ABSTRACT
Carbohydrate epitopes are capable of binding human IgE from allergic subjects and these epitopes play a role in the cross-reactivity between allergens from unrelated sources. A monoclonal antibody (5E6), specific for a carbohydrate epitope detectable on components of Cupressus arizonica pollen extract, has been produced and characterized. To study the relationship between the epitopes recognized by the monoclonal antibody and by IgE from allergic subjects. To investigate the presence of such carbohydrate IgE determinant in extracts from 21 pollen species belonging to 16 taxonomically related and unrelated families, by means of the monoclonal antibody. IgG-depleted fraction from protein G-purified human allergic serum was obtained. The monoclonal antibody and the IgE from the purified fraction were tested on two glycoproteins, polyamine oxidase and ascorbate oxidase, adsorbed on the ELISA plates. The relationship between the monoclonal- and the IgE-recognized epitopes was investigated by ELISA-competition experiments. Analysis of the distribution of this carbohydrate epitope was performed by direct binding of the monoclonal antibody onto the various extracts. The monoclonal antibody and the IgE were able to bind carbohydrate epitopes on the two plant glycoproteins, ascorbate oxidase and polyamine oxidase. Polyamine oxidase shows only one N-glycosilation site whose carbohydrate moiety seems to be composed of a branched chain of seven ordered sugars, i.e. two N-acetyl-D-glucosamine-, three mannose-, one fucose- and one xylose-residues. This structure bears the epitope recognized by mAb 5E6. Human IgE from the IgG-depleted fraction were found capable of inhibiting the monoclonal antibody binding. The allergenic epitope identified was shared by a large number of extracts with different levels of reactivity (OD490 ranging from 0.110 to 2.060). Our data support the finding that a monoclonal antibody specific for a carbohydrate epitope of Cupressus arizonica pollen extract detects an epitope which is also recognized by IgE from allergic subjects. This characterized reagent could be a useful tool for studying distribution of cross-reactive carbohydrate determinants in allergenic pollen extracts and their components.
Subject(s)
Allergens/immunology , Antibodies, Monoclonal/immunology , Carbohydrates/immunology , Immunoglobulin E/immunology , Pollen/immunology , Animals , Antibody Specificity , Humans , Immunodominant Epitopes/immunology , MiceABSTRACT
The gene coding for nitrite reductase of Pseudomonas aeruginosa has been cloned and its sequence determined. The coding region is 1707 bp long and contains information for a polypeptide chain of 568 amino acids. The sequence of the mature protein has been confirmed independently by extensive amino acid sequencing. The amino-terminus of the mature protein is located at Lys-26; the preceding 25 residue long extension shows the features typical of signal peptides. Therefore the enzyme is probably secreted into the periplasmic space. The mature protein is made of 543 amino acid residues and has a molecular mass of 60,204 Da. The c-heme-binding domain, which contains the only two Cys of the molecule, is located at the amino-terminal region. Analysis of the protein sequence in terms of hydrophobicity profile gives results consistent with the fact that the enzyme is fully water soluble and not membrane bound; the most hydrophilic region appears to correspond to the c-heme domain. Secondary structure predictions are in general agreement with previous analysis of circular dichroic data.
Subject(s)
Genes, Bacterial , Genes , NADH, NADPH Oxidoreductases/genetics , Nitrite Reductases/genetics , Pseudomonas aeruginosa/enzymology , Amino Acid Sequence , Amino Acids/isolation & purification , Base Sequence , Molecular Sequence Data , Nitrite Reductases/isolation & purification , Peptides/isolation & purification , Pseudomonas aeruginosa/genetics , Restriction MappingABSTRACT
The complete amino acid sequence was determined for the Cu,Zn superoxide dismutase from the shark Prionace glauca. The active site region shows the substitution of an Arg for Lys at position 134, which is important for electrostatic facilitation of the diffusion of O2- to the catalytically active copper. This change may be related to observed alterations of electrostatic parameters of the enzyme (pK of the pH dependence of the enzyme activity, rate of inactivation by H2O2), although it preserves a high efficiency of dismutation at neutral pH.
Subject(s)
Arginine , Lysine , Sharks/metabolism , Superoxide Dismutase/metabolism , Amino Acid Sequence , Animals , Binding Sites , Cattle , Electrochemistry , Fishes , Molecular Sequence Data , Protein Conformation , Sequence Homology, Nucleic Acid , Species Specificity , Superoxide Dismutase/geneticsABSTRACT
The presence of new hypotensive peptides, possibly not related to ACE inhibition, has been investigated on 66 snake venoms from crotalid, viperid and elapid families. Only the venom of Crotalus atrox showed a substantial amount of a new decapeptide, called POL-236, with the following aminoacid sequence: PYR-LEU-TRP-PRO-ARG-PRO-GLN-ILE-PRO-PRO. Pharmacological assays performed on the synthesized peptide revealed effects on blood pressure, probably derived from vascular and cardiac interferences.
