Functional and structural analysis of GMI, a fungal immunomodulatory protein from Ganoderma microsporum
Date Issued
2011
Date
2011
Author(s)
Weng, Jui-Yun
Abstract
Fungal immunomodulatory proteins (FIPs) were immune functional proteins existed in the medical fungus. Based on the protein structural alignment of different species, the first loop from C terminus showed great difference in the conformation. Therefore, in this study, we tried to investigate that whether the stereo conformational difference in this loop area contributes to the immune activity.
Here we used the mutated recombinant FIP, GMI-L6C, as the topic to study the relationship between immune activity and function. GMI-L6C was a mutated protein from GMI, which was the FIP from Ganoderma microsporum. The sixth amino acid was mutated from leucine to cysteine. The disulfide bond stabilized the homo dimer structure of GMI-L6C. Using GMI-L6C as the base to conduct the site-specific mutagenesis in the final loop amino acid sequence, the effect was reduced by its shift between dimer and momer form while changing the resudues. In addition, the mutations were carried out in 2 sets of single amino acid mutation based on either residue charge or size and sequence deletion. For deletion, the b-sheet sequence downstream the final loop was deleted to let the loop structure as a free form tail, or delete the amino acid on the loop region to shorten the loop size. Human T cell lymphoblast-like cell line (Jurkat cell line) was used as the assay cells to test the immune activity mutated GMI-L6C protein. IL-2 secretion by Jurkat T cell after protein stimulation indicated the mutagenesis effect on the loop structure and its immune functional change.
On the other side, to further investigate the importance of FIP dimer structure, the sixth amino acid mutagenesis protein GMI-L6K and GMI-L6F were used to express the proteins, and the unstable dimer FIP were expected to be obtained.
Taken together the recombinant protein GMI and GMI-L6C were not able to stimulation Jurkat T cell to secrete IL-2 in E. coli expression system. In methyltrophic yeast Pichia pastoris system, the IL-2 secretion was up to 709.7±199.1 pg/ml in the recombinant protein GMI-L6C. The result of GMI-L6K and GMI-L6F were lower than 10 pg/ml and the 98th amino acid mutated protein GMI-L6C&D98N and GMI-L6C&D98A were lower than 50 pg/ml in the secretion of IL-2. The results suggested that the unstable dimer form of GMI decreased the immune activity and showed the importance of the 98th amino acid in modulating immune function.
Subjects
Fungal immunomodulatory proteins (FIPs)
Ganoderma microsporum
cytokine expression
Type
thesis
File(s)![Thumbnail Image]()
Loading...
Name
ntu-100-R98b47401-1.pdf
Size
23.32 KB
Format
Adobe PDF
Checksum
(MD5):1d43b53bfa76fcf365e843ccdceb4c40