Glycoproteomic analysis of seven major allergenic proteins reveals novel post-translational modifications.
Allergenic proteins such as grass pollen and house dust mite (HDM) proteins are known to trigger hypersensitivity reactions of the immune system, leading to what is commonly known as allergy. Key allergenic proteins including sequence variants have been identified but characterization of their post-translational modifications (PTMs) is still limited. Here, we present a detailed PTM(1) characterization of a series of the main and clinically relevant allergens used in allergy tests and vaccines. We employ Orbitrap-based mass spectrometry with complementary fragmentation techniques (HCD/ETD) for site-specific PTM characterization by bottom-up analysis. In addition, top-down mass spectrometry is utilized for targeted analysis of individual proteins, revealing hitherto unknown PTMs of HDM allergens. We demonstrate the presence of lysine-linked polyhexose glycans and asparagine-linked N-acetylhexosamine glycans on HDM allergens. Moreover, we identified more complex glycan structures than previously reported on the major grass pollen group 1 and 5 allergens, implicating important roles for carbohydrates in allergen recognition and response by the immune system. The new findings are important for understanding basic disease-causing mechanisms at the cellular level, which ultimately may pave the way for instigating novel approaches for targeted desensitization strategies and improved allergy vaccines.
From the ‡Copenhagen Center for Glycomics and Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, DK-2200 Copenhagen N, Denmark;
Adnan
A
Halim
From the ‡Copenhagen Center for Glycomics and Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, DK-2200 Copenhagen N, Denmark;
Michael C
MC
Carlsson
From the ‡Copenhagen Center for Glycomics and Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, DK-2200 Copenhagen N, Denmark;
Caroline Benedicte
CB
Madsen
§Global Research, ALK, Hørsholm, Denmark;
Stephanie
S
Brand
From the ‡Copenhagen Center for Glycomics and Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, DK-2200 Copenhagen N, Denmark; ¶Department of Plant and Environmental Biology, Glyco section, Faculty of Science, University of Copenhagen 1871 Frederiksberg C, Denmark.
Svenning Rune
SR
Møller
¶Department of Plant and Environmental Biology, Glyco section, Faculty of Science, University of Copenhagen 1871 Frederiksberg C, Denmark.
Carl Erik
CE
Olsen
From the ‡Copenhagen Center for Glycomics and Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, DK-2200 Copenhagen N, Denmark;
Sergey Y
SY
Vakhrushev
§Global Research, ALK, Hørsholm, Denmark;
Jens
J
Brimnes
§Global Research, ALK, Hørsholm, Denmark;
Peter Adler
PA
Wurtzen
§Global Research, ALK, Hørsholm, Denmark;
Henrik
H
Ipsen
From the ‡Copenhagen Center for Glycomics and Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, DK-2200 Copenhagen N, Denmark; ¶Department of Plant and Environmental Biology, Glyco section, Faculty of Science, University of Copenhagen 1871 Frederiksberg C, Denmark.
Bent L
BL
Petersen
From the ‡Copenhagen Center for Glycomics and Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, DK-2200 Copenhagen N, Denmark; hhw@sund.ku.dk blp@plen.ku.dk.
Hans H
HH
Wandall
D000485:Allergens / Q000378:metabolism*; D039741:Antigens, Dermatophagoides / Q000378:metabolism*; D052179:Antigens, Plant / Q000378:metabolism*; D029662:Betula; D013058:Mass Spectrometry; D031784:Phleum; D010940:Plant Proteins / Q000378:metabolism*; D011058:Pollen; D011134:Polysaccharides / Q000378:metabolism*; D011499:Protein Processing, Post-Translational
D000485:Allergens; D039741:Antigens, Dermatophagoides; D052179:Antigens, Plant; C076049:Phl p V protein, Phleum pratense; D010940:Plant Proteins; D011134:Polysaccharides; C061312:Bet v 1 allergen, Betula; C090396:PHLPI protein, Phleum pratense