Characterization of allergens from Penicillium oxalicum and P. notatum by immunoblotting and N‐terminal amino acid sequence analysis

Shen,; J, Lin; Tâm,; Wang, Hong; Tzean,; Huang, Biao; Han, Sang-Min

doi:10.1046/j.1365-2222.1999.00509.x

Cited by 53 publications

(100 citation statements)

References 18 publications

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“…Protein components in the crude fungal extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or by 2D gel electrophoresis and transferred electrophoretically onto polyvinylidene difluoride (PVDF) membrane (0.45 µm, Millipore, Bedford, Mass., USA) as described [12, 13, 14, 15, 16]. The membrane was blocked with 1% skim milk, then incubated with diluted human serum samples (1:5 dilution in Tris-buffered saline, pH 7.5, 0.05% Tween 20 and 1% skim milk) for 16 h at 4°C.…”

Section: Methodsmentioning

confidence: 99%

“…We have shown previously that the alkaline/vacuolar serine proteases are major allergens in eight prevalent Penicillium and Aspergillus species (Penicillium citrinum, Penicillium brevicompactum, Penicillium notatum, Penicillium oxalicum, Aspergillus oryzae, Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger) [12, 13, 14, 15, 16, 17]. IgE cross-reactivity among these serine protease allergens was also detected [13, 14, 15, 16].…”

Section: Introductionmentioning

confidence: 99%

“…IgE cross-reactivity among these serine protease allergens was also detected [13, 14, 15, 16]. In addition, we have documented that a heat shock protein in the hsp70 family [18]and an 18-kD peroxisomal membrane protein [19]are also important allergens of P. citrinum.…”

Section: Introductionmentioning

confidence: 99%

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cDNA Cloning and Immunological Characterization of a Newly Identified Enolase Allergen from Penicillium citrinum and Aspergillus fumigatus

Lai¹,

Tam

Tang

et al. 2002

Int Arch Allergy Immunol

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Background:Penicillium citrinum and Aspergillus fumigatus are prevalent indoor airborne fungal species that have been implicated in human respiratory allergic disorders. It is important to understand the allergenic profile of these fungal species. The purpose of the present study is to characterize a newly identified enolase allergen from P. citrinum and A. fumigatus. Methods: Fungal proteins were separated by two-dimensional (2D) gel electrophoresis and blotted onto polyvinylidene difluoride membranes. Protein spots that reacted with IgE antibodies in serum samples from asthmatic patients were identified and the N-terminal amino acid sequences were determined by Edman degradation. The peptide sequences obtained were utilized in cloning the cDNA of the allergen genes by reverse transcriptase-polymerase chain reaction and the 5′- and 3′-rapid amplification cDNA end reactions. Results: Our results from 2D immunoblotting identified a 47-kD IgE-reactive component in the extracts of P. citrinum and A. fumigatus. The N-terminal amino acid sequences of the 47-kD proteins are homologous to those of fungal enolases. The corresponding enolase cDNA from P. citrinum contains 1,552 bp and encodes a protein of 438 residues. In A. fumigatus, the isolated enolase cDNA has 1,649 bp and contains a 438-amino acid open reading frame. The deduced amino acid sequences of these two enolases have 94% identity. These enolases from P. citrinum and A. fumigatus were expressed in Escherichia coli as a His-tagged protein and designated as rPen c 22 and rAsp f 22, respectively. Sera from 7 (30%) of the 23 Penicillium-sensitized asthmatic patients showed IgE binding to the 47-kD P. citrinum component (Pen c 22) and rPen c 22. In addition, six of seven Pen c 22-positive serum samples have IgE immunoblot reactivity to the 47-kD A. fumigatus component (Asp f 22) and rAsp f 22. A polyclonal rabbit antiserum generated against the N-terminal peptide of Pen c 22 can react with Pen c 22, rPen c 22, Asp f 22 and rAsp f 22. In addition, the presence of IgE cross-reactivity between rPen c 22 and rAsp f 22 and between enolases from A. fumigatus and Alternaria alternata was also detected by immunoblot inhibition. Conclusions: These results demonstrated that a novel enolase allergen from P. citrinum (Pen c 22) and A. fumigatus (Asp f 22) was identified. In addition, IgE cross-reactivity between enolase allergens from A. fumigatus and P. citrinum and between enolases from A. fumigatus and A. alternata was also detected. Results obtained provide more information on fungal enolase allergens.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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cDNA Cloning and Immunological Characterization of a Newly Identified Enolase Allergen from Penicillium citrinum and Aspergillus fumigatus

Lai¹,

Tam

Tang

et al. 2002

Int Arch Allergy Immunol

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“…We have demonstrated by immunoblotting and N-terminal amino acid sequence analysis that alkaline and vacuolar serine proteases are major allergens in eight prevalent Penicillium and Aspergillus species [9, 10, 11, 12, 13]. They have been designated as the group 13 (alkaline serine protease) and the group 18 (vacuolar serine protease) allergens, respectively, for both fungal genera by the Allergen Nomenclature Subcommittee [14].…”

Section: Introductionmentioning

confidence: 99%

“…In this study, we isolated a full-length cDNA clone of the 34-kD alkaline serine protease major allergen (Pen ch 13, also known as Pen n 13 before) from P. chrysogenum [10]. The deduced N-terminal amino acid sequence of the cDNA clone correlated with that reported previously for the native protease from P. chrysogenum [10]. In addition, we report here the isolation and characterization of the 34-kD major allergen from the culture medium of P. chrysogenum .…”

Section: Introductionmentioning

confidence: 99%

cDNA Cloning, Biological and Immunological Characterization of the Alkaline Serine Protease Major Allergen from Penicillium chrysogenum

Chou

Lai

Tam

et al. 2002

Int Arch Allergy Immunol

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Background:Penicillium chrysogenum (Penicillium notatum) is a prevalent airborne Penicillium species. A 34-kD major IgE-reacting component from P. chrysogenum has been identified as an alkaline serine protease (Pen ch 13, also known as Pen n 13 before) by immunoblot and N-terminal amino acid sequence analysis. Methods: In the present study, Pen ch 13 was further characterized in terms of cDNA cloning, protein purification, enzymatic activity, histamine release and IgE cross-reactivity with alkaline serine protease allergens from two other prevalent fungal species – P. citrinum (Pen c 13) and Aspergillus flavus (Asp fl 13). Results: A 1,478-bp cDNA (Pen ch 13) that encodes a 398-amino-acid alkaline serine protease from P. chrysogenum was isolated. This fungal protease has pre- and pro-enzyme sequences. The previously determined N-terminal amino acid sequence of the P. chrysogenum 34-kD major allergen is identical to that of residues 116–125 of the cDNA. Starting from Ala116, the deduced amino acid sequence (283 residues) of the mature alkaline serine protease has a calculated molecular mass of 28.105 kD with two cysteines and two putative N-glycosylation sites. It has 83 and 49% sequence identity with the alkaline serine proteases from P. citrinum and A. fumigatus, respectively. The recombinant Pen ch 13 was recovered from inclusion bodies and isolated under denaturing condition. This recombinant protein reacted with IgE antibodies in serum from an asthmatic patient and with monoclonal antibodies (PCM8, PCM10, PCM39) that reacted with the 34-kD component from P. chrysogenum. The N-terminal amino acid sequence of the purified native Pen ch 13 is identical to that determined previously for the 34-kD major allergen in crude P. chrysogenum extracts. The purified native Pen ch 13 has proteolytic activity with casein as the substrate at pH 8.0. This enzymatic activity was inhibited by phenylmethylsulfonyl fluoride or diethylpyrocarbonate. Pen ch 13 was also able to degrade gelatin and collagen but not elastin. Basophils from 5 asthmatic patients released histamine (12–73%) when exposed to the purified Pen ch 13. In ELISA (enzyme-linked immunosorbent assay) experiments, IgE for Pen ch 13 was able to compete with purified Pen ch 13, Pen c 13 or Asp fl 13 in a dose-related manner. Conclusions: These results demonstrated that the 34-kD major allergen of P. chrysogenum is an alkaline serine protease. These results also indicated that atopic patients primarily sensitized by either of these prevalent fungal species may develop allergic symptoms by exposure to other environmental fungi due to cross-reacting IgE antibodies against this protease.

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Characterization of Pen n 13, a Major Allergen from the Mold Penicillium notatum

Chow

Chiou

Hsiao

et al. 2000

Biochemical and Biophysical Research Communications

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Characterization of allergens from Penicillium oxalicum and P. notatum by immunoblotting and N‐terminal amino acid sequence analysis

Cited by 53 publications

References 18 publications

cDNA Cloning and Immunological Characterization of a Newly Identified Enolase Allergen from <i>Penicillium citrinum</i> and <i>Aspergillus fumigatus</i>

cDNA Cloning and Immunological Characterization of a Newly Identified Enolase Allergen from <i>Penicillium citrinum</i> and <i>Aspergillus fumigatus</i>

cDNA Cloning, Biological and Immunological Characterization of the Alkaline Serine Protease Major Allergen from <i>Penicillium chrysogenum</i>

Characterization of Pen n 13, a Major Allergen from the Mold Penicillium notatum

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