| Summary: | 碩士 === 國立臺灣大學 === 生物化學暨分子生物學研究所 === 93 === BG60 is an important allergen of Bermuda grass (Cynodon dactylon) pollen, which causes allergic diseases such as asthma, hey fever and allergic rhinitis. According to previous reports, BG60 has been identified as an N-linked glycoprotein and suggested that its carbohydrate moiety may be relevant to allergic diseases. Furthermore, the major structures of carbohydrate moiety were characterized as a paucimannosidic-type. In particular, a 24-residue internal peptide sequence was determined by N-terminal protein sequencing, which showed similarity to a conserved flavin-binding motif. Spectroscopic analysis also indicated BG60 was a flavoprotein. The aim of this study is to analyze these post-translational modification sites of BG60 including glycosylation and flavin-binding site.
The analysis of N-linked glycosylation sites was performed by comparing the profile of MALDI-TOF MS spectra of protease-digested (trypsin and lys-C, respectively) peptide mixture with and without PNGase A treatment. The sequence of deglycosylated peptides were confirmed by MALDI MS/MS analysis. Four N-linked glycosylation sites were identified in BG60 : Asn88, Asn325, Asn354, Asn477. Furthermore, the four N-linked glycopeptides were purified by reverse-phase HPLC. The glycosylation pattern of each site was determined by MALDI-TOF MS. Our results may provide an insight into the microheterogeneity of glycoproteins.
In order to identify the putative flavin-binding site, purified BG60 was limiting digested with trypsin and separated by SDS-PAGE, only the protein band which showed yellow-green fluorescence under UV illumination was subjected to in-gel tryptic digestion. The fluorescent peptide was purified using reverse-phase HPLC by monitoring of excitation at 450 nm and emission at 520 nm; which is the characterization of flavin. N-terminal protein sequence analysis of the fluorescent peptide indicated that the fluorescent compound is linked to His113. However, it was not proved by the result of MALDI-TOF MS and MS/MS analysis that the fluorescent compound purified from BG60 is a flavin. It was suggested that an unknown compound covalently linked to BG60 such as flavin derivatives that might result in similar fluorescent properties as flavin.
By combination of enzymatic digestion, HPLC separation and mass spectrometric analysis, we have identified four N-linked glycosylation sites and an unknown fluorescent compound binding site. This strategy may provide an efficient and useful tool for the analysis of post-translational modification sites of protein.
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