreatic-type ribonucleases. hRI has 32 cysteine residues. The oxidation of these cysteine residues to form disulfide bonds is a rapid, cooperative process that inactivates hRI. The most proximal cysteine residues in native hRI are two pairs that are adjacent in sequence: Cys94 and Cys95, and Cys328 and Cys329. A cystine formed from such adjacent cysteine residues would likely contain a perturbing cis peptide bond within its eight-membered ring, which would disrupt the structure of hRI and could facilitate further oxidation. We find that replacing Cys328 and Cys329 with alanine residues has little effect on the affinity of hRI for bovine pancreatic ribonuclease A (RNase A), but increases its resistance to oxidation by 10- to 15-fold. Similar effects are observed for the single variants, C328A hRI and C329A hRI, suggesting that oxidation resistance arises from the inability to form a Cys328-Cys329 disulfide bond. Replacing Cys94 and Cys95 with alanine residues increases oxidation resistance to a lesser extent, and decreases the affinity of hRI for RNase A. The C328A, C329A, and C328A/C329A variants are likely to be more useful than wild-type hRI for inhibiting pancreatic-type ribonucleases in vitro and in vivo. We conclude that replacing adjacent cysteine residues can confer oxidation resistance in a protein. 相似文献
5.
Van't Klooster JW Van der Kamp MW Vervoort J Beekwilder J Boeren S Joosten MH Thomma BP De Wit PJ 《Molecular Plant Pathology》2011,12(1):21-30
The Cladosporium fulvum Avr2 effector is a novel type of cysteine protease inhibitor with eight cysteine residues that are all involved in disulphide bonds. We have produced wild-type Avr2 protein in Pichia pastoris and determined its disulphide bond pattern. By site-directed mutagenesis of all eight cysteine residues, we show that three of the four disulphide bonds are required for Avr2 stability. The six C-terminal amino acid residues of Avr2 contain one disulphide bond that is not embedded in its overall structure. Avr2 is not processed by the tomato cysteine protease Rcr3 and is an uncompetitive inhibitor of Rcr3. We also produced mutant Avr2 proteins in which selected amino acid residues were individually replaced by alanine, and, in one mutant, all six C-terminal amino acid residues were deleted. We determined the inhibitory constant (K(i) ) of these mutants for Rcr3 and their ability to trigger a Cf-2-mediated hypersensitive response (HR) in tomato. We found that the two C-terminal cysteine residues and the six amino acid C-terminal tail of Avr2 are required for both Rcr3 inhibitory activity and the ability to trigger a Cf-2-mediated HR. Individual replacement of the lysine-17, lysine-20 or tyrosine-21 residue by alanine did not affect significantly the biological activity of Avr2. Overall, our data suggest that the affinity of the Avr2 mutants for Rcr3 correlates with their ability to trigger a Cf-2-mediated HR. 相似文献
6.
Conversion of cysteine to serine residues alters the activity, stability, and heparin dependence of acidic fibroblast growth factor. 总被引:10,自引:0,他引:10
S Ortega M T Schaeffer D Soderman J DiSalvo D L Linemeyer G Gimenez-Gallego K A Thomas 《The Journal of biological chemistry》1991,266(9):5842-5846
Acidic fibroblast growth factor (aFGF) is a broad spectrum mitogen that is stabilized by complexation with heparin and heparan proteoglycans. The monomeric human protein contains 3 reduced cysteine residues of unknown function, the first 2 of which are conserved among all seven known fibroblast growth factors. The influence of these free sulfhydryl groups on the level, stability, and heparin dependence of the mitogenic activity at physiological temperature and pH is characterized using a complete set of site-directed mutants in which either any 1, 2, or all 3 of the cysteine residues are converted to serines. Mutants of aFGF in which either any 2 or all 3 cysteine residues are substituted by serines are more active, have longer activity half-lives, and are less heparin dependent than wild-type aFGF. In contrast, wild-type aFGF and the three mutants that each retain 2 cysteine residues inactivate more rapidly in the absence of heparin by a nonproteolytic mechanism but are markedly stabilized by heparin. This cysteine-mediated destabilization of aFGF not only diminishes its activity in the absence of heparin in tissue culture but also could functionally restrict its activity in vivo to the vicinity of mast cell-derived heparins and heparan proteoglycans associated with cell surfaces and basement membranes. 相似文献
7.
Galectin-1: a small protein with major functions 总被引:13,自引:0,他引:13
Galectins are a family of carbohydrate-binding proteins with an affinity for beta-galactosides. Galectin-1 (Gal-1) is differentially expressed by various normal and pathological tissues and appears to be functionally polyvalent, with a wide range of biological activity. The intracellular and extracellular activity of Gal-1 has been described. Evidence points to Gal-1 and its ligands as one of the master regulators of such immune responses as T-cell homeostasis and survival, T-cell immune disorders, inflammation and allergies as well as host-pathogen interactions. Gal-1 expression or overexpression in tumors and/or the tissue surrounding them must be considered as a sign of the malignant tumor progression that is often related to the long-range dissemination of tumoral cells (metastasis), to their dissemination into the surrounding normal tissue, and to tumor immune-escape. Gal-1 in its oxidized form plays a number of important roles in the regeneration of the central nervous system after injury. The targeted overexpression (or delivery) of Gal-1 should be considered as a method of choice for the treatment of some kinds of inflammation-related diseases, neurodegenerative pathologies and muscular dystrophies. In contrast, the targeted inhibition of Gal-1 expression is what should be developed for therapeutic applications against cancer progression. Gal-1 is thus a promising molecular target for the development of new and original therapeutic tools. 相似文献
8.
Rebecca K. Donegan Shannon E. Hill Katherine C. Turnage Susan D. Orwig Raquel L. Lieberman 《The Journal of biological chemistry》2012,287(52):43370-43377
Myocilin is a protein found in the trabecular meshwork extracellular matrix tissue of the eye that plays a role in regulating intraocular pressure. Both wild-type and certain myocilin variants containing mutations in the olfactomedin (OLF) domain are linked to the optic neuropathy glaucoma. Because calcium ions are important biological cofactors that play numerous roles in extracellular matrix proteins, we examined the calcium binding properties of the myocilin OLF domain (myoc-OLF). Our study reveals an unprecedented high affinity calcium binding site within myoc-OLF. The calcium ion remains bound to wild-type OLF at neutral and acidic pH. A glaucoma-causing OLF variant, myoc-OLF(D380A), is calcium-depleted. Key differences in secondary and tertiary structure between myoc-OLF(D380A) and wild-type myoc-OLF, as well as limited access to chelators, indicate that the calcium binding site is largely buried in the interior of the protein. Analysis of six conserved aspartate or glutamate residues and an additional 18 disease-causing variants revealed two other candidate residues that may be involved in calcium coordination. Our finding expands our knowledge of calcium binding in extracellular matrix proteins; provides new clues into domain structure, function, and pathogenesis for myocilin; and offers insights into highly conserved, biomedically relevant OLF domains. 相似文献
9.
The Thomsen-Friedenreich (TF or T) antigen, Galβ1-3GalNAcα1-O-Ser/Thr, is the core 1 structure of O-linked mucin type glycans appearing in tumor-associated glycosylation. The TF antigen occurs in about 90% of human cancer cells and is a potential ligand for the human endogenous galectins. It has been reported that human galectin-1 (Gal-1) and galectin-3 (Gal-3) can perform their cancer-related functions via specifically recognizing TF antigen. However, the detailed binding properties have not been clarified and structurally characterized. In this work, first we identified the distinct TF-binding abilities of Gal-1 and Gal-3. The affinity to TF antigen for Gal-3 is two orders of magnitude higher than that for Gal-1. The structures of Gal-3 carbohydrate recognition domain (CRD) complexed with TF antigen and derivatives, TFN and GM1, were then determined. These structures show a unique Glu-water-Arg-water motif-based mode as previously observed in the mushroom galectin AAL. The observation demonstrates that this recognition mode is commonly adopted by TF-binding galectins, either as endogenous or exogenous ones. The detailed structural comparisons between Gal-1 and Gal-3 CRD and mutagenesis experiments reveal that a pentad residue motif ((51)AHGDA(55)) at the loop (g1-L4) connecting β-strands 4 and 5 of Gal-1 produces a serious steric hindrance for TF binding. This motif is the main structural basis for Gal-1 with the low affinity to TF antigen. These findings provide the intrinsic structural elements for regulating the TF-binding activity of Gal-1 in some special conditions and also show certain target and approach for mediating some tumor-related bioactivities of human galectins. 相似文献
10.
The first example of the use of a reporter affinity label (NNA) that contains a fluorogenic naphthalene dialdehyde moiety to identify neighboring lysine and cysteine residues at a recognition site is described. The opioid receptors have served as the proof-of-concept because they contain multiple lysine and cysteine residues. The kinetics of isoindole formation resulting from covalent binding of NNA to wild-type and mutant opioid receptors were followed in cultured cells using flow cytometry. The finding that NNA bound to mutant mu opioid receptors (K233R and C235S) without producing specific fluorescence enhancement suggested that covalent bonding occurred at these positions to produce an isoindole fluorophore in the wild-type mu receptor. The similar kinetics of fluorophore formation for wild-type mu, delta, and kappa opioid receptors suggest that these conserved residues are the cross-linking sites in all three types of opioid receptors. The combined utilization of a reporter affinity label and site-directed mutagenesis offers a more expeditious method of identifying cross-linking at a recognition site when compared to classical procedures. 相似文献
11.
We have produced human fibrinogen gamma 259-411 in Escherichia coli in order to study the relationship between the calcium binding activity of the polypeptide and the integrity of the disulfide bond cysteine326-cysteine339. The polypeptide was produced from a plasmid expression vector at approximately 5 micrograms per milliliter of bacterial culture. The identity of the polypeptide was confirmed by N-terminal amino acid sequence analysis. The expression vector was modified by oligonucleotide directed mutagenesis to remove the nucleotides encoding cysteines gamma 326 and gamma 339. The calcium binding activity of wild-type and altered polypeptides were compared using a solid phase assay. Our results indicate that removal of the two cysteine residues had no appreciable effect on calcium binding activity. We conclude that the integrity of the disulfide bond cysteine326-cysteine339 is not critical for calcium binding to gamma 259-411. 相似文献
12.
Preparation, characterization and application of interleukin-1 beta mutant proteins with surface-accessible cysteine residues 总被引:2,自引:0,他引:2
P Wingfield P Graber A R Shaw A M Gronenborn G M Clore H R MacDonald 《European journal of biochemistry》1989,179(3):565-571
Two mutants of interleukin-1 beta (K27C and K138C) were produced using site-specific mutagenesis in which lysine residues at positions 27 and 138 of the mature protein sequence were substituted by cysteine residues. The conformations of the mutant proteins were studied by 1H-NMR spectroscopy and shown to be similar to the wild-type protein. The receptor-binding affinity and biological activity of K27C and K138C were also similar to wild-type protein. The substituted cysteines in both mutant proteins were shown to be solvent-accessible as judged by their reactivity towards sulfhydryl reagents. As the wild-type protein contains two cysteines, which are both solvent-inaccessible in the native state, the mutants offer the opportunity to introduce probes in a sequence-specific manner via reaction with sulfhydryl groups. Examples of this are described in which the K138C was disulfide-linked to phycobiliproteins. The highly fluorescent conjugates had similar receptor-binding affinities to that of the wild-type unconjugated protein and were found suitable for flow-cytometric analysis. 相似文献
13.
Nuclear export of phosphorylated galectin-3 regulates its antiapoptotic activity in response to chemotherapeutic drugs 总被引:11,自引:0,他引:11
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Takenaka Y Fukumori T Yoshii T Oka N Inohara H Kim HR Bresalier RS Raz A 《Molecular and cellular biology》2004,24(10):4395-4406
Galectin-3 (Gal-3), a member of the beta-galactoside binding protein family containing the NWGR antideath motif of the Bcl-2 protein family, is involved in various aspects of cancer progression. Previously, it has been shown that the antiapoptotic activity of Gal-3 is regulated by the phosphorylation at Ser(6) by casein kinase 1 (CK1). Here we questioned how phosphorylation at Ser(6) regulates Gal-3 function. We have generated serine-to-alanine (S6A) and serine-to-glutamic acid (S6E) Gal-3 mutants and transfected them into the BT-549 human breast carcinoma cell line, which does not express Gal-3. BT-549 cell clones expressing wild-type (wt) and mutant Gal-3 were exposed to chemotherapeutic anticancer drugs. In response to the apoptotic insults, phosphorylated wt Gal-3 was exported from the nucleus to the cytoplasm and protected the BT-549 cells from drug-induced apoptosis while nonphosphorylated mutant Gal-3 neither was exported from the nucleus nor protected BT-549 cells from drug-induced apoptosis. Furthermore, leptomycin B, a nuclear export inhibitor, increased the cisplatin-induced apoptosis of Gal-3 expressing BT-549 cells. These results suggest that Ser(6) phosphoryaltion acts as a molecular switch for its cellular translocation from the nucleus to the cytoplasm and, as a result, regulates the antiapoptotic activity of Gal-3. 相似文献
14.
Begg GE Holman SR Stokes PH Matthews JM Graham RM Iismaa SE 《The Journal of biological chemistry》2006,281(18):12603-12609
Transglutaminase type 2 (TG2; also known as G(h)) is a multifunctional protein involved in diverse cellular processes. It has two well characterized enzyme activities: receptor-stimulated signaling that requires GTP binding and calcium-activated transamidation or cross-linking that is inhibited by GTP. In addition to the GDP binding residues identified from the human TG2 crystal structure (Liu, S., Cerione, R. A., and Clardy, J. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 2743-2747), we have previously implicated Ser171 in GTP binding, as binding is lost with glutamate substitution (Iismaa, S. E., Wu, M.-J., Nanda, N., Church, W. B., and Graham, R. M. (2000) J. Biol. Chem. 275, 18259-18265). Here, we have shown that alanine substitution of homologous residues in rat TG2 (Phe174 in the core domain or Arg476, Arg478, or Arg579 in barrel 1) does not affect TG activity but reduces or abolishes GTP binding and GTPgammaS inhibition of TG activity in vitro, indicating that these residues are important in GTP binding. Alanine substitution of Ser171 does not impair GTP binding, indicating this residue does not interact directly with GTP. Arg579 is particularly important for GTP binding, as isothermal titration calorimetry demonstrated a 100-fold reduction in GTP binding affinity by the R579A mutant. Unlike wild-type TG2 or its S171E or F174A mutants, which are sensitive to both trypsin and mu-calpain digestion, R579A is inherently more resistant to mu-calpain, but not trypsin, digestion, indicating reduced accessibility and/or flexibility of this mutant in the region of the calpain cleavage site(s). Basal TG activity of intact R579A stable SH-SY5Y neuroblastoma cell transfectants was slightly increased relative to wild-type transfectants and, in contrast to the TG activity of the latter, was further stimulated by muscarinic receptor-activated calcium mobilization. Thus, loss of GTP binding sensitizes TG2 to intracellular calcium concentrations. These findings are consistent with the notion that intracellularly, under physiological conditions, TG2 is maintained largely as a latent enzyme, its calcium-activated cross-linking activity being suppressed allosterically by guanine nucleotide binding. 相似文献
15.
The PI-SceI protein is a member of the LAGLIDADG family of homing endonucleases that is generated by a protein splicing reaction. PI-SceI has a bipartite domain structure, and the protein splicing and endonucleolytic reactions are catalyzed by residues in domains I and II, respectively. Structural and mutational evidence indicates that both domains mediate DNA binding. Treatment of the protein with trypsin breaks a peptide bond within a disordered region of the endonuclease domain situated between residues Val-270 and Leu-280 and interferes with the ability of this domain to bind DNA. To identify specific residues in this region that are involved in DNA binding and/or catalysis, alanine-scanning mutagenesis was used to create a set of PI-SceI mutant proteins that were assayed for activity. One of these mutants, N281A, was >300-fold less active than wild-type PI-SceI, and two other proteins, R277A and N284A, were completely inactive. These decreases in cleavage activity parallel similar decreases in substrate binding by the endonuclease domains of these mutant proteins. We mapped the approximate position of the disordered region to one of the ends of the 31 base pair PI-SceI recognition sequence using mutant proteins that were substituted with cysteine at residues Asn-274 and Glu-283 and tethered to the chemical nuclease FeBABE. These mutational and affinity cleavage data strongly support a model of PI-SceI docked to its DNA substrate that suggests that one or more residues identified here are responsible for contacting base pair A/T(-)(9), which is essential for substrate binding. 相似文献
16.
Frändberg PA Doufexis M Kapas S Chhajlani V 《Biochemical and biophysical research communications》2001,281(4):851-857
Reduction of disulfide bonds in human melanocortin 1 receptor (hMC1R) with increasing concentrations of DTT (dithiothreitol) resulted in a decrease in the binding of [125I]-ACTH (adrenocorticotropic hormone, L-isomer) in an uniphasic manner and a decrease in [125I]-NDP-MSH ([Nle(4),D-Phe(7)]-alpha-melanocyte stimulating hormone; D-isomer) binding in a biphasic manner. Pretreatment of hMC1R with 10 mM DTT resulted in a 36-fold loss of affinity for alpha-MSH (L-isomer) without affecting the affinity of NDP-MSH (D-isomer). To characterize the role of individual cysteine residues, we employed site-directed mutagenesis to substitute cysteine by glycine at all fourteen positions in hMC1R and analysed wild-type and mutant receptors for ligand binding and cAMP signalling. Single point mutation of four cysteine residues in extracellular loops to glycine (C35G, C267G, C273G, and C275G) resulted in a complete loss of binding for [125I]-NDP-MSH. Moreover, mutants with normal ligand binding, at positions C191G (transmembrane segment 5), C215G (third intracellular loop), and C315G (C-terminal loop) failed to generate cAMP signal in response to both agonists alpha-MSH and NDP-MSH. Mutant at position C78G (with wild-type binding to alpha-MSH as well as NDP-MSH) generated a cAMP signal in response to alpha-MSH (identical to wild-type hMC1R) but interestingly could not be stimulated by NDP-MSH. Moreover, this single amino acid substitution converted NDP-MSH from being an agonist to antagonist at the C78G mutant receptor. These findings demonstrate that (i) alpha-MSH and ACTH (L-isomers) are different from D-isomer NDP-MSH in their sensitivity to DTT for receptor binding, (ii) cysteine residues in N-terminus and extracellular loop three make disulfide bridges and are needed for structural integrity of hMC1R, (iii) cysteine residues in transmembrane segments and intracellular loops are required for receptor-G-protein coupling, (iv) C78 in transmembrane segment two is required for generating a functional response by D-isomer agonist (NDP-MSH) but not by L-isomer agonist (alpha-MSH), and (v) wild-type receptor agonist NDP-MSH is an antagonist at the mutant C78G receptor. 相似文献
17.
Tie JK Jin DY Loiselle DR Pope RM Straight DL Stafford DW 《The Journal of biological chemistry》2004,279(52):54079-54087
The enzymatic activity of the vitamin K-dependent proteins requires the post-translational conversion of specific glutamic acids to gamma-carboxy-glutamic acid by the integral membrane enzyme, gamma-glutamyl carboxylase. Whether or not cysteine residues are important for carboxylase activity has been the subject of a number of studies. In the present study we used carboxylase with point mutations at cysteines, chemical modification, and mass spectrometry to examine this question. Mutation of any of the free cysteine residues to alanine or serine had little effect on carboxylase activity, although C343A mutant carboxylase had only 38% activity compared with that of wild type. In contrast, treatment with either thiol-reactive reagent 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, disodium salt, or sodium tetrathionate, caused complete loss of activity. We identified the residues modified, using matrix-assisted laser desorption/ionization time of flight mass spectrometry, as Cys(323) and Cys(343). According to our results, these residues are on the cytoplasmic side of the microsomal membrane, whereas catalytic residues are expected to be on the lumenal side of the membrane. Carboxylase was partially protected from chemical modification by factor IXs propeptide. Although all mutant carboxylases bound propeptide with normal affinity, chemical modification caused a >100-fold decrease in carboxylase affinity for the consensus propeptide. We conclude that cysteine residues are not directly involved in carboxylase catalysis, but chemical modification of Cys(323) and Cys(343) may disrupt the three-dimensional structure, resulting in inactivation. 相似文献
18.
Reiji Kojima Tatsukuni Ohno Motoyasu Iikura Toshiro Niki Mitsuomi Hirashima Keichi Iwaya Hitoshi Tsuda Shigeaki Nonoyama Akio Matsuda Hirohisa Saito Kenji Matsumoto Susumu Nakae 《PloS one》2014,9(1)
Galectin-9 (Gal-9), a lectin having a β-galactoside-binding domain, can induce apoptosis of Th1 cells by binding to TIM-3. In addition, Gal-9 inhibits IgE/Ag-mediated degranulation of mast cell/basophilic cell lines by binding to IgE, thus blocking IgE/Ag complex formation. However, the role of Gal-9 in mast cell function in the absence of IgE is not fully understood. Here, we found that recombinant Gal-9 directly induced phosphorylation of Erk1/2 but not p38 MAPK in a human mast cell line, HMC-1, which does not express FcεRI. Gal-9 induced apoptosis and inhibited PMA/ionomycin-mediated degranulation of HMC-1 cells. On the other hand, Gal-9 induced cytokine and/or chemokine production by HMC-1 cells, dependent on activation of ERK1/2 but not p38 MAPK. In addition, the lectin activity of Gal-9 was required for Gal-9-mediated cytokine secretion by HMC-1 cells. These observations suggest that Gal-9 has dual properties as both a regulator and an activator of mast cells. 相似文献
19.
Mutational analysis of baculovirus phosphatase identifies structural residues important for triphosphatase activity in vitro and in vivo 总被引:1,自引:0,他引:1
Baculovirus phosphatase (BVP) and mammalian capping enzyme (Mce1) are members of the RNA triphosphatase branch of the cysteine phosphatase superfamily. Although RNA triphosphatases have a core alpha/beta fold similar to other cysteine phosphatases, there is little conservation of primary structure outside of the cysteine-containing P-loop motif, HCxxxxxR(S/T), that comprises the active site. However, there is extensive primary structure conservation between members of the RNA triphosphatase branch, whether from cellular or viral sources and whether they are bifunctional capping enzymes such as Mce1 or monofunctional RNA phosphatases such as BVP. To evaluate the functional significance of such sequence conservation, we performed a mutational analysis of 14 residues of BVP. We identified three side chains (Trp6, Lys25, and Arg153) as essential for triphosphatase activity in vitro, i.e., W6A, K25A, and R153A were <0.1% as active as wild-type BVP, and were unable to complement a yeast RNA triphosphatase null mutant in vivo. Six other BVP residues (Thr62, Tyr67, Tyr68, Lys82, Glu158, and Arg159) were deemed functionally important, i.e., Ala mutations reduced triphosphatase activity to <20% of wild-type. On the basis of the locations of the equivalent amino acids in the Mce1 crystal structure, we surmise that the essential/important BVP residues ensure proper conformation of the catalytic P-loop (e.g., Arg153 and Tyr68) or other elements of the tertiary structure. Our results highlight a conserved Trp6-Lys25 pi-cation pair essential for BVP function. 相似文献
20.
Toshiro Niki Shoko Tsutsui Shigeru Hirose Sachiko Aradono Yasushi Sugimoto Keisuke Takeshita Nozomu Nishi Mitsuomi Hirashima 《The Journal of biological chemistry》2009,284(47):32344-32352
Galectin (Gal)-9 was first described as an eosinophil chemoattractant. With the progress in research, Gal-9 has come to be known as a versatile immunomodulator that is involved in various aspects of immune regulations, and the entire picture of the function still remains elusive. To uncover as-yet unknown activity of Gal-9, we have been examining the effect of the protein in various disease animal models. Here we show that Gal-9 attenuated asthmatic reaction in guinea pigs and suppressed passive-cutaneous anaphylaxis in mice. These results indicate the mast cell stabilizing effect of Gal-9. In vitro studies of mast cell degranulation involving RBL-2H3 cells demonstrated that Gal-9 suppressed degranulation from the cells stimulated by IgE plus antigen and that the inhibitory effect was completely abrogated in the presence of lactose, indicating lectin activity of Gal-9 is critical. We found that Gal-9 strongly and specifically bound IgE, which is a heavily glycosylated immunoglobulin, and that the interaction prevented IgE-antigen complex formation, clarifying the mode of action of the anti-degranulation effect. Gal-9 is expressed by several mast cells including mouse mast cell line MC/9. The fact that immunological stimuli of MC/9 cells augmented Gal-9 secretion from the cells implies that Gal-9 is an autocrine regulator of mast cell function to suppress excessive degranulation. Collectively, these findings shed light on a novel function of Gal-9 in mast cells and suggest a beneficial utility of Gal-9 for the treatment of allergic disorders including asthma. 相似文献