Dimerization of the N-terminal amphipathic alpha-helix domain of the fungal immunomodulatory protein from Ganoderma tsugae (Fip-gts) defined by a yeast two-hybrid system and site-directed mutagenesis.
A fungal immunomodulatory protein (Fip-gts) was purified from Ganoderma tsugae. The DNA encoding Fip-gts was isolated from a cDNA library of G. tsugae by reverse transcriptase-polymerase chain reaction. The complete amino acid sequence of Fip-gts, deduced from the nucleotide sequence of the cDNA, was the same as LZ-8 isolated from Ganodermn lucidum. Recombinant Fip-gts was expressed as a glutathione S-transferase fusion protein in Escherichia coli with a yield of 20 mg/liter of culture. Recombinant Fip-gts, purified to homogeneity, had the same blast formation stimulatory activity to human peripheral blood lymphocytes as native Fip-gts. The yeast two-hybrid system and site-directed mutagenesis were used to determine whether dimerization of Fip-gts occurred. Deletion analysis of the N-terminal amphipathic alpha-helix domain of Fip-gts identified a sequence of about 10 amino acids responsible for inducing immunomodulatory activity. Non-functional Fip-gts deletion mutants did not form dimers, whereas wild type Fip-gts did as determined by gel filtration. A mutant with deletions at Leu-5, Phe-7, and Leu-9 lost the amphipathic characteristics of the N-terminal domain and the ability to form dimers as well as its immunomodulatory activity. Fusion of Fip-gts with the DNA binding and the transactivation domains of GAL4 resulted in the activation of the lacZ activator gene, indicating the interaction of Fip-gts with it itself. The dimerization domain was further defined by analyzing the ability of the N-terminal 13 amino acids or Leu-5, Phe-7, and Leu-9 deletion mutants of Fip-gts to interact with the wild type Fip-gts. These experiments confirmed the N-terminal amphipathic alpha-helix as the dimerization domain and suggest that the dimerization of Fip-gts may play an important role in Fip-gts immunomodulatory activity.
Lin
W H
WH
Institute of Biochemistry, College of Medicine, National Taiwan University, Taipei 100, Taiwan, Republic of China.
Hung
C H
CH
Hsu
C I
CI
Lin
J Y
JY
D000276:Adjuvants, Immunologic / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001487:Basidiomycota; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents / Q000378:metabolism; D016207:Cytokines / Q000096:biosynthesis; D019281:Dimerization; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D037102:Lectins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D016415:Sequence Alignment
D000276:Adjuvants, Immunologic; D003432:Cross-Linking Reagents; D016207:Cytokines; D005656:Fungal Proteins; D037102:Lectins; D011994:Recombinant Proteins