Substitution of Pichia pastoris-derived recombinant proteins with mannose containing O- and N-linked glycans decreases specificity of diagnostic tests.
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.


CONCLUSIONS
Production of recombinant molecules in yeast (or moulds) can introduce IgG-binding glycans that negatively affect the specificity of diagnostic tests.
Sanquin, Department of Immunopathology and Laboratory for Experimental and Clinical Immunology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
Erica
E
van Oort
Patrice
P
Lerouge
Pleuni G
PG
de Heer
Martial
M
Séveno
Laurent
L
Coquet
Piet W
PW
Modderman
Loïc
L
Faye
Rob C
RC
Aalberse
Ronald
R
van Ree
D000485:Allergens / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D000595:Amino Acid Sequence; D039741:Antigens, Dermatophagoides / Q000235:genetics / Q000276:immunology; D052179:Antigens, Plant; D060829:Arthropod Proteins; D001229:Aspergillosis, Allergic Bronchopulmonary / Q000175:diagnosis / Q000276:immunology; D001324:Autoantigens / Q000276:immunology; D003546:Cysteine Endopeptidases; D004797:Enzyme-Linked Immunosorbent Assay; D006031:Glycosylation; D014890:Granulomatosis with Polyangiitis / Q000175:diagnosis* / Q000276:immunology; D006801:Humans; D007073:Immunoglobulin E / Q000097:blood / Q000276:immunology; D007074:Immunoglobulin G / Q000097:blood; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry / Q000276:immunology*; D011852:Radioallergosorbent Test / Q000592:standards*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D012680:Sensitivity and Specificity
D000485:Allergens; D039741:Antigens, Dermatophagoides; D052179:Antigens, Plant; D060829:Arthropod Proteins; D001324:Autoantigens; C444200:Dac g 5 allergen; D007074:Immunoglobulin G; D011134:Polysaccharides; D011994:Recombinant Proteins; D007073:Immunoglobulin E; D003546:Cysteine Endopeptidases; C451162:Dermatophagoides pteronyssinus antigen p 1