title: 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.
authors.affiliation: CLB Department of Immunopathology and Laboratory for Experimental and Clinical Immunology, Academic Medical Center, University of Amsterdam, the Netherlands.
authors.forename: Erica
authors.initials: E
authors.lastname: van Oort
authors.forename: Pleuni G
authors.initials: PG
authors.lastname: de Heer
authors.forename: W Astrid
authors.initials: WA
authors.lastname: van Leeuwen
authors.forename: Ninotska I L
authors.initials: NI
authors.lastname: Derksen
authors.forename: Marcel
authors.initials: M
authors.lastname: Müller
authors.forename: Stephan
authors.initials: S
authors.lastname: Huveneers
authors.forename: Rob C
authors.initials: RC
authors.lastname: Aalberse
authors.forename: Ronald
authors.initials: R
authors.lastname: van Ree
mesh_terms: D000485:Allergens / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D002942:Circular Dichroism; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D006031:Glycosylation; D006868:Hydrolysis; D007073:Immunoglobulin E / Q000276:immunology; D008925:Mites; D010843:Pichia / Q000235:genetics*; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D000485:Allergens; D017931:DNA Primers; D011994:Recombinant Proteins; D007073:Immunoglobulin E; D003546:Cysteine Endopeptidases