Relationship between Propeptide pH Unfolding and Inhibitory Ability during ProDer p 1 Activation Mechanism

Abstract

The major allergen Der p 1 of the house dust mite Dermatophagoides pteronyssinus is a papain-like cysteine protease (CA1) produced as an inactive precursor and associated with allergic diseases. The propeptide of Der p 1 exhibits a specific fold that makes it unique in the CA1 propeptide family. In this study, we investigated the activation steps involved in the maturation of the recombinant protease Der p 1 expressed in Pichia pastoris and the interaction of the full-length and truncated soluble propeptides with their parent enzyme in terms of activity inhibition and BIAcore interaction analysis. According to our results, the activation of protease Der p 1 is a multistep mechanism that is characterized by at least two intermediates. The propeptide strongly inhibits unglycosylated and glycosylated recombinant Der p 1 (K D = 7 nM) at neutral pH. This inhibition is pH dependent. It decreases from pH 7 to pH 4 and can be related to conformational changes of the propeptide characterized by an increase of its flexibility and formation of a molten globule state. Our results indicate that activation of the zymogen at pH 4 is a compromise between activity preservation and propeptide unfolding.

Der p 1 is the primary activator of Der p 3, Der p 6 and Der p 9 the proteolytic allergens produced by the house dust mite Dermatophagoides pteronyssinus

“…Unglycosylated recombinant mature Der p 1 (rDer p 1) was obtained from proDer p 1 produced in P. pastoris as described previously [10,11]. The reaction mixtures contained 8 μM substrate (S1-S9) and rDer p 1 (0.04 μM for S3 and S6 and 2.32 μM for S1 and S9) or HDM extract (1 mg/ml) in polybuffer (mixture of 50 mM Tris, phosphate, citrate, acetate and KCl containing 1 mM DTT and 1 mM EDTA, adjusted to pH 7.4), with or without the addition of the cysteine protease inhibitor L-trans-epoxysuccinyl-leucylamido (4-guanidino)butane (E-64, 100 μM) or the serine protease inhibitor soybean trypsin inhibitor (SBTI, 10 μM).…”

“…The recombinant proDer p 1 protease (N-64A, N52A), fused with a C-terminal six-histidine sequence, was produced in P. pastoris and then purified and activated to a mature protease as described previously [10]. This enzyme was incubated with MagneHis™ Ni-Particles (Promega, Madison, USA) overnight at 37°C, and the particles with coated Der p 1 were washed with PBS containing 1 mM DTT and EDTA prior to incubation with proDer p 6 for 8 h at 37°C.…”

“…The E64 inhibitor (100 μM), SBTI (10 μM) and/or the Der p 1 propeptide (10 μM, produced and purified as previously described [10]) were also added to the HDM extract prior to incubation with proDer p 6. The samples were analysed via SDS-PAGE, and Der p 6 activity was measured in 20 mM Tris, pH 7, using the Ala-Ala-Pro-Phe-AMC substrate (200 μM).…”

“…To further confirm the role of cysteine protease Der p 1 in the activation mechanism of proDer p 6, HDM extracts were pre-incubated with the highly specific inhibitory propeptide of Der p 1 (K D 7 nM [10]). The rate of activation of proDer p 6 in the presence of such HDM extracts was drastically delayed (Fig.…”

“…The Der p 1 propeptide (80 amino acids) plays an essential role in zymogen folding (as an intramolecular chaperone) and in the inhibition of its own enzymatic activity. ProDer p 1 was shown to activate itself through a multistep mechanism at an acidic pH [10].…”