Maturation of Pichia pastoris‐derived recombinant pro‐Der p 1 induced by deglycosylation and by the natural cysteine protease Der p 1 from house dust mite

Abstract

The mature cysteine protease from Dermatophgoides pteronyssinus, Der p 1, is a major house dust mite allergen. Its enzymatic activity has been shown to have pro‐inflammatory effects that could also negatively influence efficacy of allergen‐specific immunotherapy. The aim of this study was to express recombinant pro‐Der p 1 (rpro‐Der p 1) in the yeast Pichia pastoris and to study its maturation. Expression was achieved at a concentration ranging from 45 mg·L−1 (methanol‐induced expression) to 168 mg·L−1 (constitutive expression). No significant spontaneous maturation of the secreted proenzyme was observed. rpro‐Der p 1 with a sequence‐based molecular mass of 34 kDa was hyperglycosylated by the yeast, migrating at 50–60 kDa on SDS/PAGE. Compared with its natural counterpart (nDer p 1), the recombinant proenzyme demonstrated decreased IgE reactivity, resulting in a 30‐fold lower capacity to induce histamine release from human basophils. Decreased immunoreactivity was also shown by competitive RIA and sandwich ELISA with Der p 1‐specific antibody reagents. CD spectra of rpro‐Der p 1 and nDer p 1 revealed significant structural differences. Deglycosylation of rpro‐Der p 1 with endoglycosidase H resulted in a decrease in apparent molecular mass from 50 kDa to 34 kDa, but did not affect nDer p 1. On removal of N‐glycans from rpro‐Der p 1, which harbours two putative N‐glycosylation sites in both propeptide and mature sequence, the mature rDer p 1 appeared. This suggests that hyperglycosylation hampers spontaneous maturation. Maturation of the recombinant pro‐enzyme was also achieved by addition of the active natural cysteine protease, nDer p 1. In conclusion, high‐level expression of rpro‐Der p 1 in P. pastoris results in a stable hypoallergenic proenzyme with potential for use in allergen‐specific immunotherapy.

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

“…This observation is in good agreement with that of previous studies of the influence of glycosylation on the activation process. This hypothesis could also explain the different results reported by Jacquet et al and van Oort et al 16,17 van Oort et al did not succeed in obtaining maturation of WT recombinant ProDer p 1 with the correct N terminus, 17 whereas Jacquet et al reported that the WT ProDer p 1 lacking the first 19 residues was easily converted into mature allergen after acidic treatment. 16 However, the fact that the ProDer p 1 N52Q mutant also presents the ATFE intermediate at the end of the production may be attributed to the heterogeneous glycosylation of the propeptide as previously proposed.…”

“…17,18 In this study, we investigated the influence of the different potential N-glycosylation sites in the maturation process and the pH influence on the propeptide association.…”

“…13,14 Finally, several experiments performed with ProDer p 1 mutants lacking the N-glycosylation sites or with endoglycosylase H-pretreated ProDer p 1 indicated that the glycosylation of recombinant zymogen expressed in P. pastoris can impair the maturation of the allergen. 17,18 Analysis of the X-ray structure of the zymogen form of the allergen reveals that the propeptide of Der p 1 adopts a new fold within the CA1 protease family 19 ( Fig. 1).…”

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

“…The proposed preference for small aliphatic residues and charged residues in subsites S2 and S1, respectively, is of particular interest because proDer p 1 is cleaved after the A79-E80 sequence during maturation. It is therefore plausible that mature Der p 1 is capable of activating other proDer p 1 molecules, which experiments using nDer p 1 to process rproDer p 1 have shown (47). The theoretical isoelectric point of mature wt Der p 1 (Swiss-Prot P08176) is 5.6 in contrast to the classical papaya cysteine endopeptidases, which have a basic theoretical pI (e.g., 9.6 for papain, P00784).…”

“…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).…”