PMID- 14510716 OWN - NLM STAT- MEDLINE DCOM- 20040218 LR - 20190823 IS - 0105-4538 (Print) IS - 0105-4538 (Linking) VI - 58 IP - 10 DP - 2003 Oct TI - Molecular and immunological characterization of Pen ch 18, the vacuolar serine protease major allergen of Penicillium chrysogenum. PG - 993-1002 AB - 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. FAU - Shen, H-D AU - Shen HD AD - Department of Medical Research and Education, Veterans General Hospital-Taipei, Taiwan, Republic of China. FAU - Chou, H AU - Chou H FAU - Tam, M F AU - Tam MF FAU - Chang, C-Y AU - Chang CY FAU - Lai, H-Y AU - Lai HY FAU - Wang, S-R AU - Wang SR LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't PL - Denmark TA - Allergy JT - Allergy JID - 7804028 RN - 0 (Allergens) RN - 0 (Fungal Proteins) RN - 0 (Pen n 18 protein, Penicillium chrysogenum) RN - 0 (Recombinant Fusion Proteins) RN - 37341-29-0 (Immunoglobulin E) RN - EC 3.4.21.- (Serine Endopeptidases) SB - IM MH - Adult MH - Allergens/*chemistry/genetics/*immunology MH - Amino Acid Sequence MH - Animals MH - Asthma/immunology MH - Epitope Mapping MH - Fungal Proteins/*chemistry/genetics/*immunology MH - Humans MH - Immunoglobulin E/immunology MH - Molecular Sequence Data MH - Recombinant Fusion Proteins/immunology MH - Sequence Alignment MH - Serine Endopeptidases/chemistry/genetics/*immunology MH - Vacuoles/enzymology EDAT- 2003/09/27 05:00 MHDA- 2004/02/19 05:00 CRDT- 2003/09/27 05:00 PHST- 2003/09/27 05:00 [pubmed] PHST- 2004/02/19 05:00 [medline] PHST- 2003/09/27 05:00 [entrez] AID - 107 [pii] AID - 10.1034/j.1398-9995.2003.00107.x [doi] PST - ppublish SO - Allergy. 2003 Oct;58(10):993-1002. doi: 10.1034/j.1398-9995.2003.00107.x.