Assessment of the immunoglobulin E-mediated immune response to milk-specific proteins in allergic patients using microarrays.

BACKGROUND: Cow's milk allergy (CMA) is one of the most widespread human allergies, especially in young children. Although CMA is intensively studied, little is known about the recognition patterns of milk allergens in allergic patients, and the determination these patterns is a prerequisite for the development of efficient diagnostic and prognostic tools. Several factors present difficulties for such a determination, because (i) milk contains a large number of potential allergens; (ii) the majority of these allergens consist of complex suspensions rather than solutions; (iii) the major allergens, such as caseins, cannot be highly purified in large amounts; and (iv) most of the time, very small amount of young patients' sera are readily available. METHODS: To overcome these difficulties, we developed a sensitive microarray assay that, in combination with near-infrared fluorescence detection, was used to study the immune response to milk and purified native milk proteins. RESULTS: This new assay allowed us to assess the binding ability of IgE to milk allergens from a large number of young patients using reduced amounts of clinical material. The data show that bovine lactoferrin can be classed as a strong milk allergen. We confirmed that bovine caseins are the main allergens in milk and that alpha(S1)-casein is more allergenic than alpha(S2)-, beta- and kappa-caseins, which were recognized with almost a similar frequency by the sera of patients. CONCLUSION: Microarray methods, in combination with near-infrared fluorescence detection, can be useful for the in vitro diagnosis of food allergies.

[Protein arrays and perspectives of medical applications]. / Puces à protéines et perspectives d'applications médicales.

Protein microarrays make it possible to detect molecular interactions with various partners (proteins, peptides, nucleic acids, sugars, etc.). Their advantages are crucial for high-throughput analysis of proteomes of different organisms. Moreover, the recent data reveal the performance of microarrays over current immunological methods. Therefore, the antigen and antibody microarrays become indispensable for medical applications, in particular, for diagnosis and prognosis of microbial infections, autoimmune and allergic diseases. The further technological progress might provide the extension of the miniaturized assays for multiparametric monitoring of human pathologies in practical medicine.

An invariant threonine is involved in self-catalyzed cleavage of the precursor protein for ornithine acetyltransferase.

In Bacillus stearothermophilus ornithine acetyltransferase is a bifunctional enzyme, catalyzing the first and the fifth steps of arginine biosynthesis; it follows a ping-pong kinetic mechanism. A single chain precursor protein is cleaved between the alanine and threonine residues in a highly conserved ATML sequence leading to the formation of alpha and beta subunits that assemble into a heterotetrameric 2alpha2beta molecule. The beta subunit has been shown to form an acetylated intermediate in the course of the transacetylation reaction. The present data show that the precursor protein synthesized in vitro or in vivo undergoes a self-catalyzed cleavage involving an invariant threonine (Thr-197). Using site-directed mutagenesis T197G, T197S, and T197C derivatives have been generated. The T197G substitution abolishes both precursor protein cleavage and catalytic activity, whereas T197S and T197C substitutions reduce precursor cleavage and catalytic activity in the order Thr-197 (wild type) --> Ser-197 --> Cys-197. A mechanism is proposed in which Thr-197 plays a crucial role in the autoproteolytic cleavage of ornithine acetyltransferase.

Characterization and kinetic mechanism of mono- and bifunctional ornithine acetyltransferases from thermophilic microorganisms.

The argJ gene coding for N2-acetyl-L-ornithine: L-glutamate N-acetyltransferase, the key enzyme involved in the acetyl cycle of L-arginine biosynthesis, has been cloned from thermophilic procaryotes: the archaeon Methanoccocus jannaschii, and the bacteria Thermotoga neapolitana and Bacillus stearothermophilus. Archaeal argJ only complements an Escherichia coli argE mutant (deficient in acetylornithinase, which catalyzes the fifth step in the linear biosynthetic pathway), whereas bacterial genes additionally complement an argA mutant (deficient in N-acetylglutamate synthetase, the first enzyme of the pathway). In keeping with these in vivo data the purified His-tagged ArgJ enzyme of M. jannaschii only catalyzes N2-acetylornithine conversion to ornithine, whereas T. neapolitana and B. stearothermophilus ArgJ also catalyze the conversion of glutamate to N-acetylglutamate using acetylCoA as the acetyl donor. M. jannaschii ArgJ is therefore a monofunctional enzyme, whereas T. neapolitana and B. stearothermophilus encoded ArgJ are bifunctional. Kinetic data demonstrate that in all three thermophilic organisms ArgJ-mediated catalysis follows ping-pong bi-bi kinetic mechanism. Acetylated ArgJ intermediates were detected in semireactions using [14C]acetylCoA or [14C]N2-acetyl-L-glutamate as acetyl donors. In this catalysis L-ornithine acts as an inhibitor; this amino acid therefore appears to be a key regulatory molecule in the acetyl cycle of L-arginine synthesis. Thermophilic ArgJ are synthesized as protein precursors undergoing internal cleavage to generate alpha and beta subunits which appear to assemble to alpha2beta2 heterotetramers in E. coli. The cleavage occurs between alanine and threonine residues within the highly conserved PXM-ATML motif detected in all available ArgJ sequences.

Thermostability, oligomerization and DNA-binding properties of the regulatory protein ArgR from the hyperthermophilic bacterium Thermotoga neapolitana.

The hexameric regulatory protein ArgR formed by arginine-mediated dimerization of identical trimers governs the expression of genes required for arginine metabolism and some other genes in mesophilic and moderately thermophilic bacteria. We have cloned the argR gene from two hyperthermophilic bacteria of the genus Thermotoga. The two-domain ArgR proteins encoded by T. neapolitana and T. maritima share a low degree of sequence similarity with other bacterial arginine repressors. The ArgR protein from T. neapolitana binds to an operator located just upstream of its coding sequence and, therefore, the argR gene may be autoregulated. The protein has extremely high intrinsic thermostability and tolerance to urea. Moreover, its binding to target DNA increases the melting temperature by approximately 15 degrees C. The formation of oligomeric ArgR-DNA complexes is a function of protein concentration, with hexameric complexes being favoured at higher concentrations. In the presence of arginine the hyperthermophilic ArgR protein binds to its own operator, argRo, only by forming hexamer ArgR-DNA complexes, whereas both trimer-DNA and hexamer-DNA complexes are detected in the absence of arginine. However, the affinity of T. neapolitana ArgR for DNA has been found to be higher for a mixture of trimers and non-bound hexamers than for arginine-bound hexamers. Our data indicate that genes for arginine biosynthesis are clustered in a putative operon, which could also be regulated by the ArgR protein, in the hyperthermophilic host.

Mutational analysis of the thermostable arginine repressor from Bacillus stearothermophilus: dissecting residues involved in DNA binding properties.

Recently the crystal structure of the DNA-unbound form of the full-length hexameric Bacillus stearothermophilus arginine repressor (ArgR) has been resolved, providing a possible explanation for the mechanism of arginine-mediated repressor-operator DNA recognition. In this study we tested some of these functional predictions by performing site-directed mutagenesis of distinct amino acid residues located in two regions, the N-terminal DNA-binding domain and the C-terminal oligomerization domain of ArgR. A total of 15 mutants were probed for their capacity to repress the expression of the reporter argC - lacZ gene fusion in Escherichia coli cells. Substitutions of highly conserved amino acid residues in the alpha2 and alpha3 helices, located in the winged helix-turn-helix DNA-binding motif, reduced repression. Loss of DNA-binding capacity was confirmed in vitro for the Ser42Pro mutant which showed the most pronounced effect in vivo. In E. coli, the wild-type B. stearothermophilus ArgR molecule behaves as a super-repressor, since recombinant E. coli host cells bearing B. stearothermophilusargR on a multicopy vector did not grow in selective minimal medium devoid of arginine and grew, albeit weakly, when l -arginine was supplied. All mutants affected in the DNA-binding domain lost this super-repressor behaviour. Replacements of conserved leucine residues at positions 87 and/or 94 in the C-terminal domain by other hydrophobic amino acid residues proved neutral or caused either derepression or stronger super-repression. Substitution of Leu87 by phenylalanine was found to increase the DNA-binding affinity and the protein solubility in the context of a double Leu87Phe/Leu94Val mutant. Structural modifications occasioned by the various amino acid substitutions were confirmed by circular dichroism analysis and structure modelling.

Structure of the arginine repressor from Bacillus stearothermophilus.

The arginine repressor (ArgR) is a hexameric DNA-binding protein that plays a multifunctional role in the bacterial cell. Here, we present the 2.5 A structure of apo-ArgR from Bacillus stearothermophilus and the 2.2 A structure of the hexameric ArgR oligomerization domain with bound arginine. This first view of intact ArgR reveals an approximately 32-symmetric hexamer of identical subunits, with six DNA-binding domains surrounding a central oligomeric core. The difference in quaternary organization of subunits in the arginine-bound and apo forms provides a possible explanation for poor operator binding by apo-ArgR and for high affinity binding in the presence of arginine.

The Bacillus stearothermophilus argCJBD operon harbours a strong promoter as evaluated in Escherichia coli cells.

We have shown that the B. stearothermophilus argCJBD genes form a single operon. In B. stearothermophilus, a specific repressor governs operon expression by binding to the argCo operator site overlapping the Parg promoter sequence (Dion et al., 1997). Therefore, the enzymatic and transcriptional analyses performed in this work did not reflect the potential strength of Parg in the native host. For evaluation of the Parg promoter strength, E. coli was used as a host since its own ArgR repressor does not interact with the B. stearothermophilus heterologous operator. Parg-promoted argC gene expression dramatically increased, reaching up to 38% of the total protein in E. coli cells. An AT-rich sequence upstream of a -35 site of Parg was found to be indispensable for the promoter strength. Plasmids carrying the B. stearothermophilus argCJBD operon linked with its Parg/argCo region were unstable in E. coli. Stabilization of plasmids was achieved by repression of B. stearothermophilus arg genes through the action of the B. subtilis AhrC repressor.

The highly thermostable arginine repressor of Bacillus stearothermophilus: gene cloning and repressor-operator interactions.

We report here the cloning of the arginine repressor gene argR of Bacillus stearothermophilus and the characterization and purification to homogeneity of its product. The deduced amino acid sequence of the 16.8-kDa ArgR subunit shares 72% identity with its mesophilic homologue AhrC of Bacilus subtilis. Sequence analysis of B. stearothermophilus ArgR and comparisons with mesophilic arginine repressors suggest that the thermostable repressor comprises an N-terminal DNA-binding and a C-terminal oligomerization and arginine-binding region. B. stearothermophilus ArgR has been overexpressed in E. coli and purified as a 48.0-kDa trimeric protein. The repressor inhibits the expression of a B. stearothermophilus argC-lacZ fusion in E. coli cells. In the presence of arginine, the purified protein binds tightly and specifically to the argC operator, which largely overlaps the argC promoter. The purified B. stearothermophilus repressor proved to be very thermostable with a half-life of approximately 30 min at 90 degrees C, whereas B. subtilis AhrC was largely inactivated at 65 degrees C. Moreover, ArgR operator complexes were found to be remarkably thermostable and could be formed efficiently at up to 85 degrees C, well above the optimal growth temperature of the moderate thermophile B. stearothermophilus. This pronounced resistance of the repressor-operator complexes to heat treatment suggests that the same type of regulatory mechanism could operate in extreme thermophiles.

Two amino acid amidohydrolase genes encoding L-stereospecific carbamoylase and aminoacylase are organized in a common operon in Bacillus stearothermophilus.

The L-carbamoylase gene (amaB) upstream of the previously detected L-aminoacylase gene (amaA) in the Bacillus stearothermophilus NCIB8224 strain was identified in this study. The amaB and amaA genes are cotranscribed as a single mRNA from the same transcriptional start. The two-ama-gene operon is conserved in B. stearothermophilus strains. A cross-activity of L-carbamoylase towards respective substrates for L-aminoacylase supports the hypothesis of a common ancestor for both amino acid amidohydrolase genes.

The arginine operon of Bacillus stearothermophilus: characterization of the control region and its interaction with the heterologous B. subtilis arginine repressor.

Mechanisms of gene regulation have not yet been extensively studied in thermophilic bacteria. In previous studies we showed that the Bacillus stearothermophilus argCJBD gene cluster is subject to specific repression by arginine. Here we report the cloning by colony hybridization, and characterization of the proximal part of the argC gene together with the adjacent control region of the cluster. The promoter was identified by primer extension mapping of the argC transcription startpoint: a sequence overlapping it was found to be similar to the arginine operators of B. subtilis and to a smaller extent of E. coli. Use of an argC-lacZ gene fusion revealed that the argC promoter is strongly repressed by the heterologous B. subtilis arginine repressor/activator AhrC in E. coli cells. Mobility shift and DNase I footprinting experiments revealed tight, specific and arginine-dependent binding of this operator-like sequence to purified AhrC. It is therefore very likely that in B. stearothermophilus the expression of the argCJBD operon is modulated by a repressor that is the thermophilic homologue of AhrC.

Divergent evolution of two plastid genes, rbcL and atpB, in a non-photosynthetic parasitic plant.

Plastid DNA (ptDNA) regions for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubiso) (rbcL) and the beta-subunit of ATP synthase (atpB) genes of the holoparasite Lathraea clandestina L. were sequenced. These regions were obtained by cloning either a Bam HI endonuclease generated fragment from the Lathraea ptDNA or polymerase chain reaction (PCR) amplified products. The Lathraea ptDNA contains the entire sequence for the rbcL gene which shares 94.5% homology with the Nicotiana tabacum gene, whereas atpB is maintained as a pseudogene. The intergenic region between divergently transcribed rbcL and atpB genes is shorter (758 bp) in L. clandestina plastid genome in comparison with N. tabacum (823 bp), however they have a noticeable similarity, mainly in the rbcL 5'-upstream region. A low level of the rbcL gene transcription was detected whereas no atpB transcripts were found in Latraea. The plasmid rbcL gene of the hemiparasite Melampyrum pratense and the autotroph Digitalis purpurea both from the Scrophulariaceae were cloned by PCR amplification and then sequenced. The L. clandestina rbcL gene is highly homologous to the M. pratense and D. purpurea genes. The data indicate that the evolution of the plastid atpB-rbcL region was different in parasites from the Scrophulariaceae and Orobanchaceae families.

Gene cloning, sequence analysis, purification, and characterization of a thermostable aminoacylase from Bacillus stearothermophilus.

A genomic DNA fragment encoding aminoacylase activity of the eubacterium Bacillus stearothermophilus was cloned into Escherichia coli. Transformants expressing aminoacylase activity were selected by their ability to complement E. coli mutants defective in acetylornithine deacetylase activity, the enzyme that converts N-acetylornithine to ornithine in the arginine biosynthetic pathway. The 2.3-kb cloned fragment has been entirely sequenced. Analysis of the sequence revealed two open reading frames, one of which encoded the aminoacylase. B. stearothermophilus aminoacylase, produced in E. coli, was purified to near homogeneity in three steps, one of which took advantage of the intrinsic thermostability of the enzyme. The enzyme exists as homotetramer of 43-kDa subunits as shown by cross-linking experiments. The deacetylating capacity of purified aminoacylase varies considerably depending on the nature of the amino acid residue in the substrate. The enzyme hydrolyzes N-acyl derivatives of aromatic amino acids most efficiently. Comparison of the predicted amino acid sequence of B. stearothermophilus aminoacylase with those of eubacterial acetylornithine deacylase, succinyldiaminopimelate desuccinylase, carboxypeptidase G2, and eukaryotic aminoacylase I suggests a common origin for these enzymes.

Primary structure, partial purification and regulation of key enzymes of the acetyl cycle of arginine biosynthesis in Bacillus stearothermophilus: dual function of ornithine acetyltransferase.

A 3.4 kb EcoRI fragment, cloned in E. coli, that carries part of a cluster of genes encoding arginine biosynthetic functions of the thermophilic bacterium Bacillus stearothermophilus, was sequenced on both strands. The sequence consists of a truncated argC gene, an argJ region encoding a polypeptide with both N-acetylglutamate synthase and ornithine acetyltransferase activities, the argB gene and the N-terminal part of argD. The argB gene encodes a 258-amino-acid polypeptide with a deduced M(r) of 26918. A very high and thermostable N-acetylglutamate 5-phosphotransferase activity was detected in extracts of E. coli arg B mutants transformed with the 3.4 kb fragment on a plasmid. A polypeptide band of M(r) 27,000 was detected by SDS-PAGE of heat-treated extract from such a strain. Both N-acetylglutamate synthase and ornithine acetyltransferase are encoded by the same 1290 bp open reading frame. The deduced sequence of 410 amino acids corresponds to a peptide of M(r) 43,349. The subcloned B. stearothermophilus argJ can complement a double argA argE E. coli mutant to prototrophy. Gel-filtration of a heat-treated extract of the complemented double mutant E. coli host showed that N-acetylglutamate synthase and ornithine acetyltransferase activities co-elute in a single peak corresponding to M(r) 110,000. Both activities were also heat-inactivated at the same temperature and strongly inhibited by ornithine. These results suggest that both activities can be ascribed to a single protein.

Acetylornithine deacetylase, succinyldiaminopimelate desuccinylase and carboxypeptidase G2 are evolutionarily related.

The nucleotide (nt) sequence of the Escherichia coli argE gene, encoding the acetylornithine deacetylase (AO) subunit, has been established and corresponds to a 43-kDa (M(r) 42,320) polypeptide. The enzyme has been purified to near homogeneity and it appears to be a dimer consisting of two 43-kDa subunits. The amino acid sequence deduced from the nt sequence was compared to that of the subunit of E. coli succinyldiaminopimelate desuccinylase (the dapE gene product involved in the diaminopimelate pathway for lysine biosynthesis), since both enzymes share functional and biochemical features. Significant similarity covering the entire sequence allows us to infer a common origin for both deacylases. This homology extends to the Pseudomonas sp. G2 carboxypeptidase (G2CP); this or a functionally related enzyme may be responsible for the minor AO activity found in organisms relying on ornithine acetyltransferase for ornithine biosynthesis.

Transformation of Bacillus thuringiensis subsp. galleria protoplasts by plasmid pBC16.

Protoplasts of the entomopathogenic bacterium Bacillus thuringiensis subsp. galleria were transformed by plasmid pBC16. The frequency of transformation was much lower than that of Bacillus subtilis. All isolated B. thuringiensis transformants were characterized by increased sensitivity to lysozyme as compared with the original strain.

Location of nodulation and nitrogen fixation genes on a high molecular weight plasmid of R. meliloti.

R. meliloti strain 41 (Rm41) was shown to harbour two indigenous plasmids with molecular weights of 140 Mdal (pRme41a) and more than 300 Mdal (pRme41b), respectively. Using a heat-treatment procedure, derivatives of Rm41 defective in nodulation (Nod-) or nitrogen fixation (Fix-) have been readily obtained. In some Nod- mutants the deletion of a segment of plasmid pRme41b was found. Based on the demonstrated homology between the nitrogen fixation (nif) genes of Klebsiella pneumoniae and of R. meliloti the Rhizobium nif region has been cloned into the cosmid vector pHC79, then recloned into pBR322 and the restriction map of the nif region has been determined. 32P-labelled nick-translated probe prepared from the cloned nif DNA fragment hybridized to pRme41b of Rm41 but for most Nod- mutants this hybridization was not detected. Hybridization of a cosmid containing Rm41 DNA to total DNA digest from the wild-type bacterium and from a series of Nod- mutants revealed that at least a 2 kb DNA fragment including the nif structural genes was missing from most of the Nod- mutants. These results, together with the genetic analyses of these symbiotic mutations suggest that some nod and fix genes are located on pRme41b.

Mapping of RP4 plasmid using deletion mutants of pAS8 hybrid (RP4--ColE1).

We used the hybrid plasmid pAS8 in order to conduct the genetic analysis of RP4 plasmid. The presence of two replicons in the hybrid plasmid permitted to expand the spectrum of deletion mutants of RP4 isolated, which are capable to autonomous replication. The shortening of the hybrid plasmid was achieved by P22 transduction, by induction of deletion mutants using mitomycin C, as well as by seletion of Tra- mutants on the basis of resistance of cells to P-specific phages. These techniques have lead to isolation of clones possessing different combinations of plasmid resistance determinants. Comparison of phenotypic characteristics of deletion plasmids pAS9, pAS10, pAS11, pAS12 and pAS10-2 permitted to propose the map for pAS8 plasmid with the following sequence of markers: tra--kam--ColE1--amp--tet... Heteroduplex analysis of deletion mutants of pAS8 permitted to construct a physical map and to elaborate in greater detail the functional map of RP4 plasmid. The correlation between the ability of mutants to replicate in polA(TS) strain at nonpermissive conditions and the length of the deleted segment permitted to map rep genes of RP4 on a region with coordinates 9.8--17.3 kb. A relationship between the manifestation of incompatibility of mutants with Inc P-1 plasmids and the length of deletions points out that inc genes are located on DNA region with coordinates 2.1--9.8 kb. The analysis of replication of deletion mutants and the manifestation of incompatibility just as of the data about the size of appropriate deletions permitted to make the conclusion about the functional and genetic independence of the replication control and incompatibility control in RP4 plasmid.