A major antigen of Alternaria alternata with potential for safe and effective immunotherapy.
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Alternaria↗
Biomedical subjects
N M Young
Publications and source records attributed to N M Young.
Purification and characterization of Alt a-29 from Alternaria alternata.
A major protein component reactive with pooled human atopic sera was isolated from a lyophilized broth extract of Alternaria alternata 34-016. By successive chromatography on Whatman DE-52, Sephadex G-100 and Mono Q HR5/5, a low molecular weight antigen was obtained. Comparison with standard proteins on Sephadex G-100 indicated its molecular weight was 31 kD. Non-reduced samples run on SDS-PAGE showed a band at 29.2 kD which reacted strongly with human IgE. After reduction, it produced a doublet pattern on SDS-PAGE with MW 14.5 and 16.0 kD. The doublet pattern was confirmed by Western blotting with pooled human atopic sera. IEF of the protein showed a major component with a PI of 4.15 and two minor components at 4.25 and 4.40. Immunoblots of the IEF bands showed all three were reactive with human IgE. Ion exchange chromatography of the protein on Mono Q HR5/5 resulted in three resolved components, all of which are immunoreactive. Together with the IEF data, this suggests that there are several conformational or structural isoforms of this protein.
Allergens↗
Analysis of sequence variation among legume lectins. A ring of hypervariable residues forms the perimeter of the carbohydrate-binding site.
Twelve plant lectins from the Papilionoideae subfamily were selected to represent a range of carbohydrate specificities, and their sequences were aligned. Two variability indices were applied to the aligned sequences and the results were analysed using the three-dimensional structures of concanavalin A and the pea lectin. The areas of greatest variability were located in the carbohydrate-binding site region, forming a perimeter around a well-conserved core. These residues are inferred to be specificity determining, in the manner of antibodies, and the most variable position corresponded to Tyr100 in concanavalin A, a known ligand contact residue. In addition to the five peptide loops known to form the binding site from crystallographic studies, a sixth segment with variable residues was located in the binding-site region, and this may contribute to oligosaccharide specificity. In their overall composition, the lectin sites resemble those of the sugar-transport proteins rather than antibodies. The prospects for modelling lectin binding sites by the methods used for antibodies were also assessed.
Amino Acid Sequence↗
Thiol groups and other chemical characteristics of rat monoclonal immunoglobulin A.
1. Three aspects of the monoclonal rat IgA IR22 were investigated: its free thiol groups, its hinge structure and its glycopeptides. 2. Exposed thiol groups, 1.5 per IgA dimer, were located in the Fc region, and buried thiol groups, 3.2 per IgA dimer, were located in the Fd region. 3. Rat IgA was cleaved to Fab fragments by pepsin, but, unlike mouse IgA, it was not cleaved at the hinge region by papain. 4. The contents of GlcNAc indicated that there were two N-linked glycopeptides in the Fab region, and one in the Fc or tail regions.
Amino Sugars↗
Bacterial expression and secretion of various single-chain Fv genes encoding proteins specific for a Salmonella serotype B O-antigen.
Active single-chain Fv molecules encoded by synthetic genes have been expressed and secreted to the periplasm of Escherichia coli using the ompA secretory signal. Four different constructs were developed to investigate the effects of peptide linker design and VL-VH orientation on expression, secretion, and binding to a Salmonella O-polysaccharide antigen. Peptide linker sequences derived from the elbow regions of the Fab molecule were used alone or in combination with the flexible (GGGGS)2 sequence. VL and VH domain order in the single chain molecules had a profound effect on the level of secretion but hardly influenced total expression levels, which were approximately 50 mg/liter, chiefly in the form of inclusion bodies. With VL in the NH2-terminal position, the amount of secreted product obtained was 2.4 mg/liter, but when VH occupied this position the yield was less than 5% of this value. Enzyme immunoassays of the four products showed domain order and linker sequence affected antigen binding by less than an order of magnitude. Attempts to express active Fv from dicistronic DNA were unsuccessful, but active Fv was obtained from single-chain Fv by enzymic cleavage at a site in the elbow linker peptide. The thermodynamic binding parameters of intact and cleaved single-chain Fvs determined by titration microcalorimetry were similar to those of bacterially produced Fab and mouse IgG.
Amino Acid Sequence↗
The amino acid sequences of jacalin and the Maclura pomifera agglutinin.
Amino acid sequences for the alpha-chains of the Moraceae lectins, jacalin and Maclura pomifera agglutinin, were determined by protein sequencing. Both are 133 residues long and contain several genetically variant positions; the overall homology is 85%. A possible site for the known glycopeptide of jacalin was located. The alpha-chains have a conserved tryptophan residue that may be part of the binding-site.
Amino Acid Sequence↗
The amino acid sequence of peanut agglutinin.
The amino acid sequence of peanut (Arachis hypogaea) agglutinin was determined from three major fragments obtained by mild acid cleavage at Asp-Pro peptide bonds. The sequence of 236 amino acids has residues identical to those that form the metal-binding site and the hydrophobic pocket in concanavalin A and other lectins, although the overall similarity is only 42%. In the segments of peanut agglutinin that correspond to the four loops that form the carbohydrate-binding site in concanavalin A and favin, several central residues are homologous, while others show changes to smaller side chains, such as Tyr----Gly. The carbohydrate-binding site of peanut agglutinin may therefore have a similar peptide-backbone architecture, but form a considerably more open cleft.
Amino Acid Sequence↗
Synthesis and expression in Escherichia coli of cistronic DNA encoding an antibody fragment specific for a Salmonella serotype B O-antigen.
A 1460-bp DNA encoding the two chains of the antigen-binding fragment (Fab) portion of a monoclonal antibody have been chemically synthesized and expressed in Escherichia coli. The antibody, Se155-4, is specific for a Salmonella serogroup B O-antigen and its crystal structure is under investigation. The genes were synthesized according to a strategy that allows for easy manipulation in genetic engineering studies of the Fab-binding site. Each gene is preceded by the ompA secretory signal and a ribosome-binding site, and has been expressed from the two-cistron DNA under the control of the lac promoter. Active Fab of 50 kDa with an inter-chain disulfide bond has been isolated from the periplasm of E. coli in a one-step affinity purification in high yield (2 micrograms/ml of cells). The bacterially produced Fab is as active as purified mouse Fab in antigen-binding and competitive immunoassays. This is the first example of a completely synthetic Fab gene and provides an ideal system to probe the nature of antigen binding by anti-carbohydrate antibodies.
Amino Acid Sequence↗
Molecular modeling of antibody-antigen complexes between the Brucella abortus O-chain polysaccharide and a specific monoclonal antibody.
A molecular model of the binding site of an anti-carbohydrate antibody (YsT9.1) has been developed using computer-assisted modeling techniques and molecular dynamics calculations. Sequence homologies among YsT9.1 and the Fv regions of McPC603, J539 and human Bence--Jones protein REI, all of which have solved crystal structures, provided the basis for the modeling. The groove-type combining site model had a topography which was complementary to low energy conformers of the polysaccharide, a Brucella O-antigen, and the site could be almost completely filled by a pentasaccharide epitope in either of two docking modes. Putative interactions between this epitope and the antibody are consistent with the known structural requirements for binding and lead to the design of oligosaccharide inhibitors that probe the veracity of the modeled docked complex. Ultimately both the Fv model and the docked complex will be compared with independent crystal structures of YsT9.1 Fab with and without pentasaccharide inhibitor, currently at the stage of refinement.
Amino Acid Sequence↗
Mycobacterium paratuberculosis antigen D: characterization and evidence that it is a bacterioferritin.
By using a combination of agarose and polyacrylamide gel electrophoresis, Mycobacterium paratuberculosis antigen D was resolved from a crude sonicated preparation of the organism and characterized as a component with a molecular mass of approximately 400,000 Da. While this component was composed mainly of protein, with unusually high proportions of glutamic acid and leucine, it was resistant to digestion with a number of proteolytic enzymes. Structural detail revealed by electron microscopy, amino acid sequence data, and the demonstration of a Soret band in its absorption spectrum indicated that antigen D was similar to an Escherichia coli bacterioferritin.
Amino Acid Sequence↗
Chromatographic purification and characterization of antigens A and D from Mycobacterium paratuberculosis and their use in enzyme-linked immunosorbent assays for diagnosis of paratuberculosis in sheep.
The protein antigens A and D were purified from culture filtrates and sonic extracts of laboratory strains of Mycobacterium paratuberculosis by salt precipitation and chromatography. The characterization of antigen A is shown here, and both antigens were evaluated along with lipoarabinomannan antigen in indirect enzyme-linked immunosorbent assays (ELISA) for the serodiagnosis of ovine paratuberculosis. After anion-exchange (DEAE-5PW) and hydrophobic (phenyl-5PW) chromatography using high-performance liquid chromatography, antigen A showed a prominant band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 31 kDa with small amounts of low-molecular-mass proteins but with no evidence of antigen D. A single precipitin arc was evident with purified antigen A in crossed immunoelectrophoresis. The determination of the N-terminal amino acid sequence showed a high degree of homology between the 31-kDa component of antigen A and antigens of the BCG85 complex of Mycobacterium bovis BCG, a total of 24 of 26 residues being identical to those of BCG85C. A prominant SDS-PAGE band at 400 kDa and a single crossed-immunoelectrophoresis arc was also evident for antigen D after gel filtration (Sephacryl S-200), anion-exchange (DEAE-Sephacel), and concanavalin A-Sepharose affinity chromatography. By ELISA, purified antigen A detected antibody in the sera of 18 of 22 paratuberculosis-infected sheep (82% sensitivity), whereas the purified antigen D detected antibody in all 22 infected animals (100% sensitivity). Combined ELISA results showed increased specificity with some loss in sensitivity.
Amino Acid Sequence↗
Scanning of key residues in antibody binding sites by two saturation-mutagenesis approaches.
The complementarity-determining region 3 of the heavy chain (CDRH3) generally contributes the most to antibody-antigen binding. His101H in CDRH3 of the antibody Se155-4, which is specific for a trisaccharide epitope of Salmonella serotype B O-antigen, was mutated systematically into all nineteen other amino acids by a double mutation approach. Enzyme immunoassay (EIA) and affinity chromatography showed that the Asn, Gln, Gly and Ser mutants exhibited moderate to strong activity. Some mutants, such as Thr and Pro, had weak binding activity, while the acidic and hydrophobic amino acid substitutions resulted in complete loss of activity. A second mutation approach which randomly changed a selected residue into all other nineteen amino acids, while precluding wild-type transformants, is also described.
Amino Acid Sequence↗
O-antigen biotin conjugates. Preparation and use in direct competitive enzyme immunoassays.
Bacterial O-antigens coupled to biotin were shown to function well as labelled antigens in direct enzyme immunoassay. O-polysaccharides released from lipopolysaccharides by mild acid hydrolysis were oxidized by sodium periodate at sites located within the lipopolysaccharide inner core region and the generated aldehyde groups were subjected to reductive amination with 1,3-diaminopropane to yield per-aminated O-polysaccharide derivatives. Biotin was coupled to the introduced amino groups by way of a N-hydroxy-succinimide ester derivative. Biotinylated polysaccharides were used in direct enzyme immunoassays, that employed monoclonal antibody coated microtitre plates and detection of bound biotinylated antigen by streptavidin/horseradish peroxidase reagent. The assay format was designed for rapid estimation of the affinity constants of monoclonal antibodies for small oligosaccharide inhibitors, but the assay is also well suited to fast and sensitive detection of bacterial O-polysaccharides at concentrations within the range 1-10 ng/ml.
Antibodies, Monoclonal↗
The glycopeptides of the mouse immunoglobulin A T15.
Cleavage of mouse IgA T15 with papain yielded (a) a glycosylated Fab fragment, (b) a non-glycosylated Fc fragment and (c) a glycosylated C-terminal peptide. The cleavage sites at the hinge and at the end of the C alpha 3 domain were located by sequencing. The two glycopeptides were prepared from the Fab and C-terminal fragments by pronase digestion. The C alpha 1 glycopeptide at Asn 155 was complex type with alpha (1-3)galactose terminal groups, and closely resembled the Asn 171 glycopeptide of mouse IgM (Anderson et al. (1985) Arch. Biochem. Biophys. 243, 605-618). In contrast, the C-terminal glycopeptide at Asn 446 was entirely different from the corresponding IgM glycopeptide, being complex rather than high-mannose type.
Amino Acid Sequence↗
Synthesis and expression in Escherichia coli of DNA encoding the murine lambda 1 chain of a monoclonal antibody specific for Salmonella serotype B O-antigen.
A 658 bp DNA sequence corresponding to the murine lambda 1 chain of a monoclonal antibody, Se155-4, specific for the Salmonella serotype B O-antigen, was designed using Escherichia coli preferred codons and chemically synthesized by ligation of synthetic fragments into a linearized plasmid followed by transformation into E. coli. A synthetic signal peptide (ompA) was fused to express the L chain as a free polypeptide into the periplasm of E. coli cells. After isolation and purification, heterologous recombination of the E. coli L chain with mouse H chain gave an active antigen-binding protein. The activity was 15-20% when compared to protein created by an equivalent association of isolated natural mouse L and H chains as measured by a direct EIA assay. In inhibition experiments with the polysaccharide antigen, the two proteins showed identical titration curves and 50% inhibition points, indicating comparable KA values.
Amino Acid Sequence↗
Crystallization and preliminary X-ray diffraction studies of the complex of Maclura pomifera agglutinin with the disaccharide Gal beta 1-3GalNAc.
Single crystals of Maclura pomifera agglutinin, a seed lectin from the Moraceae family, complexed with the disaccharide Gal beta 1-3GalNAc have been obtained by the method of vapor diffusion with Li2SO4 as precipitant at pH 4.5. The crystals belong to the trigonal space group P3(1)21 or P3(2)21, with a = b = 67.4 A, c = 149.3 A. They contain two subunits per asymmetric unit and diffract beyond 2.7 A. This and other evidence indicate that both this lectin and the Artocarpus integrifolia lectin, jacalin, have dimeric structures rather than the tetrameric structures previously proposed.
Antigens, Surface↗
Homology of the D-galactose-specific lectins from Artocarpus integrifolia and Maclura pomifera and the role of an unusual small polypeptide subunit.
The Maclura pomifera agglutinin (MPA) was purified by affinity chromatography from a seed extract and its properties were compared with those of the Artocarpus integrifolia lectin, jacalin. Reverse-phase high-performance liquid chromatography showed both proteins had multiple forms of a small approximately 20-residue polypeptide chain in addition to the major 12,000 Mr subunit. The amino acid sequences of the small chains and the N-terminal sequences of the large subunits showed considerable similarity between the two proteins, approximately 60% identical residues. The homology of the proteins was confirmed by the similarity of their circular dichroism and fluorescence emission spectra. MPA showed much greater spectral changes upon binding methyl alpha-D-galactoside, suggesting it has complete activity rather than the partial activity found for jacalin. The binding of methyl alpha-D-galactoside by MPA was measured by fluorescence titration; the KA was 1.9 X 10(4) M-1 compared to 3.4 X 10(4) M-1 for jacalin. MPA also precipitated human IgA1 in the same manner as jacalin. The spectra indicate the involvement of tryptophan and tyrosine residues in the binding site of these lectins. Since a tryptophan residue is conserved in all the small subunits, they may form part of the binding site.
Amino Acid Sequence↗
Age considerations in acoustic neuroma surgery: the horns of a dilemma.
Experience has shown that early removal of acoustic tumors results in less morbidity because the rate of surgical complications increases with tumor size. Nevertheless, acoustic tumors are benign and grow slowly--facts that support conservatism in elderly patients. We managed 21 patients with acoustic tumors over the age of 65. Eleven patients underwent immediate surgical excision. Ten patients were monitored clinically and with serial magnetic resonance imaging (MRI) or computed tomographic (CT) scans. To date, two of these patients have required surgical excision because of continued tumor growth. Our experience managing these patients both surgically and with "watchful waiting" is the subject of this report.
Adolescent↗