Substrate-binding lipoprotein of the cyanobacterium Synechococcus sp. strain PCC 7942 involved in the transport of nitrate and nitrite

Of the four genes (nrtABCD) required for active transport of nitrate in the cyanobacterium Synechococcus sp. strain PCC 7942, nrtBCD encode membrane components of an ATP-binding cassette transporter involved in the transport of nitrite as well as of nitrate, whereas nrtA encodes a 45-kDa cytoplasmic membrane protein, the biochemical function of which remains unclear. Characterization of the nrtA deletional mutants showed that the 45-kDa protein is essential for the functioning of the nitrate/nitrite transporter. A truncated NrtA protein lacking the N-terminal 81 amino acids, expressed in Escherichia coli cells as a histidine-tagged soluble protein, was shown to bind nitrate and nitrite with high affinity (Kd = 0.3 microM). Immunoblotting analysis using the antibody against the 45-kDa protein revealed a 48-kDa precursor of the protein, which accumulated in the cyanobacterial cells treated with globomycin, an antibiotic that specifically inhibits cleavage of the signal peptide of lipoprotein precursors. These findings indicated that the nrtA gene product is a nitrate- and nitrite-binding lipoprotein. The N-terminal sequences of putative cyanobacterial substrate-binding proteins suggested that lipoprotein modification of substrate-binding proteins of ATP-binding cassette transporters is common in cyanobacteria.     

The interaction of coumarin antibiotics with fragments of DNA gyrase B protein

DNA gyrase is the target of the coumarin group of antibacterial agents. The drugs are known to inhibit the ATPase activity of gyrase and bind to the 24-kDa N-terminal subdomain of gyrase B protein. Supercoiling assays with intact DNA gyrase and ATPase assays with a 43-kDa N-terminal fragment of the B protein suggest that the drugs bind tightly, with Kd values <10(-7) M. In addition, the ATPase data suggest that 1 coumermycin molecule interacts with 2 molecules of the 43-kDa protein while the other coumarins form a 1:1 complex. This result is confirmed by cross-linking experiments. Rapid gel-filtration experiments show that the binding of ADPNP(5'-adneylyl beta,gamm-imidodiphosphate) and coumarins to the 43-kDa protein is mutally exclusive, consistent with a competitive mode of action for the drugs. Rapid gel-filtration binding experiments using both the 24-and 43-kDa proteins also show that the drugs bind with association rate constants of >10(5) M-1.s-1, and dissociation rate constants of approximately 3x10(-3)s-1 and approximately 4x10(-3)s-1 for the 43-and 24-kDa proteins, respectively. Titration calorimetry shows that the Kd values for coumarins binding to both proteins are approximately 10-8M and that binding is enthalpy driven.     

Insulin-like growth factor-I (IGF-I) concentration in 150-kilodalton complexes containing human IGF-binding protein-3 (hIGFBP-3) after intravenous injection of adult rats with hIGFBP-3

After i.v. injection into adult rats, human insulin-like growth factor-binding protein-3 (hIGFBP-3) forms 150-kDa complexes with excess endogenous rat acid-labile subunit (ALS) within 2 min (Lewitt et al., 1993, Endocrinology 133:1797). Because their previous in vitro studies indicated that hIGFBP-3 only bound to ALS in the presence of IGF-I, and because little free IGF-I is present in plasma, the authors postulated that IGF-I had been mobilized to the circulation to saturate the 150-kDa hIGFBP-3 complexes. We examined this hypothesis by determining whether the hIGFBP-3 that appears in 150-kDa complexes 2 min after i.v. injection is accompanied by an increase in IGF-I. Within 2 min, some of the injected hIGFBP-3 (approximately 30% as much as endogenous intact rat IGFBP-3) is present in complexes that are cleared slowly from the circulation and presumed to be 150-kDa complexes. Gel filtration and immunoprecipitation studies performed on blood collected 2 min after injection confirmed that the injected hIGFBP-3 (46-82% as much as rat IGFBP-3) was associated with ALS in 150-kDa complexes. The formation of 150-kDa complexes containing hIGFBP-3 was not accompanied by a corresponding change in the IGF-I content (determined by RIA) of whole serum or 150-kDa serum fractions, suggesting that the hIGFBP-3 had rapidly associated with rALS in vivo without mobilizing IGF-I. Surprisingly, however, hIGFBP-3 was cleared much more rapidly from 150-kDa complexes formed after injection of hIGFBP-3 than after injection of hIGFBP-3:IGF-I complexes, suggesting that the complexes observed after hIGFBP-3 injection might not have formed in vivo. In fact, 150-kDa complexes formed to a similar extent when hIGFBP-3 was added ex vivo to blood collected from rats that had not received hIGFBP-3. We conclude that hIGFBP-3 can rapidly associate with rALS to form 150-kDa complexes in vivo without the mobilization of IGF-I. Because 150-kDa complexes also are formed ex vivo, however, we are unable to resolve whether the complexes that formed in vivo formed as binary or ternary complexes.     

Physical genome map of the unicellular cyanobacterium Synechococcus sp. strain PCC 7002

A physical restriction map of the genome of the cyanobacterium Synechococcus sp. strain PCC 7002 was assembled from AscI, NotI, SalI, and SfiI digests of intact genomic DNA separated on a contour-clamped homogeneous electric field pulsed-field gel electrophoresis system. An average genome size of 2.7 x 10(6) bp was calculated from 21 NotI, 37 SalI, or 27 SfiI fragments obtained by the digestions. The genomic map was assembled by using three different strategies: linking clone analysis, pulsed-field fragment hybridization, and individual clone hybridization to singly and doubly restriction-digested large DNA fragments. The relative positions of 21 genes or operons were determined, and these data suggest that the gene order is not highly conserved between Synechococcus sp. strain PCC 7002 and Anabaena sp. strain PCC 7120.     

Purification and characterization of a novel stress protein, the 150-kDa oxygen-regulated protein (ORP150), from cultured rat astrocytes and its expression in ischemic mouse brain

As the most abundant cell type in the central nervous system, astrocytes are positioned to nurture and sustain neurons, especially in response to cellular stresses, which occur in ischemic cerebrovascular disease. In a previous study (Hori, O., Matsumoto, M., Kuwabara, K., Maeda, M., Ueda, H., Ohtsuki, T., Kinoshita, T., Ogawa, S., Kamada, T., and Stern, D. (1996) J. Neurochem., in press), we identified five polypeptide bands on SDS-polyacrylamide gel electrophoresis, corresponding to molecular masses of about 28, 33, 78, 94, and 150 kDa, whose expression was induced/enhanced in astrocytes exposed to hypoxia or hypoxia followed by replacement into the ambient atmosphere (reoxygenation). In the current study, the approximately 150-kDa polypeptide has been characterized. Chromatography of lysates from cultured rat astrocytes on fast protein liquid chromatography Mono Q followed by preparative SDS-polyacrylamide gel electrophoresis led to isolation of a approximately 150-kDa band only observed in hypoxic cells and which had a unique N-terminal sequence of 15 amino acids. Antisera raised to either the purified approximately 150-kDa band in polyacrylamide gels or to a synthetic peptide comprising the N-terminal sequence detected the same polypeptide in extracts of cultured rat astrocytes exposed to hypoxia; expression was not observed in normoxia but was induced by hypoxia within 24 h, augmented further during early reoxygenation, and thereafter decreased to the base line by 24 h in normoxia. ORP150 expression in hypoxic astrocytes resulted from de novo protein synthesis, as shown by inhibition in the presence of cycloheximide. In contrast to hypoxia-mediated induction of the approximately 150-kDa polypeptide, neither heat shock nor a range of other stimuli, including hydrogen peroxide, cobalt chloride, 2-deoxyglucose, or tunicamycin, led to its expression, suggesting selectivity for production of ORP150 in response to oxygen deprivation, i.e. it was an oxygen-regulated protein (ORP150). Northern and nuclear run-off analysis confirmed the apparent selectivity for ORP150 mRNA induction in hypoxia. Subcellular localization studies showed ORP150 to be present intracellularly within endoplasmic reticulum and only in hypoxic astrocytes, not cultured microglia, endothelial cells, or neurons subject to hypoxia. Consistent with these in vitro results, induction of cerebral ischemia in mice resulted in expression of ORP150 (the latter was not observed in normoxic brain). These data suggest that astroglia respond to oxygen deprivation by redirection of protein synthesis with the appearance of a novel stress protein, ORP150. This polypeptide, selectively expressed by astrocytes, may contribute to their adaptive response to ischemic stress, thereby ultimately contributing to enhanced survival of neurons.     

Cloning and sequencing of the gene encoding a 31-kilodalton antigen of Haemophilus somnus

Immunoblots using bovine antibody against Haemophilus somnus as the primary antibody consistently identified 31-, 40- and 78-kDa proteins in Sarkosyl-insoluble extracts of H. somnus. A genomic library of H. somnus 8025 DNA was constructed in plasmid pUC19, and 45 recombinants expressed proteins which were recognized by bovine antiserum in Western blots (immunoblots). Ten of the recombinants expressing a 31-kDa protein caused the lysis of bovine erythrocytes. Restriction endonuclease mapping indicated that the hemolytic recombinants shared an approximately 1.7-kb BglII fragment. Southern blot analysis using the BglII fragment as a probe revealed homology among the recombinants and the presence of an identically sized BglII fragment in the chromosome of all H. somnus isolates tested. Sequence analysis indicated the presence of an 822-bp open reading frame within the 1.7-kb BglII fragment. Deletion of this open reading frame resulted in the loss of hemolytic activity and protein expression in recombinant Escherichia coli, suggesting the possible role of the 31-kDa protein as a hemolysin. An amino acid sequence deduced from the DNA sequence shared homology with outer membrane protein A of E. coli, Salmonella typhimurium, and Shigella dysenteriae, with P6 of Haemophilus influenzae, and with PIII of Neisseria gonorrhoeae. An amino acid analysis of the recombinant 31-kDa protein agreed with the amino acid composition deduced from the DNA sequence.     

N-linked glycosylation of the human Ca2+ receptor is essential for its expression at the cell surface

The human Ca2+ receptor (hCaR) is a member of the superfamily of G protein-coupled receptors. Its large (approximately 600 residue) amino-terminal extracellular domain contains 9 potential N-linked glycosylation sites. Immunoblot of cell membranes derived from HEK-293 cells, stably transfected with the hCaR, showed two major immunoreactive bands of approximately 150 and 130 kDa, respectively. Complete digestion of the membranes with PN-glycosidase F yielded a single major immunoreactive band of approximately 115 kDa, confirming the presence of N-linked glycosylation. Treatment of these cells with tunicamycin, which blocks N-linked glycosylation, inhibited signal transduction in response to Ca2+. Flow cytometric analysis showed decreased expression of the hCaR on the cell membrane in tunicamycin-treated cells. Immunoblot of tunicamycin-treated cells showed a reduction in the amount of the 150-kDa band and conversion of the 130-kDa band to the presumptively nonglycosylated 115-kDa form. Tunicamycin treatment of cells, transfected with a mutant hCaR complementary DNA containing a nonsense codon at position 599 preceding the 1st transmembrane domain, blocked the secretion of a 95-kDa protein, representing the amino-terminal extracellular domain, into the medium. These results demonstrate that N-linked glycosylation is required for normal expression of the hCaR at the cell surface.     

Recurrence of blackwater fever: triggering of relapses by different antimalarials

A monoclonal antibody that reacts with a 150-kDa protein of Entamoeba histolytica on Western immunoblotting under nonreducing conditions inhibits the adherence and cytotoxicity of the ameba to mammalian cells in vitro. Affinity purification of solubilized trophozoites using the monoclonal antibody and electrophoresis yielded three glycoproteins with molecular masses of 150, 170, and 260 kDa, suggesting the existence of either a common epitope or the close association of these proteins. The 260-kDa fraction was identified as the well-known galactose (Gal)- and N-acetyl-D-galactosamine (GalNAc)-inhibitable lectin. The 150- and 170-kDa fractions seemed to exist as part of a 380-kDa native protein with an isoelectric point of pH 6.9. The N-terminal amino acid sequence of the 150-kDa protein was unique, indicating that the protein was not a degraded product of the 260-kDa lectin. By gel filtration, the 260-kDa lectin and the 150/170-kDa protein could be separated. When Chinese hamster ovary cells were pretreated with the fraction consisting of the 150/170-kDa protein the adherence of trophozoites to Chinese hamster ovary cells was competitively inhibited to a level equivalent to that observed for the 260-kDa lectin. The inhibitory effect was lost in the presence of Gal and GalNAc but was not influenced by the presence of glucose. These results demonstrate that the 150/170-kDa protein is a Gal/GalNAc-inhibitable lectin. The existence of a sugar-binding domain in the protein was confirmed by Gal-affinity chromatography.     

Molecular and immunological analyses of the Mycobacterium avium homolog of the immunodominant Mycobacterium leprae 35-kilodalton protein

The analysis of host immunity to mycobacteria and the development of discriminatory diagnostic reagents relies on the characterization of conserved and species-specific mycobacterial antigens. In this report, we have characterized the Mycobacterium avium homolog of the highly immunogenic M. leprae 35-kDa protein. The genes encoding these two proteins were well conserved, having 82% DNA identity and 90% identity at the amino acid level. Moreover both proteins, purified from the fast-growing host M. smegmatis, formed multimeric complexes of around 1000 kDa in size and were antigenically related as assessed through their recognition by antibodies and T cells from M. leprae-infected individuals. The 35-kDa protein exhibited significant sequence identity with proteins from Streptomyces griseus and the cyanobacterium Synechoccocus sp. strain PCC 7942 that are up-regulated under conditions of nutrient deprivation. The 67% amino acid identity between the M. avium 35-kDa protein and SrpI of Synechoccocus was spread across the sequences of both proteins, while the homologous regions of the 35-kDa protein and the P3 sporulation protein of S. griseus were interrupted in the P3 protein by a divergent central region. Assessment by PCR demonstrated that the gene encoding the M. avium 35-kDa protein was present in all 30 M. avium clinical isolates tested but absent from M. intracellulare, M. tuberculosis, or M. bovis BCG. Mice infected with M. avium, but not M. bovis BCG, developed specific immunoglobulin G antibodies to the 35-kDa protein, consistent with the observation that tuberculosis patients do not recognize the antigen. Strong delayed-type hypersensitivity was elicited by the protein in guinea pigs sensitized with M. avium.     

Bacillus isolates from the spermosphere of peas and dwarf French beans with antifungal activity against Botrytis cinerea and Pythium species

ClpP functions as the proteolytic subunit of the ATP-dependent Clp protease in eubacteria, mammals and plant chloroplasts. We have cloned a clpP gene, designated clpP1, from the cyanobacterium Synechococcus sp. PCC 7942. The monocistronic 591 bp gene codes for a protein 80% similar to one of four putative ClpP proteins in another cyanobacterium, Synechocystis sp. PCC 6803. The constitutive ClpP1 content in Synechococcus cultures was not inducible by high temperatures, but it did rise fivefold with increasing growth light from 50 to 175 micromol photons m(-2) s(-1). A clpP1 inactivation strain (delta clpP1) exhibited slower growth rates, especially at the higher irradiances, and changes in the proportion of the photosynthetic pigments, chlorophyll a and phycocyanin. Many mutant cells (ca. 35%) were also severely elongated, up to 20 times longer than the wild type. The stress phenotype of delta clpP1 when grown at high light was confirmed by the induction of known stress proteins, such as the heat shock protein GroEL and the alternate form of PSII reaction center D1 protein, D1 form 2. ClpP1 content also rose significantly during short-term photoinhibition, but its loss in delta clpP1 did not exacerbate the extent of inactivation of photosynthesis, nor affect the inducible D1 exchange mechanism, indicating ClpP1 is not directly involved in D1 protein turnover.     

Raman and CD spectroscopy of recombinant 68-kDa DNA human topoisomerase I and its complex with suicide DNA-substrate

N-terminally truncated recombinant 68-kDa human topoisomerase (topo) I exhibits the same DNA-driving activities as the wild-type protein. In the present study, Raman and circular dichroism techniques were employed for detailed structural characterization of the 68-kDa human topo I and its transformations induced by the suicide sequence-specific oligonucleotide (solig) binding and cleavage. Spectroscopic data combined with statistical prediction techniques were employed to construct a model of the secondary structure distribution along the primary protein structure in solution. The 68-kDa topo I was found to consist of ca. 59% alpha-helix, 24% beta-strand and/or sheets, and 17% other structures. A secondary structure transition of the 68-kDa topo I was found to accompany solig binding and cleavage. Nearly 15% of the alpha-helix of 68-kDa topo I is transferred within the other structures when in the complex with its DNA substrate. Raman spectroscopy analysis also shows redistribution of the structural rotamers of the 68-kDa topo I disulfide bonds and significant changes in the H-bonding of the Tyr residues and in the microenvironment/conformation of the Trp side chains. No structural modifications of the DNA substrate were detected by spectroscopic techniques. The data presented provide the first direct experimental evidence of the human topo I conformational transition after the cleavage step in the reaction of binding and cleavage of DNA substrate by the enzyme. This evidence supports the model of the enzyme function requiring the protein conformational transition. The most probable location of the enzyme transformations was the core and the C-terminal conservative 68-kDa topo I structural domains. By contrast, the linker domain was found to have an extremely low potential for solig-induced structural transformations. The pattern of redistribution of protein secondary structures induced by solig binding and covalent suicide complex formation supports the model of an intramolecular bipartite mode of topo I/DNA interaction in the substrate binding and cleavage reaction.     

Induction of the heat shock protein ClpB affects cold acclimation in the cyanobacterium Synechococcus sp. strain PCC 7942

The heat shock protein ClpB is essential for acquired thermotolerance in cyanobacteria and eukaryotes and belongs to a diverse group of polypeptides which function as molecular chaperones. In this study we show that ClpB is also strongly induced during moderate cold stress in the unicellular cyanobacterium Synechococcus sp. strain PCC 7942. A fivefold increase in ClpB (92 kDa) content occurred when cells were acclimated to 25 degrees C over 24 h after being shifted from the optimal growth temperature of 37 degrees C. A corresponding increase occurred for the smaller ClpB' (78 kDa), which arises from a second translational start within the clpB gene of prokaryotes. Shifts to more extreme cold (i.e., 20 and 15 degrees C) progressively decreased the level of ClpB induction, presumably due to retardation of protein synthesis within this relatively cold-sensitive strain. Inactivation of clpB in Synechococcus sp. increased the extent of inhibition of photosynthesis upon the shift to 25 degrees C and markedly reduced the mutant's ability to acclimate to the new temperature regime, with a threefold drop in growth rate. Furthermore, around 30% fewer delta clpB cells survived the shift to 25 degrees C after 24 h compared to the wild type, and more of the mutant cells were also arrested during cell division at 25 degrees C, remaining attached after septum formation. Development of a cold thermotolerance assay based on cell survival clearly demonstrated that wild-type cells could acquire substantial resistance to the nonpermissive temperature of 15 degrees C by being pre-exposed to 25 degrees C. The same level of cold thermotolerance, however, occurred in the delta clpB strain, indicating ClpB induction is not necessary for this form of thermal resistance in Synechococcus spp. Overall, our results demonstrate that the induction of ClpB contributes significantly to the acclimation process of cyanobacteria to permissive low temperatures.     

Engineering multi-domain redox proteins containing flavodoxin as bio-transformer: preparatory studies by rational design

Protein tyrosine phosphorylation is associated with sperm capacitation and the acrosome reaction in several mammalian species. Changes in phosphorylation of a 95-kDa protein in human, mouse, and domestic cat spermatozoa are known to be influenced by capacitation and exposure to zona pellucida (ZP) proteins. We previously reported diminished phosphorylation of 95- and 160-kDa proteins in spermatozoa from teratospermic cats, compared with normospermic domestic cats. To determine if these proteins and mechanisms are present in other species in the phenotypically diverse Felidae family, we examined the relationship between tyrosine-phosphorylated sperm proteins and sperm morphology in the leopard cat (approximately 65% normal sperm/ejaculate), tiger (approximately 65%), clouded leopard (approximately 15%), and cheetah (approximately 30%). Furthermore, we investigated the involvement of cyclic adenosine monophosphate (cAMP) in the regulation of sperm protein tyrosine phosphorylation. Specifically, we assessed the following: 1) presence of tyrosine-phosphorylated proteins in sperm extracts; 2) changes in protein tyrosine phosphorylation after sperm capacitation and swim-up separation; 3) impact of tyrosine kinase inhibition on leopard cat sperm protein phosphorylation and ZP penetration; and 4) involvement of a cAMP-dependent pathway in the regulation of protein tyrosine phosphorylation. Immunoblotting analysis with anti-phosphotyrosine antibody (PY20) indicated that a 95-kDa protein was present in all four species. Additional phosphorylated proteins were detected in the leopard cat (145- and 175-kDa proteins), tiger (185-kDa protein), clouded leopard (160- and 190-kDa proteins), and cheetah (115- and 155-kDa proteins). Sperm capacitation in vitro increased phosphorylation of one or more proteins in the leopard cat, tiger and clouded leopard, but not in the cheetah. Although swim-up separation increased the proportion of morphologically normal spermatozoa in the clouded leopard and cheetah, no changes were observed in phosphorylation of the 95-kDa sperm protein. Thus, phosphorylation of the 95-kDa protein appeared to be related to the condition of teratospermia. Exposing leopard cat spermatozoa to the tyrosine kinase inhibitor, tyrphostin, reduced (P < 0.05) phosphorylation of the 95- and 145-kDa proteins, as well as ZP penetration, without affecting sperm motility. Similarly, when spermatozoa were incubated in the presence of cAMP analogs or active and inactive stereoisomers of cAMP, phosphorylation of sperm proteins was either stimulated or inhibited. Together, these data suggest that protein tyrosine kinase mechanisms appear conserved within the family Felidae and are regulated by a cAMP/protein kinase A pathway.     

Role of signal peptides in targeting of proteins in cyanobacteria

Proteins of cyanobacteria may be transported across one of two membrane systems: the typical eubacterial cell envelope (consisting of an inner membrane, periplasmic space, and an outer membrane) and the photosynthetic thylakoids. To investigate the role of signal peptides in targeting in cyanobacteria, Synechococcus sp. strain PCC 7942 was transformed with vectors carrying the chloramphenicol acetyltransferase reporter gene fused to coding sequences for one of four different signal peptides. These included signal peptides of two proteins of periplasmic space origin (one from Escherichia coli and the other from Synechococcus sp. strain PCC 7942) and two other signal peptides of proteins located in the thylakoid lumen (one from a cyanobacterium and the other from a higher plant). The location of the gene fusion products expressed in Synechococcus sp. strain PCC 7942 was determined by a chloramphenicol acetyltransferase enzyme-linked immunosorbent assay of subcellular fractions. The distribution pattern for gene fusions with periplasmic signal peptides was different from that of gene fusions with thylakoid lumen signal peptides. Primary sequence analysis revealed conserved features in the thylakoid lumen signal peptides that were absent from the periplasmic signal peptides. These results suggest the importance of the signal peptide in protein targeting in cyanobacteria and point to the presence of signal peptide features conserved between chloroplasts and cyanobacteria for targeting of proteins to the thylakoid lumen.