Determination of N-Glycosylation Site and Glycan Structures of Pectin Methylesterase in Jelly Fig (Ficus awkeotsang) Achenes
2009
Hsiao, Eric S.L. | Chen, Jeff C.F. | Tsai, Hsien-Yu | Khoo, Kay-Hooi | Chen, Shui-Tein | Tzen, Jason T.C.
Pectin methylesterase (PME) in jelly fig (Ficus awkeotsang) achenes is an N-glycosylated enzyme responsible for the gelation of jelly curd. A recombinant jelly fig PME was overexpressed in Escherichia coli and confirmed by immunodetection and LC-nanoESI-MS/MS analysis. To identify the N-glycosylation site, native PME and its deglycosylated and recombinant forms, which lacked glycan, were purified and subjected to comparative MALDI-MS mapping of the corresponding tryptic fragments. The results showed that N-glycosylation occurred at Asn153 of the mature jelly fig PME, the only glycosylation site predicted by its sequence analysis. The major N-glycans released from the native PME by PNGase F were identified by MS/MS analyses as xylosylated, noncore fucosylated pauci-mannose, and complex-type structures. Molecular modeling of the 3D structure of jelly fig PME indicated that the N-glycan was putatively attached to the back region of the active site of this enzyme.
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