Two conserved tryptophan residues of tumor necrosis factor and lymphotoxin are not involved in the biological activity

Each of the two highly conserved tryptophan residues in hTNF (positions 28 and 114) was converted into phenylalanine by site-directed mutagenesis and the mutant proteins were partially purified. A cytotoxicity assay on mouse L929 cells showed only a slight reduction in biological activity, strongly suggesting that neither of the two amino acids is involved in the active site.     

Little Heterogeneity among Genes Encoding Heat-Labile and Heat-Stable Toxins of Enterotoxigenic Escherichia coli Strains Isolated from Diarrheal Pigs

To examine whether the heat-labile enterotoxin gene in porcine enterotoxigenic Escherichia coli (ETEC) strains is as divergent as in human ETEC strains, we sequenced the heat-labile and heat-stable toxin genes from 52 and 33 porcine ETEC strains, respectively. We found that the STa gene is identical, that the LT gene has only two mutations in 4 (of 52) strains, and that both mutations cause a reduction in GM1 binding and toxicity.Enterotoxigenic Escherichia coli (ETEC) strains that colonize small intestines and produce enterotoxins are the major cause of diarrheal disease in humans and animals (8, 16, 18, 21). The key virulence factors of ETEC in diarrhea include enterotoxins and colonization factors or adhesins. Colonization factors or adhesins mediate the attachment of bacteria to host epithelium cells and facilitate bacterial colonization. Enterotoxins disrupt fluid homeostasis and stimulate fluid hyper-secretion in the intestinal epithelial cells that results in diarrhea. Heat-labile toxin (LT) and heat-stable toxin (ST) are the main enterotoxins associated with diarrhea in humans and farm animals, but different LT and ST are produced by human and animal ETEC strains (9, 16).The LT produced by porcine ETEC strains (pLT) or human ETEC strains (hLT) is a holotoxin-structured protein that has one LT_A subunit and five LT_B subunits. Although pLT and hLT are highly homologous in structure and function, these two proteins differ antigenetically (9). Sequence comparative studies showed that the following seven amino acids are different between pLT and hLT: the 4th, 213th, and 237th amino acids of the A subunits and the 4th, 13th, 46th, and 102nd amino acids of the B subunits (6, 7). Similarly, STa (ST type 1) carried by human and porcine ETEC strains is also different. The STa associated with porcine diarrhea (pSTa) is a peptide of 18 amino acids, whereas the STa produced by human ETEC strains (hSTa) is 19 amino acids in length (5, 19). Despite the fact that ETEC constructs expressing pLT or hLT, and pSTa or hSTa, are equivalently virulent in causing diarrhea in gnotobiotic pigs (25), pLT and pSTa are typically expressed by porcine ETEC strains that only cause diarrhea in pigs, whereas hLT and hSTa are exclusively produced by human ETEC strains associated with diarrhea in humans. Although pLT and STb, another porcine-specific ST, were occasionally detected in ETEC strains isolated from human diarrheal patients (3), only infections with hSTa⁺, hLT⁺, or hSTa⁺/hLT⁺ ETEC strains cause diarrhea in humans (17).Interspecies LT have been intensively compared for molecular and immunological characteristics (4, 10, 20, 23). In contrast, intraspecies LT has not been studied much. For a long time, both pLT and hLT were assumed to be highly conserved. However, a very recent study showed that the hLT gene carried by human ETEC strains is considerably divergent (12). After restriction fragment length polymorphism analysis and DNA sequencing of 51 human ETEC strains, Lasaro et al. reported that the human LT gene had seven polymorphic restriction fragment length polymorphism types and 30 nucleotide polymorphic sites and recognized 16 different hLT types (12). To examine whether the LT gene carried by porcine ETEC strains has a similar heterogeneity, we PCR amplified and DNA sequenced the LT genes and also the STa genes of various ETEC strains isolated from diarrheal pigs and analyzed gene sequence conformity.Fifty-two porcine ETEC strains that express LT alone or LT together with other toxins (LT⁺/STb⁺, LT⁺/STb⁺/STa⁺, LT⁺/STb⁺/EAST1⁺, and LT⁺/STa⁺/STb⁺/EAST1⁺) and K88ac or F18 fimbria were selected for the sequencing of the LT gene. Those porcine ETEC strains were isolated from pigs with postweaning diarrhea at different farms in South Dakota, Iowa, Minnesota, Nebraska, and North Dakota. The eltAB gene encoding LT from these 52 strains was PCR amplified with primers pLT-F (5′-ATCCTCGCTAGCATGTTTTAT-3′) and pLT-R (5′-CCCCTCCGGCCGAGCTTAGTT-3′) (25). PCRs were performed in an MJ PT-100 thermocycler (Bio-Rad, Hercules, CA) in a reaction of 50 μl containing 1× Taq DNA polymerase buffer (with Mg²⁺), 0.2 mM deoxynucleoside triphosphate, 0.5 μM each forward and reverse primers, 100 ng of total genomic DNA, and 1 unit Taq DNA polymerase (Applied Biosystems, Foster City, CA). The PCR program contained one cycle of 2 min at 94°C; 30 cycles of 35 s at 94°C, 35 s at 52°C, and 2 min at 72°C; and an extension of 6 min at 72°C. The amplified PCR products were separated on 1% agarose gels (FMC Bioproducts, Rockland, MA) by electrophoresis and purified using a QIAquick gel extraction kit according to the manufacturer''s instructions (Qiagen, Valencia, CA). A mixture of purified PCR product (100 to 150 ng) and 10 pmol primer was sent to the Nevada Genomic Center at the University of Nevada for sequencing. Three primers, pLT-F, LT₁₉₂-F (5′-GATTCATCAAGAACAATCCACAGGTG-3′), and LT₁₉₂-R (5′-CCTGTGATTGTTCTTGATGAATC-3′), were used for sequencing the entire eltAB gene.The sequences of the eltAB gene from all 52 porcine ETEC strains were aligned and visually examined. We found that the eltAB gene was nearly identical among the sequenced porcine ETEC strains. Forty-eight (of 52) ETEC strains had identical gene sequences, and only four strains showed heterogeneity. The pathotypes of these four strains were K88/LT/STb, K88/LT/STb/STa, K88/LT/STb/EAST1, and F18/LT/STa/STb/Stx2e. Furthermore, only nucleotides coding two amino acids, the 44th (S44N) and the 60th (S60T) of the eltB gene encoding the B subunit, differed among these four strains. To our surprise, neither of these two substitutions were homologous to the hLT gene nor to any of the hLT types recognized by Lasaro et al. (12). Lasaro et al. showed that 11 of the 15 different hLT types shared some homology with pLT, and some hLT types had as many as four amino acids (K4R and K213E of LT_A and S4T, R13H, or A46E of LT_B; out of seven heterogeneous amino acids) homologous to pLT. Indeed, the hLT6 type differed from the LT of human ETEC prototype {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 in four amino acids (K4R and K213E of LT_A and S4T and A46E of LT_B) (12), but all four of these heterogeneous amino acids were homologous to pLT. Similarly, four of the five amino acids that differed from the prototype hLT in the hLT4 type were identical to pLT. That means that the hLT4 and hLT6 types had only three amino acids heterogeneous to pLT but four different residues compared to the hLT prototype. It seems that hLT4 and hLT6 are more likely pLT rather than hLT. Given that the divergence of the pLT and hLT genes is assumed to be a very recent evolutionary event that occurred 0.9 million years ago (23), it is likely that the hLT gene retains some pLT gene characters (amino acids) that could be of their common ancestor. However, a high homology in the pLT gene certainly seems unparallel to the evolution of the hLT gene. Our further sequence comparison indicated that S44N-substituted pLT_B [pLT_B(S44N)] is homologous to cholera toxin (CT). It has been suggested that the CT and LT genes were derived from the same ancestor but diverged to two lineages about 130 million years ago (23). Then, it is more likely that this pLT_B(S44N) represents a plesiomorphic character, meaning a primitive character that belongs to the common ancestor of CT and LT. The retention of this primitive pLT_B(S44N) by some porcine ETEC strains suggests that the pLT gene could have evolved at a relatively lower rate. Whether such a lower substitution rate of the LT gene in porcine ETEC strains is associated with a lower host exchange rate or a limited travel range in pigs is unclear to us. However, future studies to determine whether an increase in sampling sizes, by including porcine ETEC strains from a greater geographic coverage, could reveal a higher heterogeneity or a greater evolution rate in the pLT gene will be worthwhile.To examine whether the heterogeneity of pS44N and pS60T at the B subunit could affect the biological function of pLT, we cloned the native pLT gene into vector pBR322 (p8458), performed site-directed mutation of the eltAB gene for a substitution of S44N or S60T, and tested these two mutated LT proteins for their binding capability to GM1 receptors and their enterotoxic activity in stimulating intracellular cyclic AMP (cAMP) in cells. Primers pBRNheI-F2 (5′-CAGCATCGCCATTCACTATG-3′) and pBREagI-R (5′-AGATGACGACCATCAGGGAC-3′) were designed to amplify the porcine eltAB gene. The amplified eltAB gene products and vector pBR322 were digested with NheI and EagI (New England Biolabs, Beverly, MA), separated by gel electrophoresis, purified with the QIAquick gel extraction kit, and then ligated with T4 DNA ligase (Promega, Madison, WI). Two microliters of the T4-ligated products were introduced into 25 μl of TOPO cells (Invitrogen, Valencia, CA) in a standard electroporation. Antibiotic-selected colonies were initially screened by PCR, and positive colonies were sequenced to ensure that the cloned gene was in the reading frame. The verified clone was selected as a pLT recombinant strain and designated strain 8458. To construct mutant strains, two pairs of primers, LT_B44-F (5′-ATCATTACATTTAAGAACGGCGAA-3′) and LT_B44-R (5′-TTCGCCGTTCTTAAATGTAATGAT-3′) and LT_B60-F (5′-CAACATATAGACACCCAGAAAAAAGCC-3′) and LT_B60-R (5′-GGCTTTTTTCTGGGTGTCTATATGTTG-3′), were used for site-directed mutation at nucleotides coding the 44th and 60th amino acids of the LT_B subunit, respectively. Briefly, the amplified products from two separate PCRs, one using pBRNheI-F2 with LT_B44-R or LT_B60-R and the other using pBREagI-R with LT_B44-F or LT_B60-F, with recombinant pLT plasmid p8458 as the DNA template, were overlapped in a third splicing overlap extension PCR to produce mutated pLT genes. The splicing overlap extension PCR products were digested with NheI and EagI restriction enzymes and ligated into vector pBR322 for the p8647 (S44N) and p8649 (S60T) plasmids. Plasmids p8647 and p8649 were separately introduced into TOPO 10 E. coli cells (Invitrogen) for mutant strains 8647 (S44N) and 8649 (S60T).Equivalent amounts of cells from overnight-grown cultures of the recombinant (8458) and two mutant (8647 and 8649) strains were used for total protein preparation by using bacterial protein extraction reagent (B-PER in phosphate buffer; Pierce, Rockford, IL). Both pelleted protein samples (periplasmic proteins) and culture supernatant samples (outer-membrane secreted proteins) were used in a GM1 enzyme-linked immunosorbent assay (ELISA) to examine whether a substitution at the 44th or 60th amino acid would affect the binding of LT to GM1 receptors. Anti-CT rabbit antiserum (1:5,000; Sigma) and horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (1:5,000; Sigma, St. Louis, MO) were used as the primary and secondary antibodies as described previously (2, 14, 24). GM1 ELISA data indicated optical density (OD) values from the pellet samples of strains 8548, 8647, and 8649 and phosphate-buffered saline of 0.677 ± 0.004, 0.616 ± 0.001, 0.647 ± 0.004, and 0.006 ± 0, whereas the OD values of the supernatant samples which were vacuum concentrated were 0.949 ± 0.008, 0.726 ± 0.004, 0.660 ± 0.005, and 0.05 ± 0.002, respectively (Fig. ​(Fig.1).1). Statistical analysis using the Student t test with two-tailed distribution indicated that the binding of the pellet samples from the native and the mutated LT to GM1 was not significantly different (P = 0.10 and P = 0.45, respectively). However, the GM1 binding from the supernatant samples of the LT mutant strains was significantly lower than that of the LT recombinant strain (P < 0.01 and P < 0.01, respectively).Open in a separate windowFIG. 1.GM1 ELISA to detect LT proteins expressed by the pLT recombinant (8458) and mutant [8647(S44N) and 8649(S60T)] strains. Protein samples from the pellet and vacuum-concentrated supernatants of overnight-grown cultures were used in the GM1 ELISA. Each sample was assayed in triplicate to calculate OD means and standard deviations. Anti-CT serum (1:5,000) was used as the primary antibody and goat anti-rabbit horseradish peroxidase-conjugated immunoglobulin G (1:5,000) was used as the secondary antibody. OD values were measured after a 20-min reaction with peroxidase substrates (KPL, Gaithersburg, MD) at a wavelength of 405 nm.Our GM1 ELISA data indicated that the supernatant sample of the recombinant strain expressing a native LT had a greater GM1 binding activity. This could suggest that the recombinant strain had more LT protein crossing the outer membrane and being secreted in the supernatant than either mutant strain or that mutations at the B subunit negatively affected the binding of LT proteins to GM1 receptors. It has been reported that a single amino acid mutation of the LT_B or CT_B subunit resulted in lower GM1 binding activity, especially mutations of residues from the binding pocket (13, 15, 22). When amino acid 33 or 88 of the CT_B subunit was replaced, both mutants failed to bind or bound poorly to GM1 (22), and when a substitution at residue 57 of its B subunit occurred, this CT mutant showed 1.5-log-lower GM1 binding than the native CT (1, 13). Similarly, when amino acid 46 or 47 of the B subunit was replaced, both LT mutants exhibited lower GM1 binding activity than the wild-type LT strain (13). However, in contrast to our observation that our 8647 and 8649 mutant strains showed lower GM1 binding activity in the supernatant, Mudrak et al. indicated that the T47A mutant strain had more LT protein detected in the supernatant than the wild-type strain (13). Whether and how a mutation at amino acid 44 or 60 of the B subunit affects the formation, stability, or secretion of the mutant LT proteins will be studied in the future.To examine whether the lower GM1 binding activity of the supernatant samples from the mutant strains was caused by a lower LT production, we conducted an ELISA by directly coating an ELISA plate with total proteins from the pellet and supernatant samples of each strain (without GM1) and by using anti-CT antiserum to quantify the LT protein. ELISA data showed that the OD values of strains 8458, 8647, and 8649 were 0.209 ± 0.005, 0.225 ± 0.009, and 0.21 ± 0 in the supernatant samples and 0.571 ± 0.025, 0.614 ± 0.060, and 0.616 ± 0.026 in the pellet samples, respectively. A Student t test indicated that there were no significant differences between the recombinant strain and the mutant strains in the OD values for the pellet and supernatant protein samples (P = 0.26 and P = 0.84, respectively, for the supernatant samples; P = 0.34 and P = 0.10, respectively, for the pellet samples). These data suggested that a similar amount of LT proteins was produced among these three strains.A single amino acid substitution of the B subunit can result in a reduction in not only GM1 binding but also toxicity for the mutated LT proteins (11, 13, 22). To study whether the mutation of S44N or S60T at the B subunit affected pLT toxicity, we measured the recombinant and mutant strains for their stimulation of intracellular cAMP levels in T-84 cells by using a cAMP competitive enzyme immunoassay (EIA) kit (Invitrogen) by following the manufacturer''s instructions. Briefly, 1 × 10⁵ T-84 cells were seeded in each well of a 24-well plate. After removing the Dulbecco''s modified Eagle medium (DMEM/F12; Gibco/Invitrogen, Grand Island, NY), 75 μl of overnight-grown (in 4AA medium) supernatant of the recombinant or each mutant strain (in triplicate) was added to each well. The cells were lysed with 100 μl of 0.1 M HCl after 2 h of incubation and then neutralized. A total of 100 μl of lysis supernatant was mixed with kit-supplied conjugates and antibody reagents, and the mixture was added to each well of the supplied EIA plate. After incubation on a shaker at 500 rpm at room temperature for 2 h, the plate was washed and dried by blotting, and p-nitrophenyl phosphate substrate solution was added. The OD was measured at 405 nm after 20 min of development. Data from the cAMP ELISA indicated that cAMP levels in T-84 cells incubated with supernatant samples from strains 8458, 8647, and 8649 (from equivalent amounts of cells) were 2.3 ± 0.1, 0.46 ± 0.05, and 0.35 ± 0.01 pmol/ml, respectively (Fig. ​(Fig.2).2). Data clearly indicated that the mutations of S44N and S60T reduced the LT toxic activity. Knowing that it is the A subunit that determines the toxicity of LT and CT, whereas the LT_B and CT_B subunits mediate the binding of the toxin to the host GM1 receptors, we thought that substitution at the B subunits would not affect toxicity. However, we believe that mutations at the B subunits could alter LT protein structure and reduce the binding of the holotoxin to the host GM1 receptors, thus resulting in the reduction of toxic activity.Open in a separate windowFIG. 2.Intracellular cAMP ELISA to detect the toxicity of native LT and mutated LT proteins. Supernatants (in 4AA medium) of overnight-grown cultures from the 8458 (recombinant), 8647 (S44N), and 8649 (S60T) strains were used to stimulate an increase in intracellular cAMP levels in T-84 cells by using a cyclic GMP EIA kit (Invitrogen).The estA gene encoding STa from 33 STa-positive porcine ETEC strains was also sequenced for conformity. This porcine estA gene was PCR amplified using primers pSTaSfcI-F2 and STaEagI-R under conditions described previously (25). The PCR products were purified and sequenced with pSTaSfcI-F2 primer. The sequencing data showed that all sampled STa genes were identical and of porcine origin.Sequence data from our study clearly indicated that both LT and STa expressed by porcine ETEC strains are porcine specific. The LT gene of porcine ETEC strains showed little heterogeneity, and the STa gene is identical. Information from this study will be helpful for a prevalence study of toxin genes among porcine ETEC strains and toxin gene evolution and possibly instructive in antitoxin vaccine development. However, future studies with increasing sampling sizes and a greater geographic coverage will be helpful to understand divergence in the LT and STa genes among porcine ETEC strains.     

Amino- and carboxy-terminal deletion mutants of Gs alpha are localized to the particulate fraction of transfected COS cells

To elucidate the structural basis for membrane attachment of the alpha subunit of the stimulatory G protein (Gs alpha), mutant Gs alpha cDNAs with deletions of amino acid residues in the amino and/or carboxy termini were transiently expressed in COS-7 cells. The particulate and soluble fractions prepared from these cells were analyzed by immunoblot using peptide specific antibodies to monitor distribution of the expressed proteins. Transfection of mutant forms of Gs alpha with either 26 amino terminal residues deleted (delta 3-28) or with 59 amino terminal residues deleted (delta 1-59) resulted in immunoreactive proteins which localized primarily to the particulate fraction. Similarly, mutants with 10 (delta 385-394), 32 (delta 353-384), or 42 (delta 353-394) amino acid residues deleted from the carboxy terminus also localized to the particulate fraction, as did a mutant form of Gs alpha lacking amino acid residues at both the amino and carboxy termini (delta 3-28)/(delta 353-384). Mutant and wild type forms of Gs alpha demonstrated a similar degree of tightness in their binding to membranes as demonstrated by treatment with 2.5 M NaCl or 6 M urea, but some mutant forms were relatively resistant compared with wild type Gs alpha to solubilization by 15 mM NaOH or 1% sodium cholate. We conclude that: (a) deletion of significant portions of the amino and/or carboxyl terminus of Gs alpha is still compatible with protein expression; (b) deletion of these regions is insufficient to cause cytosolic localization of the expressed protein. The basis of Gs alpha membrane targeting remains to be elucidated.     

人肿瘤坏死因子β(hTNFβ)在变铅青链霉菌中的表达研究

以变铅青链霉菌为宿主研究了人INFβ(hTNFβ)的异源表达。应用链霉菌S.VENEZUELAC cbs762.70分泌产生的枯草杆菌蛋白酶抑制剂vsi基因的启动子、表达调控序列和分泌信号肽序列,分别对hTNFβ进行了直接分泌表达、分泌融合表达和胞内表达。将hTNFβ的cDNA分别直接融合于vsi信号肽序列下游2个氨基酸处、vsi全长基因之后以及vsi起始密码子ATG的下游,获得的表达盒分别克隆至链霉菌高拷贝质粒pIJ486,转化Streptomyces lividans TK24,获得了重组菌株S.lividans(pIJ486-hTNFβ),s.LIVIDANS(PIJ486-vsi-hTNFβ)和S.lividans(pIVPA-hTNFβ)。分别对不同的重组菌株进行摇瓶培养,对其培养的上清液和细胞裂解液进行SDS—PAGE和Westen杂交,结果表明：hTNFβ在重组菌株中均获得了表达,且直接分泌产物和胞内表达产物均具有生物学活性。hTNFβ直接分泌表达产物的分子量约为16kDa,NB培养基中培养48h时表达水平约为0．7mg／L。胞内表达产物分子量与对照重组hTNFβ一致(18．7kDa),但随培养时间的延长远步降解为16kDa,NB培养基中培养48h时的表达水平(25．1mg／L)远高于其直接分泌表达水平。     

On the structure of platelet-derived growth factor AA: C-terminal processing, epitopes, and characterization of cysteine residues.

The complete amino acid sequence analysis of the "short" form of rPDGF-AA expressed in baby hamster kidney cells revealed the absence of posttranslationally modified amino acid. Approximately 50% of the proteins were shortened by two to three amino acid residues at the C-terminus. Trypsin treatment of BHK rPDGF-AA lead to the identification of two internal epitopes that correspond to the two previously described domains in rPDGF-BB [Vogel, S., & Hoppe, J. (1989) Biochemistry 28, 2961-2966]. Cysteine residues at positions 37, 46, 47, and 93, respectively, were converted by site-directed mutagenesis into serine residues, and the monomeric proteins were prepared through expression in Escherichia coli. None of the mutant proteins was able to dimerize, but all of them exhibited to various extents a reversible conformational change which may reflect an intermediate prefolded monomer. An intramolecular disulfide bridge between Cys-10 and Cys-91/93 was identified in these monomers. From a mixture of the mutant proteins 37 and 46, an active dimer was reconstituted, suggesting an intermolecular cysteine bridge between these two residues.     

Extracellular secretion of Escherichia coli heat-stable enterotoxin I across the outer membrane.

Escherichia coli heat-stable enterotoxin Ip (STIp) is an extracellular toxin consisting of 18 amino acid residues that is synthesized as a precursor of pre (amino acid residues 1 to 19), pro (amino acid residues 20 to 54), and mature (amino acid residues 55 to 72) regions. The precursor synthesized in the cytoplasm is translocated across the inner membrane by the general export pathway consisting of Sec proteins. The pre region functions as a leader peptide and is cleaved during translocation. However, it remains unknown how the resulting peptide (pro-mature peptide) translocates across the outer membrane. In this study, we investigated the structure of the STIp that passes through the outer membrane to determine how it translocates through the outer membrane. The results showed that the pro region is cleaved in the periplasmic space. The generated peptide becomes the mature form of STIp, which happens to have disulfide bonds, which then passes through the outer membrane. We also showed that STIp with a carboxy-terminal peptide consisting of 3 amino acid residues passes through the outer membrane, whereas STIp with a peptide composed of 37 residues does not. Amino acid analysis of mutant STIp purified from culture supernatant revealed that the peptide composed of 37 amino acid residues was cleaved into fragments of 5 amino acid residues. In addition, analyses of STIps with a mutation at the cysteine residue and the dsbA mutant strain revealed that the formation of an intramolecular disulfide bond within STIp is not absolutely required for the mature region of STIp to pass through the outer membrane.     

Expression and subcellular location of native and mutant hTNF alpha proteins in Escherichia coli.

Several mutant hTNF alpha genes were constructed by deletion and stepwise reconstitution of regions coding for C-terminal sequences. The mutant hTNF alpha proteins behaved differently from native hTNF alpha when expressed in Escherichia coli. They were either sensitive to proteolytic degradation or formed insoluble aggregates depending on the strains and conditions used for expression. By contrast, native hTNF alpha was always present in a soluble form and had a tendency to associate with the cytoplasmic membrane. It was even transported to the periplasmic space in E. coli as shown by both cell fractionation and immunoelectron microscopy. The different behaviour of mutant hTNF alpha proteins probably results from a disturbance of protein folding.     

Herpes simplex virus alpha protein ICP27 possesses separable positive and negative regulatory activities.

The HSV-1 alpha (immediate-early) protein ICP27 expressed in transfected cells can activate the expression of certain HSV-1 promoters as well as inhibit the transactivated expression of others. We constructed a set of plasmids encoding mutant ICP27 molecules truncated at their carboxyl termini and used transfection assays to determine the functional properties of the mutant proteins. A polypeptide containing the amino-terminal 263 amino acid residues of ICP27 retained partial ability to activate gene expression but was unable to inhibit transactivation. Mutant proteins possessing 406 or 504 amino acids of ICP27 were unable to activate gene expression but retained full ability to inhibit transactivation. These results define two separable regulatory activities of ICP27, one positive and one negative, which can modulate gene expression in transfected cells. Immunoblot and immunofluorescence experiments were used to study the immunological reactivities and intracellular localizations of the mutant proteins. All proteins possessing the amino-terminal 263 amino acids of ICP27 reacted with an ICP27-specific monoclonal antibody and were localized to the cell nucleus. The mutant proteins, however, exhibited a number of phenotypes with regard to intranuclear localization. A mutant possessing 504 residues of ICP27 was similar to the wild-type protein in apparently localizing to all regions of the nucleus. A mutant containing 406 residues of ICP27, on the other hand, was mostly excluded from the nucleolar regions, while a 263-residue mutant was localized predominantly in the nucleoli. Thus, some aspect of ICP27 structure or function can dramatically affect its intranuclear distribution.     

Chemical characterization of recombinant human leukocyte interferon A using fast atom bombardment mass spectrometry

Proteolytic digests of biologically active fractions of recombinant human leukocyte interferon A expressed in large quantities in Escherichia coli were analyzed by fast atom bombardment mass spectrometry and high-performance liquid chromatography. The values observed in the mass spectra of digests of the major fraction of recombinant human leukocyte interferon A with trypsin and Staphylococcus aureus protease V8 accounted for 93% of the amino acid sequences of human leukocyte interferon A predicted from the nucleotide sequence of the gene encoding the protein, indicating that the major fraction of recombinant human leukocyte interferon A was expressed with the same amino acid sequence as that translated from the nucleotide sequence of the gene encoding the protein. Mass spectrometry of proteolytic digests of two minor fractions of recombinant human leukocyte interferon A and mass and amino acid analyses of their high-performance liquid chromatography fractions showed that the amino group of the N-terminal amino acid residue of interferon was in part acetylated, and the Cys-1 and Cys-98 residues were oxidized to cysteic acid or linked to glutathione. These findings suggest that amino acid residues in recombinant proteins prepared in large quantities in E. coli are modified post-translationally.     

Sequence of heat-labile enterotoxin of Escherichia coli pathogenic for humans.

We determined the complete nucleotide sequence of the toxB gene (375 base pairs in length), which encodes the B subunit of heat-labile enterotoxin produced from Escherichia coli pathogenic for humans (hLT). The amino acid sequence of the B subunit of hLT was deduced from the nucleotide sequence. Consequently, it has become possible to study the homology between the B subunits of three similar toxins: hLT, LT produced from E. coli pathogenic for piglets (pLT), and cholera toxin (the latter two sequences have been reported by others). The three B subunits are all 103 amino acids in length. A comparison of the toxB gene and the eltB gene, which encodes the B subunit of pLT, showed a 98% homology at the nucleotide level and a 95% homology at the amino acid (of a precursor) level, indicating the possibility that the two genes share a common ancestor. With respect to the B-subunit sequences, the homologies between hLT and pLT, between hLT and cholera toxin, and between pLT and cholera toxin were 96, 81, and 79%, respectively. Several large common sequences are conserved by the three peptides. In contrast, no sequences are present in both pLT and cholera toxin but missing in hLT.     

Control of protein synthesis by a temperature-sensitive mutant of reovirus 3. I. Temperature-sensitive function of ts261-b mutant.

The ability of a temperature-sensitive (ts) mutant of reovirus, ts261-b, to synthesize virus-specific RNAs and proteins during infection at the nonpermissive temperature (37 degrees C) was investigated. The relative amounts of the mutant virus-specific single-stranded (ss) RNA''s and double-stranded (ds) RNA''s synthesized in cells at 37 degrees C were 20 to 25% as much as those synthesized in the wild-type virus-infected cells. The 10 segments of the mutant ds RNAs and the three size classes of the ss RNAs were synthesized in the usual proportions. The methylation of the mutant viral mRNA''s (ss RNAs) was not blocked at 37 degrees C in infected cells. A striking temperature-sensitive restricted function of the ts261-b mutant was expressed in the synthesis of the viral proteins. This study, which uses an in vitro protein-synthesizing system reconstituted with an endogenous polysomal fraction and a postribosomal supernatant from reovirus-infected cells, has demonstrated that the endogenous polysomes obtained from ts261-b mutant-infected cells at 37 degrees C are not active in the synthesis of the viral polypeptides of known molecular weights, and the amounts of the mutant viral polypeptides synthesized in vitro by these polysomes are 5 to 9% of those synthesized by the corresponding fraction from wild-type-infected cells. The impaired protein-synthesizing capacity of the mutant virus-specific polysomes can be restored during maintenance of the infected cells at 30 degrees C after shift-down from 37 degrees C. The in vitro synthesis of viral polypeptides of known size by the active endogenous polysomes derived from cells infected at the permissive temperature is accelerated by the addition of the postribosomal supernatant obtained from cells infected at the permissive temperature. The postribosomal supernatant from mutant-infected cells at 37 degrees C did not have a stimulatory effect, but rather, it inhibited in vitro viral protein synthesis.     

Identification of a discrete intermediate in the assembly/disassembly of physalis mottle tymovirus through mutational analysis.

Assembly intermediates of icosahedral viruses are usually transient and are difficult to identify. In the present investigation, site-specific and deletion mutants of the coat protein gene of physalis mottle tymovirus (PhMV) were used to delineate the role of specific amino acid residues in the assembly of the virus and to identify intermediates in this process. N-terminal 30, 34, 35 and 39 amino acid deletion and single C-terminal (N188) deletion mutant proteins of PhMV were expressed in Escherichia coli. Site-specific mutants H69A, C75A, W96A, D144N, D144N-T151A, K143E and N188A were also constructed and expressed. The mutant protein lacking 30 amino acid residues from the N terminus self-assembled to T=3 particles in vivo while deletions of 34, 35 and 39 amino acid residues resulted in the mutant proteins that were insoluble. Interestingly, the coat protein (pR PhCP) expressed using pRSET B vector with an additional 41 amino acid residues at the N terminus also assembled into T=3 particles that were more compact and had a smaller diameter. These results demonstrate that the amino-terminal segment is flexible and either the deletion or addition of amino acid residues at the N terminus does not affect T=3 capsid assembly. In contrast, the deletion of even a single residue from the C terminus (PhN188Delta1) resulted in capsids that were unstable. These capsids disassembled to a discrete intermediate with a sedimentation coefficent of 19.4 S. However, the replacement of C-terminal asparagine 188 by alanine led to the formation of stable capsids. The C75A and D144N mutant proteins also assembled into capsids that were as stable as the pR PhCP, suggesting that C75 and D144 are not crucial for the T=3 capsid assembly. pR PhW96A and pR PhD144N-T151A mutant proteins failed to form capsids and were present as heterogeneous aggregates. Interestingly, the pR PhK143E mutant protein behaved in a manner similar to the C-terminal deletion protein in forming unstable capsids. The intermediate with an s value of 19.4 S was the major assembly product of pR PhH69A mutant protein and could correspond to a 30mer. It is possible that the assembly or disassembly is arrested at a similar stage in pR PhN188Delta1, pR PhH69A and pR PhK143E mutant proteins.     

Site-directed mutation of the active site of influenza neuraminidase and implications for the catalytic mechanism

Different isolates of influenza virus show a high degree of amino acid sequence variation in their surface glycoproteins. Conserved residues located in the substrate-binding pocket of the influenza virus neuraminidase are therefore likely to be involved in substrate binding or enzyme catalysis. In order to study the structure and function of the active site of this protein, a full-length cDNA clone of the neuraminidase gene from influenza A/Tokyo/3/67 was subcloned into aN M13 vector and amino acid substitutions were made in selected residues by using the oligonucleotide mismatch technique. The mutant neuraminidase genes were expressed from a recombinant SV40 vector, and the proteins were analyzed for synthesis, transport to the cell surface, and proper three-dimensional folding by internal and surface immunofluorescence. The mutant neuraminidase proteins were then assayed to determine the effect of the amino acid substitution on enzyme activity. Twelve of the 14 mutant proteins were correctly folded and were transported to the cell surface in a manner identical with that of the wild type. Two of these have full enzyme activity, but seven mutants, despite correct three-dimensional structure, have completely lost neuraminidase activity. Two mutants were active at low pH. The properties of the mutant enzymes suggest a possible mechanism of neuraminidase action.     

Recombinant TNF-binding protein from variola virus as a novel potential TNF antagonist

Gel-filtration chromatographic separation of the lysate of Sf21 insect cells infected with recombinant baculovirus BVi67 containing the gene for TNF-binding protein (CrmB) of variola virus (VARV) revealed that hTNF-cytotoxicity neutralization activity is associated with a fraction corresponding mainly to high molecular weight proteins (above 500 kDa) and less with fractions corresponding to proteins of 270 or 90 kDa. The recombinant VARV-CrmB protein has been purified by affinity chromatography. Difference in the experimentally determined and estimated (according to amino acid composition) VARV-CrmB molecular weight is due to glycosylation of the recombinant protein expressed in the insect cells. VARV-CrmB neutralizes in vitro the cytotoxic effect of hTNF and hLTα, and its TNF-neutralizing activity is two to three orders of magnitude higher compared to the analogous effects of type I and II soluble TNF receptors, comparable with the activity of mAb MAK195, and somewhat lower than the effect of the commercial drug Remicade.     

Preparation of polyclonal antibody specific for AtPLC4, an Arabidopsis phosphatidylinositol-specific phospholipase C in rabbits

Phosphoinositide-specific phospholipase Cs (PI-PLCs) are important enzymes in eukaryotes, which catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate into the two second messengers inositol 1,4,5-trisphosphate and diacylglycerol. The Arabidopsis genome contains nine putative PI-PLC genes. AtPLC4, an abiotic stress induced gene, has been reported to encode an active PI-PLC isoform. However, the exact roles of putative AtPLC4 in plant remain to be elicited. The first 108 amino acid residues of the N-terminal of AtPLC4, referred to as AtPLC4 N, was expressed as a recombinant protein in Escherichia coli and used as antigen in generating antibody. Purified recombinant proteins including AtPLC1 to AtPLC5, AtPLC8, AtPLC9 and AtPLC4 N were transferred onto the same blot to test specificity of the prepared antibody. Western blot result shows that only AtPLC4 and AtPLC4 N can be recognized by the antibody. The antibody recognized a protein of approximately 68kDa in the plasma membrane fraction and cytosolic fractions prepared from Arabidopsis thaliana plants. This corresponds very well with the calculated molecular weight of AtPLC4. The results suggest that AtPLC4 may encode a plasma membrane-associated protein.     

Synthesis of Escherichia coli heat-stable enterotoxin STp as a pre-pro form and role of the pro sequence in secretion.

Escherichia coli heat-stable enterotoxin STp is presumed from its DNA sequence to be synthesized in vivo as a 72-amino-acid residue precursor that is cleaved to generate mature STp consisting of the 18 carboxy-terminal amino acid residues. There are two methionine residues in the inferred STp sequence in addition to the methionine residue at position 1. In order to confirm production of the STp 72-amino-acid residue precursor, we substituted the additional methionine residues by oligonucleotide-directed site-specific mutagenesis. Since these substitutions did not cause a significant change in STp production, it can be concluded that STp is normally synthesized as the 72-amino-acid residue precursor. The length of the STp precursor indicated the existence of a pro sequence between the signal peptide and the mature protein. In order to identify the pro sequence and determine its role in protein secretion, deletion and fusion proteins were made. A deletion mutant in which the gene fragment encoding amino acid residues 22 to 53 of STp was removed was made. STp activity was found in the culture supernatant of cells. Amino acid sequence analysis of the purified STp deletion mutant revealed that the pro sequence encompasses amino acid residues 20 to 54. A hybrid protein consisting of STp amino acids 1 to 53 fused in frame from residue 53 to nuclease A was not secreted into the culture supernatant. These results indicate that the pro sequence does not function to guide periplasmic protein into the extracellular milieu.     

Directed evolution of a beta-galactosidase from <Emphasis Type="Italic">Pyrococcus woesei</Emphasis> resulting in increased thermostable beta-glucuronidase activity

We performed directed evolution on a chemically synthesized 1,533-bp recombinant beta-galactosidase gene from Pyrococcus woesei. More than 200,000 variant colonies in each round of directed evolution were screened using the pYPX251 vector and host strain Rosetta-Blue (DE3). One shifted beta-galactosidase to beta-glucuronidase mutant, named YG6762, was obtained after four rounds of directed evolution and screening. This mutant had eight mutated amino acid residues. T29A, V213I, L217M, N277H, I387V, R491C, and N496D were key mutations for high beta-glucuronidase activity, while E414D was not essential because the mutation did not lead to a change in beta-glucuronidase activity. The amino acid site 277 was the most essential because mutating H back to N resulted in a 50% decrease in beta-glucuronidase activity at 37°C. We also demonstrated that amino acid 277 was the most essential site, as the mutation from N to H resulted in a 1.5-fold increase in beta-glucuronidase activity at 37°C. Although most single amino acid changes lead to less than a 20% increase in beta-glucuronidase activity, the YG6762 variant, which was mutated at all eight amino acid sites, had a beta-glucuronidase activity that was about five and seven times greater than the wild-type enzyme at 37 and 25°C, respectively. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Expression of genes for orthopoxviral TNF-binding proteins and study resulted recombinant proteins

Genes for TNF-binding proteins (CrmBs) of variola virus (VARV), monkeypox virus (MPXV) or cowpox (CPXV) were isolated with PCR from viral genomes and expressed within baculovirus DNAs in Sf21 insect cell line. Properties of resulted recombinant proteins were studied with physical-chemical and immunological methods. It was shown with solid phase enzyme-linked immunoassay that viral proteins inhibited hTNF binding with polyclonal hTNF-antibodies. The strongest inhibitor was VARV-CrmB, the less one was MPXV-CrmB. Biological activity of recombinant protein preparations was studied in the test of neutralization of TNF cytotoxicity for L929 murine fibroblast cells. It was shown that recombinant CrmBs neutralized cytotoxicity of hTNF, mTNF or rTNF in species-specific manner. It was shown also that effectiveness of hTNF cytotoxicity inhibition in vitro with VARV-CrmB exceeded the same effect of polyclonal hTNF-antibody. A possibility of the elaboration of new therapeutics for anti-TNF therapy on the base of CrmB-like proteins is discussed.     

GDNF缺失突变体的设计及其结构与功能关系(英文)

研究了GDNF结构与功能的关系 .基于鼠源GDNF的晶体结构 ,利用计算机SGIIndigo2(R4 4 0 0 )工作站和InsightⅡ (95.5)蛋白质分析软件模拟了人GDNF三维结构 ,设计了GDNF分子的两个缺失突变体ΔN1 2 8和ΔN78 90 .以野生型GDNFcDNA作为模板 ,用PCR法得到编码缺失突变体的DNA片段 .将大肠杆菌作为表达系统 ,使缺失突变体GDNF在大肠杆菌中表达 ,对表达产物纯化和复性后进行生物活性测定 .两株突变体在大肠肝菌中获得了高效表达 ,纯化后的GDNF突变体ΔN1 2 8可以与存在于KG 1a细胞表面的受体结合 ,但不能促进 8日龄鸡胚背根节突起的生长 .突变体ΔN78 90既不能与受体结合 ,同时也失去了促背根节突起生长的功能 .说明GDNF分子的N端氨基酸对分子的生物学活性很重要 ,但对分子与受体GDNFR α的结合并不是必需的 ,而分子中的螺旋区对分子与受体的结合以及生物学活性都必不可少 .     

Leishmania major thialysine Nepsilon-acetyltransferase: identification of amino acid residues crucial for substrate binding

Thialysine N(epsilon)-acetyltransferases and spermidine/spermine N-acetyltransferases (SSAT) are closely related members of the GCN5-related N-acetyltransferase superfamily. Accordingly, a putative orthologue from the human protozoan parasite Leishmania major exhibits an almost equal similarity to human SSAT and thialysine N(epsilon)-acetyltransferase. Characterisation of the recombinantly expressed L. major protein indicated that it represents a thialysine N(epsilon)-acetyltransferase, preferring thialysine (S-aminoethyl-l-cysteine) and structurally related amino acids as acceptor molecules. The known thialysine N(epsilon)-acetyltransferases contain five conserved amino acid residues that are replaced in SSAT sequences. Kinetic analyses of the respective recombinant mutant proteins suggest that Ser(82) and Thr(83) of L. major thialysine N(epsilon)-acetyltransferase are key residues for acceptor binding. In addition, the conserved Leu(130) is tentatively involved in specific interaction with the sulphur-containing side chain of thialysine. The presence of these three amino acid residues is suggested to be a means by which thialysine N(epsilon)-acetyltransferases can be distinguished from SSAT sequences.