Allergy Asthma Immunol Res.  2016 Sep;8(5):438-444. 10.4168/aair.2016.8.5.438.

Vacuolar Serine Protease Is a Major Allergen of Fusarium proliferatum and an IgE-Cross Reactive Pan-Fungal Allergen

• Yeh CC
• Tai HY
• Chou H
• Wu KG
• Shen HD

• KMID: 2295153
• DOI: http://doi.org/10.4168/aair.2016.8.5.438

Abstract

PURPOSE
Fusarium species are among prevalent airborne fungi and causative agents of human respiratory atopic disorders. We previously identified a 36.5-kDa F. proliferatum component recognized by IgE antibodies in 9 (53%) of the 17 F. proliferatum-sensitized atopic serum samples. The purpose of this study is to characterize the 36.5-kDa allergen of F. proliferatum.
METHODS
Characterization of allergens and determination of IgE cross-reactivity were performed by cDNA cloning/expression and immunoblot inhibition studies.
RESULTS
Based on the finding that the 36.5-kDa IgE-binding component reacted with the mouse monoclonal antibody FUM20 against fungal vacuolar serine protease allergens, the cDNA of F. proliferatum vacuolar serine protease (Fus p 9.0101) was subsequently cloned. Nine serum samples from respiratory atopic patients with IgE binding to the vacuolar serine protease allergen of Penicillium chrysogenum (Pen ch 18) also showed IgE-immunoblot reactivity to rFus p 9.0101. The purified rFus p 9.0101 can inhibit IgE and FUM20 binding to the 36.5-kDa component of F. proliferatum. Thus, a novel and important Fus p 9.0101 was identified. The rPen ch 18 can inhibit IgE binding to Fus p 9.0101. It indicates that IgE cross-reactivity between Fus p 9.0101 and Pen ch 18 also exists. Furthermore, neither rFus p 9.0101 K88A nor rPen ch 18 K89A mutants inhibited IgE binding to rFus p 9.0101. Lys88 was considered a critical core amino acid in IgE binding to r Fus p 9.0101 and a residue responsible for IgE cross-reactivity between Fus p 9.0101 and Pen ch 18 allergens.
CONCLUSIONS
Results obtained from this study indicate that vacuolar serine protease may be a major allergen of F. proliferatum and an important IgE cross-reactive pan-fungal allergen, and provide important bases for clinical diagnosis of fungal allergy.

Keyword

F. proliferatum; allergen; vacuolar serine protease; IgE cross-reactivity

MeSH Terms

Allergens
Animals
Antibodies
Clone Cells
Diagnosis
DNA, Complementary
Fungi
Fusarium*
Humans
Hypersensitivity
Immunoglobulin E
Mice
Penicillium chrysogenum
Serine Proteases*
Serine*
Allergens
Antibodies
DNA, Complementary
Immunoglobulin E
Serine
Serine Proteases

Figure

• 

  Fig. 1 (A) The nucleotide and deduced amino acid sequences of the vacuolar serine protease protein of F. proliferatum (Fus p 9.0101, GenBank accession no. KJ462778). Numbers to the right indicate the positions of the nucleotides and the deduced amino acid residues of the sequences. The amino acid triad (D45, H77 and S243) which is characteristic of serine proteases is depicted in bold type and shaded. Three potential N-glycosylation sites are in bold letters and underlined. The vertical arrow marks the proposed amino terminus of the mature vacuolar serine protease. The stop codon TAA is denoted with an asterisk. Nucleotides in gray correspond to those synthesized and used as primers for PCR in the cDNA cloning of the vacuolar serine protease of F. proliferatum as shown in Table. The sequences corresponding to primers FuVSP-5'Sma I and FuVSP-3'Hind III used in the preparation of recombinant mature Fusarium vacuolar serine protease protein and its K88A mutant (FVSP K88A-f and FVSP K88A-r) are boxed. (B) Composite alignment of the deduced amino acid sequences of the proposed mature Fus p 9.0101, Pen ch 18, Pen o 18 and the Pen ch 13 fungal serine protease allergens. Dashes denote spaces introduced to optimize the alignment. Identical amino acid residues are denoted with asterisks.

• 

  Fig. 2 Antigenicity of the wild-type and mutant fungal vacuolar serine protease allergens. (A) Coomassie blue-stained protein profiles of the recombinant wild-type Fus p 9.0101 and Pen ch 18 allergens as well as the Fus p 9.0101K88A and Pen ch 18 K89A mutants on PVDF membranes. (B) Immunoblot reactivities to MoAb FUM20. (C) Immunoblot reactivities to IgE antibodies in 9 sera from asthmatic patients (strip nos. 1-9) and 1 serum from 1 house dust mite-sensitized asthmatic patient (strip no. 10).

• 

  Fig. 3 Immunoblot inhibition of IgE- (panel B) and MoAb FUM20- (panel C) binding to nFus p 9.0101 in crude F. proliferatum extracts with purified rFus p 9.0101 and BSA as inhibitors. (A) Coomassie blue-stained protein profile of F. proliferatum extracts and protein molecular weight markers. (B) IgE binding to the 36.5-kDa component using serum no. 1 from Fig. 2C (lane 1); this binding activity was inhibited dose-dependently by 20 µg (lane 2) and 5 µg (lane 3) of rFus p 9.0101, but not 20 µg of BSA (lane 4). (C) MoAb FUM20 binding to the 36.5-kDa component of F. proliferatum extracts (lane 1); this binding activity was inhibited by 20 µg (lane 2) of rFus p 9.0101, but not 20 µg of BSA (lane 3).

• 

  Fig. 4 Inhibition of IgE-immunoblot reactivity to Fus p 9.0101. IgE-immunoblot experiment was carried out with serum no. 9 from Fig. 2C (lane 9). Serum was pre-absorbed with 10 µg of the wild-type Fus p 9.0101, the wild-type Pen ch 18, the Fus p 9.0101K88A mutant, the Pen ch 18 K89A mutant, or BSA as indicated.

Reference

1. Sharpe RA, Bearman N, Thornton CR, Husk K, Osborne NJ. Indoor fungal diversity and asthma: a meta-analysis and systematic review of risk factors. J Allergy Clin Immunol. 2015; 135:110–122.

2. Twaroch TE, Curin M, Valenta R, Swoboda I. Mold allergens in respiratory allergy: from structure to therapy. Allergy Asthma Immunol Res. 2015; 7:205–220.

3. Esch RE. Manufacturing and standardizing fungal allergen products. J Allergy Clin Immunol. 2004; 113:210–215.

4. Jeong KY, Lee JH, Kim EJ, Lee JS, Cho SH, Hong SJ, et al. Current status of standardization of inhalant allergen extracts in Korea. Allergy Asthma Immunol Res. 2014; 6:196–200.

5. Shen HD, Tam MF, Chou H, Han SH. The importance of serine proteinases as aeroallergens associated with asthma. Int Arch Allergy Immunol. 1999; 119:259–264.

6. Chou H, Lai HY, Tam MF, Chou MY, Wang SR, Han SH, et al. cDNA cloning, biological and immunological characterization of the alkaline serine protease major allergen from Penicillium chrysogenum. Int Arch Allergy Immunol. 2002; 127:15–26.

7. Shen HD, Chou H, Tam MF, Chang CY, Lai HY, Wang SR. Molecular and immunological characterization of Pen ch 18, the vacuolar serine protease major allergen of Penicillium chrysogenum. Allergy. 2003; 58:993–1002.

8. Shen HD, Lin WL, Tam MF, Chou H, Wang CW, Tsai JJ, et al. Identification of vacuolar serine proteinase as a major allergen of Aspergillus fumigatus by immunoblotting and N-terminal amino acid sequence analysis. Clin Exp Allergy. 2001; 31:295–302.

9. Shen HD, Tam MF, Tang RB, Chou H. Aspergillus and Penicillium allergens: focus on proteases. Curr Allergy Asthma Rep. 2007; 7:351–356.

10. Chou H, Tam MF, Lee LH, Chiang CH, Tai HY, Panzani RC, et al. Vacuolar serine protease is a major allergen of Cladosporium cladosporioides. Int Arch Allergy Immunol. 2008; 146:277–286.

11. Chou H, Wu KG, Yeh CC, Tai HY, Tam MF, Chen YS, et al. The transaldolase, a novel allergen of Fusarium proliferatum, demonstrates IgE cross-reactivity with its human analogue. PLoS One. 2014; 9:e103488.

12. Lin WL, Chou H, Tam MF, Huang MH, Han SH, Shen HD. Production and characterization of monoclonal antibodies to serine proteinase allergens in Penicillium and Aspergillus species. Clin Exp Allergy. 2000; 30:1653–1662.

13. Sams JT, Smith RE. Cross-antigenicity of common mold antigens. Ann Allergy. 1968; 26:55–60.

14. Prince HE, Morrow MB. Skin reaction patterns to dematiaceous mold allergens. Ann Allergy. 1971; 29:535–538.

15. Cheng TT, Tam MF, Chou H, Tai HY, Shen HD. Lys89, Lys90, and Phe91 are critical core amino acid residues of the Pen ch 18 major fungal allergen recognized by human IgE antibodies. Biochem Biophys Res Commun. 2008; 375:671–674.

16. Shen HD, Wang CW, Lin WL, Lai HY, Tam MF, Chou H, et al. cDNA cloning and immunologic characterization of Pen o 18, the vacuolar serine protease major allergen of Penicillium oxalicum. J Lab Clin Med. 2001; 137:115–124.

17. Moehle CM, Tizard R, Lemmon SK, Smart J, Jones EW. Protease B of the lysosomelike vacuole of the yeast Saccharomyces cerevisiae is homologous to the subtilisin family of serine proteases. Mol Cell Biol. 1987; 7:4390–4399.

18. Nebes VL, Jones EW. Activation of the proteinase B precursor of the yeast Saccharomyces cerevisiae by autocatalysis and by an internal sequence. J Biol Chem. 1991; 266:22851–22857.

19. Frederick GD, Rombouts P, Buxton FP. Cloning and characterisation of pepC, a gene encoding a serine protease from Aspergillus niger. Gene. 1993; 125:57–64.

20. Shen HD, Chua KY, Lin WL, Hsieh KH, Thomas WR. Characterization of the house dust mite allergen Der p 7 by monoclonal antibodies. Clin Exp Allergy. 1995; 25:416–422.

21. Chou H, Tam MF, Lee SS, Tai HY, Chang CY, Chou CT, et al. A vacuolar serine protease (Rho m 2) is a major allergen of Rhodotorula mucilaginosa and belongs to a class of highly conserved pan-fungal allergens. Int Arch Allergy Immunol. 2005; 138:134–141.

22. Gupta R, Sharma V, Sridhara S, Singh BP, Arora N. Identification of serine protease as a major allergen of Curvularia lunata. Allergy. 2004; 59:421–427.

23. Tai HY, Tam MF, Chou H, Peng HJ, Su SN, Perng DW, et al. Pen ch 13 allergen induces secretion of mediators and degradation of occludin protein of human lung epithelial cells. Allergy. 2006; 61:382–388.

24. Kurup VP, Xia JQ, Shen HD, Rickaby DA, Henderson JD Jr, Fink JN, et al. Alkaline serine proteinase from Aspergillus fumigatus has synergistic effects on Asp-f-2-induced immune response in mice. Int Arch Allergy Immunol. 2002; 129:129–137.

25. Tai HY, Tam MF, Chou H, Perng DW, Shen HD. Pen ch 13 major fungal allergen decreases CD44 expression in human bronchial epithelial cells. Int Arch Allergy Immunol. 2010; 153:367–371.

26. Tripathi P, Kukreja N, Singh BP, Arora N. Serine protease activity of Cur l 1 from Curvularia lunata augments Th2 response in mice. J Clin Immunol. 2009; 29:292–302.

27. Namvar S, Warn P, Farnell E, Bromley M, Fraczek M, Bowyer P, et al. Aspergillus fumigatus proteases, Asp f 5 and Asp f 13, are essential for airway inflammation and remodelling in a murine inhalation model. Clin Exp Allergy. 2015; 45:982–993.