Purification and structural analysis of the novel glycoprotein allergen Cyn d 24, a pathogenesis‐related protein PR‐1, from Bermuda grass pollen

Open Access
Publisher Website
Google Scholar
Abstract
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 α(3)‐fucosylated pauci‐mannose (Man3GlcNAc2) structure, without a Xyl β‐(1,2)‐linked to the branching β‐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.