Purification and structural analysis of the novel glycoprotein allergen Cyn d 24, a pathogenesis-related protein PR-1, from Bermuda grass pollen.
Bermuda grass pollen (BGP) contains a very complex mixture of allergens, but only a few have been characterized. One of the allergens, with an apparent molecular mass of 21 kDa, has been shown to bind serum IgE from 29% of patients with BGP allergy. A combination of chromatographic techniques (ion exchange and reverse phase HPLC) was used to purify the 21 kDa allergen. Immunoblotting was performed to investigate its IgE binding and lectin-binding activities, and the Lysyl-C endopeptidase digested peptides were determined by N-terminal sequencing. The cDNA sequence was analyzed by RACE PCR-based cloning. The protein mass and the putative glycan structure were further elucidated using MALDI-TOF mass spectrometry. The purified 21 kDa allergen was designated Cyn d 24 according to the protocol of International Union of Immunological Societies (IUIS). It has a molecular mass of 18,411 Da by MALDI-TOF analysis and a pI of 5.9. The cDNA encoding Cyn d 24 was predicted to produce a 153 amino acid mature protein containing tow conserved sequences seen in the pathogen-related protein family. Carbohydrate analysis showed that the most abundant N-linked glycan is a alpha(3)-fucosylated pauci-mannose (Man3GlcNAc2) structure, without a Xyl beta-(1,2)-linked to the branching beta-Man. Thus, Cyn d 24 is a glycoprotein and the results of the sequence alignment indicate that this novel allergen is a pathogenesis-related protein 1. To the best of our knowledge, this is the first study to identify any grass pollen allergen as a pathogenesis-related protein 1.
Chow
Lu-Ping
LP
Graduate Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan.
Chiu
Li-Li
LL
Khoo
Kay-Hooi
KH
Peng
Ho-Jen
HJ
Yang
Sue-Yee
SY
Huang
Shih-Wen
SW
Su
Song-Nan
SN
D000485:Allergens / Q000737:chemistry* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D052179:Antigens, Plant / Q000737:chemistry* / Q000302:isolation & purification; D002241:Carbohydrates / Q000032:analysis; D002845:Chromatography; D031744:Cynodon / Q000276:immunology*; D006023:Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification; D007073:Immunoglobulin E / Q000378:metabolism; D037102:Lectins / Q000378:metabolism; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification; D011058:Pollen / Q000276:immunology*; D016415:Sequence Alignment
D000485:Allergens; D052179:Antigens, Plant; D002241:Carbohydrates; C507393:Cyn d 24 allergen, Cynodon dactylon; D006023:Glycoproteins; D037102:Lectins; D010940:Plant Proteins; C053376:pathogenesis-related proteins, plant; D007073:Immunoglobulin E