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

Abstract

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.

Vacuolar Serine Protease Is a Major Allergen of <i>Cladosporium cladosporioides</i>

“…The fungal alkaline/vacuolar serine proteases are translated as a precursor with an N-terminal preproenzyme sequence [23,29,30,31,32, 36]. The N-terminal prosequence of these proteases seems to be involved in protein folding and processing of the precursor into an active conformation [31].…”

A Vacuolar Serine Protease (Rho m 2) Is a Major Allergen of <i>Rhodotorula mucilaginosa </i>and Belongs to a Class of Highly Conserved Pan-Fungal Allergens

“…In addition to the vacuolar serine proteases, the group 13 alkaline serine proteases are also major allergens of prevalent Penicillium and Aspergillus species [5, 7, 14, 22]. IgE cross-reactivity between the group 18 (Pen ch 18) and the group 13 (Pen ch 13) major allergens from P. chrysogenum has been reported [20].…”

“…The Pen ch 18 allergen was isolated from a P. chrysogenum strain CCRC 30568 [14]. The lyophilized crude extracts prepared from this strain were dissolved in a 20 m M Tris-buffer (20 m M Tris-HCl, 0.5 M NaCl, 1 m M CaCl 2 , 1 m M MnSO 4 , pH 7.2) and purified with the Con A Sepharose and the DEAE-Sepharose columns as described above.…”

“…The cDNA encoding the R. mucilaginosa vacuolar serine protease was isolated as described previously using the ProSTAR TM First Strand RT-PCR kit (Stratagene, La Jolla, Calif., USA) [11, 14]. The first set of polymerase chain reaction (PCR) utilized Primers 255 and AP.…”

Isolation and Characterization of a cDNA Clone Encoding One IgE-Binding Fragment of <i>Penicillium brevicompactum</i>

“…These includes Pen c 1 [21], Pen c 2 [22] and Pen c 3 [23] of P. citrinum , Pen o 18 of P. oxalicum [24], Pen ch 13 (a.k.a. Pen n 13) [25, 26], Pen ch 18 [27] ] of P. chrysogenum . However, not all Penicillium species are well-studied and a number of allergens from Penicillium species are awaiting discovery.…”