Identification of the post-translational modification sites and allergenic determinants of BG60

• Main Authors: Ming-Sian Gu, 古明憲
• Other Authors: Lu-Ping Chow
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/48788997779441174772

碩士 === 國立臺灣大學 === 生物化學暨分子生物學研究所 === 91 === Abstract Approximately 20% of the human population in industrialized countries suffer IgE-mediated (Type I ) allergic diseases such as asthma, conjunctivitis and rhinitis. The major cause of outdoor allergy is from airbone grass pollen. Bermuda grass (Cynodon dactylon) is the dominant source of allergen and contains a very complex mixture of antigen. BG60 has been demonstrated to be an important allergen of Bermuda grass pollen and identified as a glycoprotein. Purification by a combination of chromatographic technique (ion exchange, gel filtration, and blue gel affinity) led to a homogenous protein fraction of BG60. The cDNA of BG60 has been cloned. Comparison of the BG60 sequence with protein database revealed that it shared high homology with berberine bridge enzyme (BBE), a covalently flavinylated oxidase, of plants. Therefore, it suggests that the cofactor FAD is covalently linked to the central domain of BG60, and BG60 will be first identified as a flavinylated allergen. Using program “ScanProsite” predicts that the BG60 sequences have many post-translational modification sites such as N-linked glycosylation site (NXS/T) and phosphorylation site. These modification sites and allergenic determinants of BG60 are investigated in the present study. (1) Identification of the BG60 allergenic determinants BG60 was digested by V8 protease to generate various peptide fragments which were then separated by HPLC and dot-blotted to PVDF membrane. IgE binding peptide fragments were detected by immunoblotting and analyzed by MALDI-TOF. Combining the MS and Protscale analysis of BG60, we found that IgE binding peptides located at the flexible or hydrophilic regions of the molecules. (2) Identification of the flavin binding site Yellow-green fraction of BG60 was digested by trypsin and the peptides with fluorescence absorption were separated by HPLC. The fluorescent peptide fractions were collected and analyzed by MALDI-TOF and ESI-ION-TRAP. According to MS analysis of the fluorescent peptide fractions, we found a fraction with m/z value close to the predicted FAD-binding fragment (S110GGHDYEGLYR121) plus FAD, and suggested that was the possible flavinylated peptide fragment. (3) Identification of glycosylation sites BG60 was limitedly digested by Trypsin or Lys-C endopeptidase. The glycosylation sites of BG60 were investigated by lectin binding assay and N-terminal sequencing. According to the analyses, we identified a N-linked glycosylation site (N354RTS357) at C-terminus domains of BG60. (4) Identification of phosphorylation sites BG60 was cleaved by treating with CNBr and Formic acid. The mixture of BG60 fragments was analyzed by MALDI-TOF MS. Combining the “NetPhos v2.0” and PSD (post-source decay) MS/MS analysis, we identified a phosphopeptide (PEDyFRNEQSIPPLL) fragment.