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Notes: Two new crystal forms of isoenzyme 3-3 of rat liver glutathione S-transferase (GST 3-3) have been obtained. They were grown under essentially the same crystallization conditions as those reported for the C2 crystal form [Fu, Rose, Chung, Tam & Wang (1991). Acta Cryst. B47, 813–814]. The new crystals belong to space group P21 with one form having cell dimensions a = 101.6, b = 69.5, c = 81.4 Å, and β = 113.6°, and the other form having cell parameters a = 97.4, b = 81.1, c = 69.4 Å and β = 109.2°. These new crystals diffract to at least 2.5 Å, resolution. The molecular packing arrangements in these P21 crystals have been found by molecular replacement studies.

Notes: Five different crystal forms of recombinant chicken-liver glutathione S-transferase CL 3-3 have been obtained by the vapor-diffusion method. The form A crystals are monoclinic C2, a = 125.56, b = 85.81, c = 52.71 Å and β = 114.64°, and diffract to 4 Å resolution. The form B crystals are monoclinic P21, a = 105.13, b = 118.54, c = 62.62 Å and β = 124.74°, and diffract to 2.8 Å resolution. The form C crystals are orthorhombic C222l, a = 101.69, b = 115.46, c = 95.40 Å, and diffract to 2.8 Å resolution. The form D crystals are tetragonal, P41212 or P43212, a = b = 115.31, c = 171.20 Å and diffract to 3.5 Å resolution. The form E crystals are hexagonal, P61 or P65, a = b = 104.23, c = 114.35 Å, diffract to 3.5 Å resolution. Forms A, C and E have one dimer of molecular weight 50 kDa, while forms B and D have two dimers per asymmetric unit, respectively.

Notes: Background The 33 kD component has been identified as a major allergen ofPenicillium citrinum, the most prevalent Penicillium species in the Taipei area of Taiwan.Objective This study analyses the isoforms, antigenic cross-reactivity and the N-terminal amino acid sequence of the 33 kD allergen of P. citrinum.Methods The composition of isoforms and antigenic cross-reactivity was analysed by SDS-PAGE and 2D-immunobIotting using MoAbs generated. The N-terminal sequence was analysed by using an automatic gas/liquid phase sequencer.Results Two MoAbs (55A and 34H) against the 33 kD allergen were generated in the present study. In addition to the 33 kD component. MoAb 34H also showed immunoblot reactivity to other components in the crude extract of P. citrinum. Analysed by 2D-immunoblotting. at least six different isoforms of the 33 kD component with pl values ranging from 6.75 to greater than 7.0 were shown to be reactive to both MoAbs and IgE antibodies in serum of an asthmatic patient. Different immunoblot patterns were observed when both MoAbs were reacted with four different strains of P. citrinum used in the present study. Among another six different Penicillium and four different Aspergillus species tested, only an immunoblot reactivity of MoAb 55 A to the 33 kD component of P. brevicompactum was observed. In 2D-immunoblotting. components of P. brevicompactum with an MW of about 33 kD and pi values similar to those of the 33 kD component of P. citrinum reacted with MoAb 55A and IgE antibodies in serum of the asthmatic patient. The N-terminal amino acid sequence of the 33 kD component of P. citrinum was determined to be ANVVQSNVP which was identical to the first 9 N-terminal amino acids of a heat-labile alkaline serine proteinase from P. citrinum.Conclusion Results obtained in the present study suggest that the 33 kD major allergen of P. citrinum may be an alkaline serine proteinase.

Notes: Aspergillus species are common airborne fungi that have been identified as causative agents of extrinsic bronchial asthma. More than 10 allergens from A. fumigatus have been recently characterized by cDNA cloning.The objective of this study is to identify A. fumigatus allergens through immunoblot analysis using sera from asthmatic patients.IgE-binding components of A. fumigatus and IgE cross-reactivity among allergens of different prevalent airborne fungal species were analysed by immunoblot and immunoblot inhibition, respectively, using sera from asthmatic patients. The N-terminal amino acid sequences of major allergens identified were determined by Edman degradation.Among two batches (70 and 41 sera) of asthmatic sera tested, 19 (27%) and 14 (34%), respectively, have IgE immunoblot reactivity towards components of A. fumigatus. A 34-kDa protein that reacts with IgE antibodies in 15 (79%) and 11 (79%) of the 19 and 14 positive samples, respectively, may be considered a major allergen of A. fumigatus. The N-terminal amino acid sequences of the 34 kDa major allergen and the 30.5 and 30 kDa IgE-binding components of A. fumigatus showed sequence identity to that of the vacuolar serine proteinase from A. fumigatus. The results from immunoblot inhibition show IgE cross-reactivity among major allergens of A. fumigatus, P. notatum and P. oxalicum.Results obtained suggest that the 34 kDa major allergen of A. fumigatus may be a vacuolar serine proteinase. There is IgE cross-reactivity among serine proteinase allergens of A. fumigatus, P. notatum and P. oxalicum.

Notes: Background The group 7 mite allergens react with IgE in 50% of sera from allergic patients.Objective To determine the molecular and antigenic characteristics and heterogeneity of Der f 7 in mite extracts.Methods Monoclonal antibodies (MoAbs) produced from mice immunized with recombinant Der f 7 were examined for crossreactivity to Der p 7 and then used for immunoblotting of 1 and 2-D gel electrophoresis. Deglycosylation was studied with N-glycosidase-F and N-terminal sequencing by Edman degradation. The epitopes of the monoclonal antibodies were compared by cross-inhibitory immunoassays.Results Immunoblotting of D. farinae extracts with all the anti Der f 7 MoAbs showed major reactivities at 31, 30 and 25 kDa. The strongest immunostaining was at 25 kDa which contrasted with Der p 7 where the 31 and 30 kDa bands were strongest. The relative strength of staining however varied between extracts. The 31 and 30 kDa components were glycosylation products of the 25 kDa form which had the N-terminal sequence predicted from cDNA analysis. Two MoAbs stained an 18 kDa band consistent with a degradation product. The 2-D gels showed that different components with pls from 5.6–6.4. Both species-specific and Der p 7 crossreactive MoAbs were produced and a two-site ELISA assay for detecting group 7 allergen was developed with MoAbs recognizing different epitopes.Conclusions Der f 7 has been defined by its natural N-terminal sequence and MoAbs. It apparently exists as different glycosylation and degradation products in mite extracts, the relative abundance of which differs with different preparations. A two-site ELISA to measure the allergen was developed.

Notes: Background Through proteomic and genomic approaches we have previously identified and characterized an alkaline serine protease that is a major allergen (88% frequency of IgE binding) of Penicillium chrysogenum (Pen ch 13).Objective The aim of the present study is to identify the linear IgE-binding epitopes of Pen ch 13.Methods IgE-binding regions were identified by dot-blot immunoassay using 11 phage-displayed peptide fragments spanning the whole molecule of Pen ch 13. The minimal epitope requirements for IgE binding were further defined with overlapping peptides synthesized on derivatized cellulose membranes using SPOTs technology. The critical residues on the immunodominant epitopes were mapped through site-directed mutagenesis. The locations of the IgE epitopes identified were correlated with a three-dimensional structure of Pen ch 13.Results IgE antibodies in 35 serum samples reacted with at least one of the 11 peptide fragments of Pen ch 13. Peptide f-2n (residues 31–61) showed a high-intensity and the highest frequency (77%) of IgE binding. The frequencies of IgE binding to peptide f-4 (residues 93–133), f-1 (residues 1–37) and f-7 (residues 168–206) were 51%, 34% and 31%, respectively. SPOTs assay narrowed down the region of IgE binding of f-2n to residues 48–55 (GHADFGGR). Three, two and one epitope(s) that are four to nine amino acids in length, within f-4, f-1 and f-7, respectively, were found. Site-directed mutagenesis of Pen ch 13 revealed that substitution of His49 and/or Phe52 on Pen ch 13 with methionine resulted in proteins with drastic loss of IgE binding in seven sera tested. Proteins with amino acid replacements at residues 15–18 (RISS), or at residues 112 (I) and 116 (D) have lower IgE-binding reactivity in one of the two patient's sera tested. Substituting residues 117 (W), 119 (V) and 120 (K) also block most of the IgE binding in one of the two patient's sera tested. In addition, replacing residues 203 (V) and 204 (D) along with a deletion at residue 206 (Y) diminished the IgE binding in two serum samples tested. A model was constructed based on the structure of P. cyclopium subtilisin protease that has 〉90% (256 out of 283 amino acids) sequence identity with Pen ch 13. The major epitope (GHADFGGR) on Pen ch 13 formed a loop-like structure and was located at the surface of the allergen.Conclusions Several linear IgE-reactive epitopes and their critical core amino acid residues were identified for the Pen ch 13 allergen. The major linear IgE-binding epitope, 48GHADFGGR55, formed a loop-like structure at the surface of the allergen. Substitution of His49 and/or Phe52 with methionine significantly reduced IgE-binding to Pen ch 13. Mapping of these results on a 3D model of the allergen provides valuable information about the molecular basis of allergenicity for Pen ch 13 and for designing specific immunotherapeutics.

Notes: Alkaline and/or vacuolar serine proteinases are major allergens in prevalent airborne Penicillium and Aspergillus species.〈section xml:id="abs1-2"〉〈title type="main"〉ObjectiveThe object of this study is to generate and characterize monoclonal antibodies against these serine proteinase allergens.〈section xml:id="abs1-3"〉〈title type="main"〉MethodsBALB/c mice were immunized individually with the Penicillium citrinum culture medium or the crude extract and culture medium preparations of Aspergillus fumigatus. Hybridoma cells that secrete monoclonal antibodies against serine proteinase allergens were selected by immunoblotting. Antigens in three different Penicillium (P. citrinum, P. notatum and P. oxalicum) and two different Aspergillus species (A. fumigatus, and A. flavus) recognized by these monoclonal antibodies were analysed by sodium dodecyl sulphate and two-dimensional polyacrylamide gel electrophoresis immunoblotting and N-terminal amino acid sequence analysis.〈section xml:id="abs1-4"〉〈title type="main"〉ResultsFour (PCM8, PCM10, PCM16 and PCM39) and one (FUM20) monoclonal antibodies against serine proteinase allergens were generated after fusion of NS-1 cells with spleen cells obtained from BALB/c mice immunized with antigens from P. citrinum and A. fumigatus, respectively. Immunoblotting results showed that PCM8 reacted with an alkaline serine proteinase allergen in P. citrinum and P. notatum. PCM10 and PCM39 reacted with the alkaline serine proteinase in two Penicillium (P. citrinum, P. notatum) and two Aspergillus species (A. fumigatus, and A. flavus) tested. PCM16 reacted with the alkaline serine proteinase allergen in P. citrinum, A. fumigatus and A. flavus but not with that in P. notatum. MoAb FUM20 reacted with the alkaline serine proteinase allergen in two Aspergillus species (A. fumigatus and A. flavus) but not with that in two different Penicillium species (P. citrinum, P. notatum) tested. Among these five monoclonal antibodies generated, only PCM39 and FUM20 can react with the vacuolar serine proteinase allergen in P. notatum, P. oxalicum and in A. fumigatus. The 35 kDa P. citrinum component that reacted with FUM20 has an N-terminal amino acid sequence of DSPSVEKNAP.〈section xml:id="abs1-5"〉〈title type="main"〉ConclusionFive monoclonal antibodies against different epitopes of the serine proteinase major allergens in prevalent Penicillium and Aspergillus species were generated in the present study. Antibodies obtained may be useful in the characterization and standardization of serine proteinase allergens in crude fungal extracts.

Notes: Background: Candida albicans has been implicated in human allergic disorders. However, many of its immunoglobulin E (IgE)-reacting components have not yet been identified. The purpose of the present study is to characterize a novel 29 kDa IgE-binding protein from C. albicans.Methods: The 29 kDa protein was partially purified and its tryptic digests subjected to mass spectrometric analysis. The cDNA encoding this protein was isolated and heterologously expressed in Escherichia coli. Monoclonal antibodies (MoAbs) were raised against the 29 kDa protein purified from C. abicans extracts.Results: We isolated a 29 kDa IgE-reacting component from C. albicans. The protein was digested on-gel with trypsin and the masses of the resulting fragments were determined in a MALDI-TOF mass spectrometer. The data were searched against protein sequences deduced from the C. albicans genome. An open reading frame that possibly encodes the 29 kDa IgE-reacting component was identified. The cDNA corresponding to the open reading frame was isolated. It encodes a 236 residues protein that has 62% sequence identity to that of a hypothetical protein (YDR533c) from Saccharomyces cerevisiae. Conserved domain search suggests that the encoded protein belongs to the ThiJ/PfpI family. The cDNA isolated was inserted into a pQE-30 vector for protein expression in Escherichia coli. The recombinant protein can react with IgE antibodies in sera from asthmatic patients and two MoAbs that were generated against the purified native 29 kDa protein from C. albicans.Conclusions: We identified and cloned a novel 29 kDa IgE-reacting component (Cand a 3) from C. albicans. The recombinant proteins produced from this clone and the MoAbs prepared may be useful in the standardization of diagnostic extracts. They are also instrumental in elucidating the role of C. albicans in clinical allergy.

Notes: Background: We have suggested previously that the 32 and 34 kDa major allergens of Penicillium chrysogenum (also known as P. notatum) are the vacuolar (Pen ch 18) and the alkaline (Pen ch 13) serine proteases, respectively, of P. chrysogenum. The purpose of this study is to characterize the 32 kDa allergen of P. chrysogenum and its immunoglobulin E (IgE)cross-reactivity with Pen ch 13 allergen.Methods: The full-length cDNA of Pen ch 18 was isolated by reverse transcriptase-polymerase chain reaction and the 5′-rapid amplification cDNA end reaction. Recombinant Pen ch 18 was expressed as his-tagged proteins in Escherichia coli. Its reactivity with IgE and monoclonal antibodies against fungal serine protease allergens was analyzed by immunoblotting. The IgE cross-reactivity between Pen ch 18 and Pen ch 13 was analyzed by immunoblot inhibition. Overlapping recombinant fragments and synthetic peptides were used to map the B cell epitopes on Pen ch 18.Results: In this study, we isolated a 1857 bp cDNA fragment containing an open reading frame of 494 amino acids that encodes the preproenzyme of Pen ch 18. Similar to other vacuolar serine proteases, this precursor appears to undergo N- and possibly C-terminal cleavage upon maturation. The his-tagged recombinant Pen ch 18 containing the putative sequence of the mature protein reacted with IgE antibodies in serum samples from asthmatic patients. In addition, IgE-binding to the 32 kDa major allergen of P. chrysogenum was inhibited when a positive serum sample was absorbed with recombinant Pen ch 18 before immunoblotting. Both inhibition and almost no inhibition of IgE-binding to the 32 kDa major allergen of Pen ch 18 were detected when eight positive serum samples were preabsorbed individually with purified Pen ch 13 before immunoblotting. The major IgE binding region was located in a fragment (PN1) encompassing the N-terminal 102 amino acid residues of the recombinant Pen ch 18. A dominant linear IgE epitope was further mapped within residues 73–95 (peptide PN1-e) of the N-terminally processed allergen. Monoclonal antibody FUM20 that reacts with Pen ch 18 but not with Pen ch 13 binds a synthetic peptide with sequence encompassing the N-terminal 23 residues of the recombinant Pen ch 18. Monoclonal antibody PCM39 that reacts with both Pen ch 13 and Pen ch 18 recognizes a peptide containing residues 132–154 of the allergen.Conclusions: Our results confirm that the Pen ch 18 allergen is a vacuolar serine protease of P. chrysogenum that matures through N- and possibly C-terminal processing. The finding that there are cross-reactive and allergen-specific IgE epitopes for Pen ch 18 and Pen ch 13 suggests that both major allergens should be included in clinically diagnostic P. chrysogenum extracts.