Biologically active recombinant forms of a major house dust mite group 1 allergen Der f 1 with full activities of both cysteine protease and IgE binding

Yasuhara, Toru; Takai, Toshiro; Yuuki, Toshifumi; Okudaira, Hirokazu; Okumura, Yasushi

doi:10.1046/j.1365-2222.2001.00945.x

Cited by 46 publications

(61 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Although detailed knowledge of the cleavage specificity of Der p 1 is lacking, a number of susceptible residues have been determined using synthetic peptides represent- (41), and synthetic substrates containing C-terminal R or K (26,42). Using this information, several possible sites in the extracellular NH 2 -terminal domain, and first and second extracellular loop regions of PAR-1 known to be involved in the activation process can be identified (reviewed in Refs.…”

Section: Discussionmentioning

confidence: 99%

House Dust Mite Allergens Induce Proinflammatory Cytokines from Respiratory Epithelial Cells: The Cysteine Protease Allergen, Der p 1, Activates Protease-Activated Receptor (PAR)-2 and Inactivates PAR-1

Asokananthan¹,

Graham²,

Stewart³

et al. 2002

The Journal of Immunology

323

247

View full text Add to dashboard Cite

In previous studies, we demonstrated that allergenic house dust mite proteases are potent inducers of proinflammatory cytokines from the respiratory epithelium, although the precise mechanisms involved were unclear. In this study, we investigated whether this was achieved through activation of protease-activated receptor (PAR)-1 or -2. Pretreatment of A549 respiratory epithelial cells with the clinically important cysteine protease allergen, Der p 1, ablated subsequent PAR-1, but not PAR-2 agonist peptide-induced IL-6 and IL-8 release. HeLa cells transfected with the plasmid coding for PAR-2, in contrast to PAR-1, released significant concentration of IL-6 after exposure to Der p 1. Exposure of HeLa cells transfected with either PAR-1/enhanced yellow fusion protein or PAR-2/enhanced yellow fusion protein to Der p 1 caused receptor internalization in the latter cells only, as judged by confocal microscopy with re-expression of the receptor within 120-min postenzyme exposure. Der p 1-induced cytokine release from both A549 and transfected HeLa cells was accompanied by changes in intracellular Ca2+ concentrations. Desensitization studies showed that Der p 1 pretreatment of the A549 cells resulted in the abolition of both trypsin- and PAR-2 agonist peptide-induced Ca2+ release, but not that induced by subsequent exposure to either thrombin or PAR-1 agonist peptide. These data indicate for the first time that the house dust mite allergen Der p 1-induced cytokine release from respiratory epithelial cells is, in part, mediated by activation of PAR-2, but not PAR-1.

show abstract

Section: Discussionmentioning

confidence: 99%

House Dust Mite Allergens Induce Proinflammatory Cytokines from Respiratory Epithelial Cells: The Cysteine Protease Allergen, Der p 1, Activates Protease-Activated Receptor (PAR)-2 and Inactivates PAR-1

Asokananthan¹,

Graham²,

Stewart³

et al. 2002

The Journal of Immunology

323

247

View full text Add to dashboard Cite

show abstract

“…Glycosylations on natural and recombinant (P. pastoris) Der p 1 and Der f 1, resulting in a large molecular mass smear for the recombinant proteins, have been reported by a number of groups (16,17,(47)(48)(49). By mutating the potential glycosylation site at N132 and N133 in the mature region of proDer p 1 and proDer f 1, respectively, this smear can be shifted into distinct bands with a lower molecular mass in SDS-PAGE (17,49).…”

Section: Discussionmentioning

confidence: 99%

The Crystal Structure of Recombinant proDer p 1, a Major House Dust Mite Proteolytic Allergen

Meno

Thorsted

Ipsen

et al. 2005

The Journal of Immunology

View full text Add to dashboard Cite

Allergy to house dust mite is among the most prevalent allergic diseases worldwide. Most house dust mite allergic patients react to Der p 1 from Dermatophagoides pteronyssinus, which is a cysteine protease. To avoid heterogeneity in the sample used for crystallization, a modified recombinant molecule was produced. The sequence of the proDer p 1 allergen was modified to reduce glycosylation and to abolish enzymatic activity. The resulting rproDer p 1 preparation was homogenous and stable and yielded crystals diffracting to a resolution of 1.61 Å. The active site is located in a large cleft on the surface of the molecule. The 80-aa pro-peptide adopts a unique fold that interacts with the active site cleft and a substantial adjacent area on the mature region, excluding access to the cleft and the active site. Studies performed using crossed-line immunoelectrophoresis and IgE inhibition experiments indicated that several epitopes are covered by the pro-peptide and that the epitopes on the recombinant mature molecule are indistinguishable from those on the natural one. The structure confirms previous results suggesting a preference for aliphatic residues in the important P2 position in substrates. Sequence variations in related species are concentrated on the surface, which explains the existence of cross-reacting and species-specific antibodies. This study describes the first crystal structure of one of the clinically most important house dust mite allergens, the cysteine protease Der p 1.

show abstract

“…Generation of the Der p 1 and Der f 1 Protein-Recombinant Der p 1 and Der f 1 proteins were generated as described before (33,34). Briefly, proforms of four recombinant house dust mite group 1 allergens, Der p 1-N52Q, Der p 1-WT, Der f 1-N53Q, and Der f 1-WT, were secreted into the culture supernatant of transfectant cells of Pichia pastoris and converted to the mature forms with prosequences removed by dialysis against an acidic buffer.…”

Section: Methodsmentioning

confidence: 99%

“…Enzyme Assays-Enzyme assays of Der p 1 and Der f 1 were performed as described before (33,35). The substrate used for enzyme assays was butyloxycarbonyl-Gln-Arg-methylcoumarin (Boc-Gln-AlaArg-MCA), purchased from Peptide Institute Inc.…”

Section: Methodsmentioning

confidence: 99%

The Squamous Cell Carcinoma Antigen 2 Inhibits the Cysteine Proteinase Activity of a Major Mite Allergen, Der p 1

Sakata¹,

Arima²,

Takai

et al. 2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

The squamous cell carcinoma antigens 1 (SCCA1) and SCCA2 belong to the ovalbumin-serpin family. Although SCCA1 and SCCA2 are closely homologous, these two molecules have distinct properties; SCCA1 inhibits cysteine proteinases such as cathepsin K, L, and S, whereas SCCA2 inhibits serine proteinases such as cathepsin G and human mast cell chymase. Although several intrinsic target proteinases for SCCA1 and SCCA2 have been found, the biological roles of SCCA1 and SCCA2 remain unknown. A mite allergen, Der p 1, is one of the most immunodominant allergens and also acts as a cysteine proteinase probably involved in the pathogenesis of allergic diseases. We have recently shown that both SCCA1 and SCCA2 are induced by two related Th2-type cytokines, IL-4 and IL-13, in bronchial epithelial cells and that SCCA expression is augmented in bronchial asthma patients. In this study, we explored the possibility that SCCA proteins target Der p 1, and it turned out that SCCA2, but not SCCA1, inhibited the catalytic activities of Der p 1. We furthermore analyzed the inhibitory mechanism of SCCA2 on Der p 1. SCCA2 contributed the suicide substrate-like mechanism without formation of a covalent complex, causing irreversible impairment of the catalytic activity of Der p 1, as SCCA1 does on papain. In addition, resistance to cleavage by Der p 1 also contributed to the inhibitory mechanism of SCCA2. These results suggest that SCCA2 acts as a crossclass serpin targeting an extrinsic cysteine proteinase derived from house dust mites and that it may have a protective role against biological reactions caused by mites.

show abstract

Biologically active recombinant forms of a major house dust mite group 1 allergen Der f 1 with full activities of both cysteine protease and IgE binding

Cited by 46 publications

References 43 publications

House Dust Mite Allergens Induce Proinflammatory Cytokines from Respiratory Epithelial Cells: The Cysteine Protease Allergen, Der p 1, Activates Protease-Activated Receptor (PAR)-2 and Inactivates PAR-1

House Dust Mite Allergens Induce Proinflammatory Cytokines from Respiratory Epithelial Cells: The Cysteine Protease Allergen, Der p 1, Activates Protease-Activated Receptor (PAR)-2 and Inactivates PAR-1

The Crystal Structure of Recombinant proDer p 1, a Major House Dust Mite Proteolytic Allergen

The Squamous Cell Carcinoma Antigen 2 Inhibits the Cysteine Proteinase Activity of a Major Mite Allergen, Der p 1

Contact Info

Product

Resources

About