Rhoptry-associated protein 1-binding monoclonal antibody raised against a heterologous peptide sequence inhibits Plasmodium falciparum growth in vitro.

Monoclonal antibodies (MAbs) specific for Plasmodium falciparum rhoptry-associated protein 1 (RAP-1) were generated and tested for inhibition of parasite growth in vitro. The majority of indirect immunofluorescence assay (IFA)-positive MAbs raised against recombinant RAP-1 positions 23 to 711 (rRAP-1(23-711)) recognized epitopes located in the immunodominant N-terminal third of RAP-1. MAbs specific for the building block 35.1 of the synthetic peptide malaria vaccine SPf66 also yielded an IFA staining pattern characteristic for rhoptry-associated proteins and reacted specifically with rRAP-1 and parasite-derived RAP-1 molecules p67 and p82. Cross-reactivity with RAP-1 was blocked by the 35.1 peptide. Epitope mapping with truncated rRAP-1 molecules and overlapping peptides identified the linear RAP-1 sequence Y218KYSL222 as a target of the anti-35.1 MAbs. This sequence lacks primary sequence similarity with the 35.1 peptide (YGGPANKKNAG). Cross-reactivity of the anti-35.1 MAbs thus appears to be associated with conformational rather than sequence homology. While the anti-35.1 MAb SP8.18 exhibited parasite growth-inhibitory activity, none of the tested anti-rRAP-1(23-711) MAbs inhibited parasite growth, independently of their fine specificity for the RAP-1 sequences at positions 33 to 42, 213 to 222, 243 to 247, 280 to 287, or 405 to 446. The growth-inhibitory activity of MAb SP8.18 was, however, accelerated by noninhibitory anti-RAP-1 MAbs. Results demonstrate that in addition to fine specificity, other binding parameters are also crucial for the inhibitory potential of an antibody.

Antigenicity of recombinant proteins derived from rhoptry-associated protein 1 of Plasmodium falciparum.

Rhoptry-associated protein 1 (RAP1) of Plasmodium falciparum is a potential component of a malaria vaccine. We have expressed in Escherichia coli eight recombinant RAP1 proteins representing almost the entire sequence of the mature protein and assessed the antigenicity of the proteins by immunization of mice. Antisera to six of the recombinant proteins reacted specifically with parasite-derived RAP1 (PfRAP1), as determined by indirect immunofluorescence and by immunoblotting. These proteins were then used in enzyme-linked immunosorbent assays to evaluate human antibody responses to RAP1 during naturally transmitted infections in The Gambia. Immunoglobulin G (IgG) antibodies specifically reactive with the recombinant RAP1 proteins are directed mostly towards fragments containing the N-terminal sequences of mature PfRAP1. The most N-terminal segment (residues 23 to 175) contains only minor epitopes, while major epitopes are outside this region. Antibodies from malaria patients do not compete for a linear epitope recognized by an inhibitory anti-RAP1 monoclonal antibody. Analysis of IgG subclass distribution shows that human anti-RAP1 antibodies are predominantly IgG1.

The toxicity of the Alzheimer's beta-amyloid peptide correlates with a distinct fiber morphology.

In an attempt to elucidate the relationship among aggregation properties, fiber morphology, and cellular toxicity several beta-amyloid peptides (A beta) were prepared according to a standardized procedure. Peptides either carried mutations inside the membrane anchor segment around amino acid position 35 or their carboxy terminus was shortened from 42 to 41, 40, or 39 amino acids. The time-dependent self-assembly of monomeric A beta into fibers was simultaneously monitored by electron microscopy, circular dichroism spectroscopy, analytical ultracentrifugation, and A beta-mediated cellular toxicity using the reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to measure cell viability. The transition of A beta monomers into fibers was analyzed by more than 600 electron micrographs. Distinct morphological changes from seed-like structures to immature and mature fibers were observed. Seeds were of spherical appearance. Immature fibers were typically elongated structures with a rough surface and with varying thickness depending on the A beta sequence. Mature fibers were characterized by a periodic variation of their thickness along the fiber axis. The proportion of these different structures and the total amount of aggregated A beta was amino acid sequence-dependent. Wild-type A beta 1-42 and its oxidized derivative carrying a methionine sulfoxide residue at position 35 showed the highest rate of fiber formation and exerted toxic activity in the MTT assay at very low nanomolar concentrations. The fibers formed by these two peptides were predominantly of the mature type. In contrast, carboxyl-terminus truncated peptides A beta 1-41, A beta 1-40, and A beta 1-39 or most A beta 1-42 derivatives mutated around amino acid position 35 showed a reduced aggregation rate, the immature fibers predominated, and the toxicity was orders of magnitude lower. Thus, a correlation can be drawn among the chemical structure, aggregation properties, fiber morphology, and cellular toxicity.

Structure and function of the dihydropteroate synthase from Staphylococcus aureus.

The gene encoding the dihydropteroate synthase of staphylococcus aureus has been cloned, sequenced and expressed in Escherichia coli. The protein has been purified for biochemical characterization and X-ray crystallographic studies. The enzyme is a dimer in solution, has a steady state kinetic mechanism that suggests random binding of the two substrates and half-site reactivity. The crystal structure of apo-enzyme and a binary complex with the substrate analogue hydroxymethylpterin pyrophosphate were determined at 2.2 A and 2.4 A resolution, respectively. The enzyme belongs to the group of "TIM-barrel" proteins and crystallizes as a non-crystallographic dimer. Only one molecule of the substrate analogue bound per dimer in the crystal. Sequencing of nine sulfonamide-resistant clinical isolates has shown that as many as 14 residues could be involved in resistance development. The residues are distributed over the surface of the protein, which defies a simple interpretation of their roles in resistance. Nevertheless, the three-dimensional structure of the substrate analogue binary complex could give important insight into the molecular mechanism of this enzyme.

A single amino acid substitution in Staphylococcus aureus dihydrofolate reductase determines trimethoprim resistance.

A single amino acid substitution, Phe98 to Tyr98, in dihydrofolate reductase (DHFR) is the molecular origin of trimethoprim (TMP) resistance in Staphylococcus aureus. This active site amino acid substitution was found in all S. aureus TMP-resistant clinical isolates tested. In order to explore the structural role of Tyr98 in TMP-resistance the ternary complexes of the chromosomal S. aureus DHFR (SaDHFR) with methotrexate (MTX) and TMP in the presence of nicotinamide adenine dinucleotide phosphate (NADPH) as well as that of mutant Phe98Tyr DHFR SaDHFR(F98Y) ternary folate-NADPH complex have been determined by X-ray crystallography. Critical evidence concerning the resistance mechanism has also been provided by NMR spectral analyses of 15N-labelled TMP in the ternary complexes of both wild-type and mutant enzyme. These studies show that the mutation results in loss of a hydrogen bond between the 4-amino group of TMP and the carbonyl oxygen of Leu5. This mechanism of resistance is predominant in both transferable plasmid-encoded and non-transferable chromosomally encoded resistance. Knowledge of the resistance mechanism at a molecular level could help in the design of antibacterials active against multi-resistant Staphylococcus aureus (MRSA), one of todays most serious problems in clinical infectology.

Automated specific IgE assay with recombinant allergens: evaluation of the recombinant Aspergillus fumigatus allergen I in the Pharmacia Cap System.

BACKGROUND: We report the results of a study comparing the recombinant Aspergillus fumigatus allergen 1 (rAsp f I) to commercial A. fumigatus extracts in serological assays. Pharmacia CAP System and skin tests. OBJECTIVE: The study was designed to test the feasibility of using recombinant allergens in an automated serology system for determination of allergen-specific IgE. METHODS: Patients with allergic bronchopulmonary aspergillosis (ABPA), asthmatics with A. fumigatus allergy and control subjects, who included allergic asthmatics without allergy to A. fumigatus and healthy subjects, were investigated. All subjects were characterized with respect to their total IgE level, radio allergosorbent test to A. fumigatus and skin test reactivity to both commercial A. fumigatus extracts and recombinant rAsp f I protein. RESULTS: All patients with ABPA (n = 30) showed positive skin test reactions with commercial A. fumigatus extracts, and 24 were sensitized to rAsp f I by the same criterion. The 10 patients with asthma and A. fumigatus allergy showed positive skin reactions to at least one commercial extract, and five reacted to rAsp f I. All control subjects (n = 19) scored negatively in skin tests to A. fumigatus extracts and rAsp f I and showed no detectable rAsp f I-specific IgE. ImmunoCAP carrying immobilized rAsp f I were evaluated using sera from all individuals described and the results compared with those obtained with the rAsp f I-specific ELISA for IgE. The data obtained with the two rAsp f I-specific detection systems correlated closely (r = 0.997) and were in perfect agreement with the skin test results. CONCLUSION: The data show that rAsp f I can be used as immobilized allergen in the Pharmacia CAP System indicating the feasibility of using recombinant allergens for an automated serological diagnosis of allergic diseases. However, every recombinant allergen needs to be evaluated individually for its performance if applied to a new diagnostic technology.

The monofunctional glycosyltransferase of Escherichia coli is a member of a new class of peptidoglycan-synthesising enzymes.

Using conserved fingerprints in the glycosyltransferase (GTase) domain of high-molecular-weight penicillin-binding proteins (PBP), a gene (mgt) encoding a putative monofunctional glycosyltransferase has been identified in Haemophilus influenzae and in other bacteria] species. Here we report the cloning of the homologous Escherichia coli gene and show that the solubilised membrane fraction of E. coli cells overexpressing the mgt gene contain a significantly increased peptidoglycan synthesis activity. In contrast to the high-molecular-weight PBPs, this activity is not inhibited by Flavomycin.

The calcium-binding protein calretinin-22k, an alternative splicing product of the calretinin gene is expressed in several colon adeno carcinoma cell lines.

An alternatively spliced mRNA for the calcium-binding protein calretinin (CR) is present in the colon adenocarcinoma cell line WiDr. As a consequence of a frame shift, the resulting protein, calretinin-22k (CR-22k), consists of the first 178 amino acids of calretinin followed by a carboxy-terminal peptide of 14 amino acids that is not present in full-length calretinin. Antibodies specific for this C-terminal region have been generated by 2 different methods. A peptide corresponding to the specific C-terminal region of CR-22k was either chemically synthesized and coupled to a carrier protein or was expressed in Escherichia coli as a carboxyterminal fusion to a carrier protein applying recombinant techniques. Both antisera produced in rabbits were tested in Western blots and immuno-histochemical experiments. The antisera recognized human recombinant CR-22k overexpressed in E. coli, but not fulllength calretinin and stained fixed WiDr cells. The presence of CR-22k was also confirmed in the colon cell lines CO115/3 in which mRNA coding for CR-22k mRNA coding for CR-22k mRNA is present as well as in the lines COLO205 and LS-180, all of which also express full-length calretinin. Although the intracellular distribution of CR-22k and CR are similar as evidenced by immunohistochemical stainings, CR-22k is preferentially localized in the nucleus in the cell lines LS-180 and Co115/3 suggesting potentially different roles for the two proteins.

A human tumor necrosis factor p75 receptor agonist stimulates in vitro T cell proliferation but does not produce inflammation or shock in the baboon.

Tumor necrosis factor (TNF) is a potentially useful adjunct to anticancer therapies. However, the clinical utility of TNF has been limited by generalized toxicity and hypotension. Recently, studies have begun to dissect the individual proinflammatory and immunologic responses that result from TNF binding to its two cellular receptors, p55 and p75, in an attempt to develop TNF receptor agonists with reduced systemic toxicity. To evaluate a p75 receptor selective TNF mutant (p75TNF), TNF and p75TNF were administered to healthy anesthetized baboons. Intravenous infusion of the p75TNF produced none of the hemodynamic changes seen after the infusion of TNF. Infusion of p75TNF also failed to induce the plasma appearance of interleukins 6 and 8. However, p75TNF enhanced in vitro baboon thymocyte proliferation to concanavalin A, and infusion of p75TNF resulted in increased soluble p55 and p75 receptor plasma concentrations. Local skin necrosis and tissue neutrophil infiltration were seen after subcutaneous injections of TNF and p55TNF. Subcutaneous injection of p75TNF did not result in skin necrosis but did result in a modest dermal infiltration of lymphocytes and macrophages. The findings suggest that p75TNF may stimulate T cell proliferation without the systemic and local toxicity seen with TNF.

A biotechnological method provides access to aggregation competent monomeric Alzheimer's 1-42 residue amyloid peptide.

Senile plaques, a neuropathological hallmark of Alzheimer's disease, consist primarily of insoluble aggregates of beta-amyloid peptide (A beta). A 42-residue peptide (A beta 1-42) appears to be the predominant form. In contrast to A beta 1-40, A beta 1-42 is characterized by its extreme tendency to aggregate into fibers or precipitate. A tailored biotechnological method prevents aggregation of A beta 1-42 monomers during its production. The method is based on a protein tail fused to the amino terminus of A beta. This tail leads to a high expression in E. coli, and a histidine affinity tag facilitates purification. Selective cleavage of the fusion tail is performed with cyanogen bromide by immobilizing the fusion protein on a reversed phase chromatography column. Cleavage then occurs only at the methionine positioned at the designed site but not at the methionine contained in the membrane anchor sequence of A beta. Furthermore, immobilization prevents aggregation of cleaved A beta. Elution from the HPLC column and all succeeding purification steps are optimized to preserve A beta 1-42 as a monomer. Solutions of monomeric A beta 1-42 spontaneously aggregate into fibers within hours. This permits the investigation of the transition of monomers into fibers and the correlation of physico-chemical properties with biological activities. Mutations of A beta 1-42 at position 35 influence the aggregation properties. Wild-type A beta 1-42 with methionine at position 35 has similar properties as A beta with a methionine sulfoxide residue. The fiber formation tendency, however, is reduced when position 35 is occupied by a glutamine, serine, leucine, or a glutamic acid residue.

Cloning and characterization of a novel, plasmid-encoded trimethoprim-resistant dihydrofolate reductase from Staphylococcus haemolyticus MUR313.

In recent years resistance to the antibacterial agent trimethoprim (Tmp) has become more widespread, and several trimethoprim-resistant (Tmpr) dihydrofolate reductases (DHFRs) have been described from gram-negative bacteria. In staphylococci, only one Tmpr DHFR has been described, the type S1 DHFR, which is encoded by the dfrA gene found on transposon Tn4003. In order to investigate the coincidence of high-level Tmp resistance and the presence of dfrA, we analyzed the DNAs from various Tmpr staphylococci for the presence of dfrA sequences by PCR with primers specific for the thyE-dfrA genes from Tn4003. We found that 30 or 33 isolates highly resistant to Tmp (MICs, > or = 512 micrograms/ml) contained dfrA sequences, whereas among the Tmpr (MICs, < or = 256 micrograms/ml) and Tmps isolates only the Staphylococcus epidermidis isolates (both Tmpr and Tmps) seemed to contain the dfrA gene. Furthermore, we have cloned and characterized a novel, plasmid-encoded Tmpr DHFR from Staphylococcus haemolyticus MUR313. The dfrD gene of plasmid pABU17 is preceded by two putative Shine-Dalgarno sequences potentially allowing for the start of translation at two triplets separated by nine nucleotides. The predicted protein of 166 amino acids, designated S2DHFR, encoded by the longer open reading frame was overproduced in Escherichia coli, purified, and characterized. The molecular size of the recombinant S2DHFR was determined by ion spray mass spectrometry to be 19,821.2 +/- 2 Da, which is in agreement with the theoretical value of 19,822 Da. In addition, the recombinant S2DHFR was shown to exhibit DHFR activity and to be highly resistant to Tmp.

Characterization of the gene for the chromosomal dihydrofolate reductase (DHFR) of Staphylococcus epidermidis ATCC 14990: the origin of the trimethoprim-resistant S1 DHFR from Staphylococcus aureus?

The gene for the chromosomally encoded dihydrofolate reductase (DHFR) of Staphylococcus epidermidis ATCC 14990 has been cloned and characterized. The structural gene encodes a polypeptide of 161 amino acid residues with a calculated molecular weight of 18,417. This trimethoprim-sensitive (Tmps) DHFR, SeDHFR, differs in only three amino acids (Val-31-->Ile, Gly-43-->Ala, and Phe-98-->Tyr) from the trimethoprim-resistant (Tmpr) S1 DHFR encoded by transposon Tn4003. Since in addition the S. epidermidis gene also forms part of an operon with thyE and open reading frame 140 as in Tn4003, the chromosomally located gene encoding the Tmps SeDHFR is likely to be the molecular origin of the plasmid-located gene encoding the Tmpr S1 DHFR. Site-directed mutagenesis and kinetic analysis of the purified enzymes suggest that a single Phe-->Tyr change at position 98 is the major determinant of trimethoprim resistance.

Improving protein solubility through rationally designed amino acid replacements: solubilization of the trimethoprim-resistant type S1 dihydrofolate reductase.

In recent years resistance to the antibacterial agent trimethoprim (Tmp) has become more widespread and several Tmp-resistant (Tmpr) dihydrofolate reductases (DHFRs) have been described from Gram-negative bacteria. In staphylococci, however, only one Tmpr DHFR (type S1 DHFR) has been found so far, and this is located on transposon Tn4003. To help understand the mechanism of resistance, we are interested in determining the 3-D structure of the recombinant enzyme produced in Escherichia coli. However, the production level of the type S1 DHFR was very low and > 95% of the total recombinant protein accumulated in inclusion bodies. Furthermore, as a result of an internal start of translation, a truncated derivative of the enzyme that copurified with the full-length enzyme was produced. We were able to increase the expression level 20-fold by changing 18 N-terminal codons and to eliminate the internal start of translation. In addition, through molecular modelling and subsequent site-directed mutagenesis to replace two amino acids, we constructed a biochemically similar but soluble derivative of the type S1 DHFR that, after production in E.coli, resulted in a 264-fold increase in DHFR activity. The highly overproduced enzyme was purified to homogeneity, characterized biochemically and crystallized.

A human tumor necrosis factor (TNF) alpha mutant that binds exclusively to the p55 TNF receptor produces toxicity in the baboon.

A number of recent studies have demonstrated that cellular responses to tumor necrosis factor (TNF) mediated by the p55 and the p75 TNF receptors are distinct. To evaluate the relative in vivo toxicities of wild-type TNF alpha (wtTNF alpha) and a novel p55 TNF selective receptor agonist, healthy, anesthetized baboons (Papio sp.) were infused with a near-lethal dose of either wtTNF alpha or a TNF alpha double mutant (dmTNF alpha) that binds specifically to the p55, but not to the p75, TNF receptor. Both wtTNF alpha and dmTNF alpha produced comparable acute hypotension, tachycardia, increased plasma lactate, and organ dysfunction in Papio. However, administration of wtTNF alpha produced a marked granulocytosis and loss of granulocyte TNF receptors, whereas little if any changes in neutrophil number or cell surface TNF receptor density were seen after dmTNF alpha mutant administration. Infusion of dmTNF alpha resulted in a plasma endogenous TNF alpha response that peaked after 90-120 min. We conclude that selective p55 TNF receptor activation is associated with early hemodynamic changes and the autocrine release of endogenous TNF alpha. Significant systemic toxicity results from p55 TNF receptor activation, but the role of the p75 TNF receptor in systemic TNF toxicity requires further study.

Characterization of the gene for chromosomal trimethoprim-sensitive dihydrofolate reductase of Staphylococcus aureus ATCC 25923.

The gene for the trimethoprim-sensitive (Tmps) chromosomal dihydrofolate reductase (DHFR) of Staphylococcus aureus ATCC 25923 was cloned and characterized. The structural gene encodes a polypeptide of 159 amino acid residues and has a calculated molecular weight of 18,251. The amino acid sequences of this Tmps DHFR and those of the trimethoprim-resistant type S1 DHFR encoded by transposon Tn4003 are 80% identical. In contrast to the trimethoprim-resistant enzyme, the Tmps DHFR can be highly overexpressed in Escherichia coli, with most of the recombinant protein occurring in a soluble and an active form.

Rat brain glyceraldehyde-3-phosphate dehydrogenase interacts with the recombinant cytoplasmic domain of Alzheimer's beta-amyloid precursor protein.

Abundant senile plaques are a histological hallmark in the brain of Alzheimer's disease patients. Such plaques consist of, among many other constituents, aggregated beta A4 amyloid peptide. This peptide is derived from an amyloid precursor protein (APP) by irregular proteolytic processing and is considered to be involved in the development of Alzheimer's disease. To study possible interactions of brain proteins with beta A4 amyloid or other fragments of APP, beta A4 amyloid and beta A4 amyloid extended to the C-terminus of APP were recombinantly produced as fusion proteins termed "Amy" and "AmyC," respectively. Using Amy and AmyC affinity chromatography, a 35-kDa protein from rat brain was isolated that bound tightly to AmyC but not to Amy, thus indicating an interaction of the protein with the C-terminus of APP. This 35-kDa protein was identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Binding of GAPDH to AmyC but not to Amy was confirmed by gel filtration. Although AmyC slightly reduced the Vmax of GAPDH, the same reduction was observed in the presence of Amy. These findings suggest that the interaction of the cytoplasmic domain of APP with GAPDH is unlikely to influence directly the rate of glycolysis but may serve another function.

Alignment of disulfide bonds of the extracellular domain of the interferon gamma receptor and investigation of their role in biological activity.

The extracellular ligand binding domain of the human interferon gamma receptor includes eight cysteine residues forming four disulfide bonds. Only the nonreduced protein binds interferon gamma. We investigated the alignment of the disulfide bonds, using an enzymatically deglycosylated form of a soluble interferon gamma receptor, produced in baculovirus-infected insect cells. The soluble receptor was digested with endoproteinase Glu-C and proteinase K, and the proteolytic fragments were characterized by amino acid sequence analysis and mass spectrometry. It was found that four consecutive disulfide bonds are formed between residues Cys60-Cys68, Cys105-Cys150, Cys178-Cys183, and Cys197-Cys218. We also investigated the role of the disulfide bonds in biological activity of the receptor, using site-directed mutagenesis and by exchanging the cysteine residues for serines. The mutated proteins were expressed in Escherichia coli and analyzed for ligand binding capacity on protein blots. The assays showed that all disulfide bonds are essential for full ligand binding capacity. Double or quadruple mutations at cysteine residues 60 and 68, and residues 178, 183, 197, and 218, respectively, resulted in complete loss of the activity, whereas double mutations at residues 105 and 150, 178 and 183, and 197 and 218, respectively, resulted in a residual activity about 1 order of magnitude lower than that of the wild type. The specific antibodies gamma R38 and gamma R99 detected conformational epitopes stabilized by disulfide bonds involving cysteine residues 60 and 68, and 178 and 183, respectively.

Expression of the trimethoprim resistant dihydrofolate reductase encoded by transposon TN4003 in a soluble form and its subsequent purification to homogeneity.

A high level expression in E. coli of the Tmpr type S1 DHFR was achieved by: (1) elimination of an internal start of translation within the RNA, and (2) optimization of gene expression by replacing nucleotides at the 5' end of the gene by nucleotides present in the highly expressible gene for SaDHFR. In addition, by replacing amino acids supposed to be on the surface of the protein, the mutein S1 DHFR[N48E,N130D] was constructed, which can be expressed in E. coli to high levels in a soluble and active form. The mutein S1 DHFR[N48E,N130D] was purified nearly to homogeneity. The enzyme is highly active and remains soluble even at a protein concentration of 10 mg/ml.