The cysteine protease activity of the major dust mite allergen Der p 1 selectively enhances the immunoglobulin E antibody response - PubMed (original) (raw)

The cysteine protease activity of the major dust mite allergen Der p 1 selectively enhances the immunoglobulin E antibody response

L Gough et al. J Exp Med. 1999.

Abstract

The house dust mite Dermatophagoides pteronyssinus allergen Der p 1 is the most immunodominant allergen involved in the expression of dust mite-specific immunoglobulin (Ig)E-mediated hypersensitivity. The reason for this potent IgE-eliciting property of Der p 1 remains unknown, but there is mounting in vitro evidence linking the allergenicity of Der p 1 to its cysteine protease activity. Here we demonstrate for the first time that immunization of mice with proteolytically active Der p 1 results in a significant enhancement in total IgE and Der p 1-specific IgE synthesis compared with animals immunized with Der p 1 that was irreversibly blocked with the cysteine protease inhibitor E-64. We conclude that the proteolytic activity of Der p 1 is a major contributor to its allergenicity.

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Figures

Figure 1

Assessment of the purity of the Der p 1 preparation. NH2-terminal sequencing showed that the sequence obtained (TNACSINGNA) matches the published sequence of Der p 1 8. (a) Silver-stained SDS-PAGE analysis showing crude extract of fecal pellets (lane 1), fraction obtained after immunoaffinity chromatography on immobilized anti–Der p 1 mAb 4C1 followed by removal of contaminating serine proteases on immobilized soybean trypsin inhibitor (lane 2), and the final product after FPLC (lane 3). Molecular mass standards are indicated at left. (b) Progression curves of the catalytic activity of Der p 1, measured in a continuous rate (kinetic) assay using the fluorogenic peptide substrate Boc-Gln-Ala-Arg-AMC 6, with or without preactivation with cysteine. (c) The addition of E-64 and iodoacetamide results in 100 and 94% inhibition of the enzymatic activity of Der p 1, respectively. Data presented in b and c are the means of duplicate experiments; SE was <5%.

Figure 1

Figure 3

Total IgE (a) and Der p 1–specific IgE (b), IgG (c), IgG1 (d), and IgG2b (e) levels in final bleeds obtained from groups of 10 mice immunized with proteolytically active or inactive (E-64–blocked) Der p 1 measured by ELISA. There was a statistically significant enhancement in total IgE (P < 0.01) and Der p 1–specific IgE (P < 0.02) responses in animals immunized with proteolytically active Der p 1; prebleeds showed no measurable levels of antibody (except for baseline levels of total IgE). Depleting serum samples of IgG by protein G treatment (to overcome competition) from animals immunized with proteolytically active Der p 1 enhances the detection of Der p 1–specific IgE (f); this effect was also observed with animals immunized with proteolytically inactive Der p 1. Error bars represent 95% confidence intervals, and the results are representative of two immunization experiments.

Figure 3

Figure 2

Proteolytically active Der p 1 (10 μg/ml) cleaves mouse CD25, but not CD3, CD4, or CD8, from spleen T cells as monitored by flow cytometry (a); cleavage of CD25 is dose dependent (b). No such effect was demonstrable when Der p 1 was inactivated by E-64. Data presented are the means of duplicate experiments; SE was <5%.

Figure 2

Figure 4

Control experiments showing that the IgE-specific effect observed in Fig. 3 is not due to E-64 exerting a suppressive influence on IgE production by a mechanism that is independent of its binding to the Der p 1 enzyme active site. Total IgE (a) and Der p 1–specific IgE (b) levels in final bleeds obtained from groups of 10 mice immunized with proteolytically active (black bar) or inactive (iodoacetamide [hatched bar]- or E-64–blocked [white bar]) Der p 1 measured by ELISA. There was a statistically significant suppression of total IgE and Der p 1–specific IgE responses in animals immunized with Der p 1 that was rendered proteolytically inactive by either iodoacetamide (P < 0.04 for total IgE and P < 0.05 for Der p 1–specific IgE) or E-64 (P < 0.01 for total IgE and P < 0.02 for Der p 1–specific IgE). Total IgE (c) and OVA-specific IgE (d) levels in final bleeds obtained from groups of 10 mice immunized with OVA (black bar) or OVA plus E-64 (white bar) were measured by ELISA. Prebleeds showed no measurable levels of antibody (except for baseline levels of total IgE). Error bars represent 95% confidence intervals.

Figure 4

References

1. Sutton B.J., Gould H.J. The human IgE network. Nature. 1993;366:421–428. - PubMed
1. Tovey E.R., Chapman M.D., Platts-Mills T.A.E. Mite faeces are a major source of house dust allergens. Nature. 1981;289:592–593. - PubMed
1. Shakib F., Schulz O., Sewell H.F. A mite subversivecleavage of CD23 and CD25 by Der p 1 enhances allergenicity. Immunol. Today. 1998;19:313–316. - PubMed
1. Schulz O., Sewell H.F., Shakib F. Proteolytic cleavage of CD25, the α subunit of the human T cell interleukin 2 receptor, by Der p 1, a major mite allergen with cysteine protease activity. J. Exp. Med. 1998;187:271–275. - PMC - PubMed
1. Abbas A.K., Murphy K.M., Sher A. Functional diversity of helper T lymphocytes. Nature. 1996;383:787–793. - PubMed

The cysteine protease activity of the major dust mite allergen Der p 1 selectively enhances the immunoglobulin E antibody response - PubMed (original) (raw)