Substitution of <i>Pichia pastoris</i>-Derived Recombinant Proteins with Mannose Containing O- and N-Linked Glycans Decreases Specificity of Diagnostic Tests

Abstract

Background: Recombinant proteins from Pichia pastoris need to be fully evaluated before used as diagnostic tools. Objective: The objective of this study was to investigate whether glycosylation by P. pastoris interferes with the specificity of diagnostic tests. Methods: An autoantigen involved in Wegener’s disease (protease 3) and 2 major inhalant allergens from grass pollen (Dac g 5) and house dust mite (Der p 1) were produced as recombinant molecules in P. pastoris. O-linked glycans on Dac g 5 were characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The immune reactivity of the recombinant proteins was compared to that of their natural counterparts by ELISA and a radio-allergosorbent test (RAST) as well as by ELISA and RAST inhibition. Results: In contrast to the non-glycosylated natural allergen, recombinant Dac g 5 was shown to carry at least 2 small mannose-containing O-glycans. We showed that both these O-glycans and the N-linked glycans on recombinant protease 3 and recombinant Der p 1 were recognized in ELISA by IgG antibodies in sera of healthy individuals. These IgG responses were closely correlated. The natural autoantigen and allergens were not recognized by IgG antibodies from healthy subjects. The carbohydrate nature of the epitopes recognized by IgG on the recombinant proteins was confirmed by inhibition studies with mannose and yeast mannan. IgE recognition of yeast glycans was observed in 2 out of 9 positive sera from patients with allergic bronchopulmonary aspergillosis. Conclusion: Production of recombinant molecules in yeast (or moulds) can introduce IgG-binding glycans that negatively affect the specificity of diagnostic tests.

Innate affairs of allergens

“…In addition, Bla g 4 is probably glycosylated [85]. Important aerollergens that are not glycosylated are Der p 2 [135], Bet v 1 [35], group 5 grass allergens [17,136] and Amb a 1 [137]. Natural Der p 2 by mass spectros-copy has precisely the molecular weight expected from its polypeptide chain and this agrees with the lack of an N-glycosylation and O-glycosylation motifs [135].…”

The purification and application of biologically active recombinant steroid cytochrome P450 21-hydroxylase: The major autoantigen in autoimmune Addison's disease

“…Still, insoluble recombinant proteins that require denaturing and refolding are frequent in yeast-based systems [26,27]. Yeast also tend to hyperglycosylate recombinant proteins, which may introduce IgGbinding glycans and lead to false-positive results [28]. The baculovirus expression system has repeatedly been shown to allow for overexpression of correctly structured and functionally active antigens, as well as a wide range of cytochrome P450 enzymes [24,29].…”

Glycosylation of Recombinant Proforms of Major House Dust Mite Allergens Der p 1 and Der f 1 Decelerates the Speed of Maturation

“…Der p 1-N52Q and Der f 1-N53Q are considered to be useful for studies in vitro and in vivo as alternatives to natural Der p 1 and Der f 1, because they exhibit similar molecular sizes, secondary structures, and allergenicities as their natural counterparts [18, 19], have enzymatic activity to cleave human protein substrates in vitro, have IgE-eliciting activity in vivo [10], and are considered free from the yeast-derived N-linked glucan-specific IgG antibodies detectable in sera of healthy individuals [28]. IgE-binding affinity and basophil histamine-releasing activity of the proforms are lower than those of the mature recombinant and natural Der p 1 and Der f 1 [18].…”