Studies on Differentiation Mechanism of Dictyostelium discoideum

Research Project

Project/Area Number	12680642
Research Category	Grant-in-Aid for Scientific Research (C)
Allocation Type	Single-year Grants
Section	一般
Research Field	Functional biochemistry
Research Institution	Kansai Medical University
Principal Investigator	FUJII Shigeru Kansai Medical University, Professor, 医学部, 教授 (60144482)
Co-Investigator(Kenkyū-buntansha)	NAKAGAWA Manabu Kansai Medical University, Assistant, 医学部, 助手 (50261053)
Project Period (FY)	2000 – 2001
Project Status	Completed (Fiscal Year 2001)
Budget Amount *help	¥3,600,000 (Direct Cost: ¥3,600,000) Fiscal Year 2001: ¥1,600,000 (Direct Cost: ¥1,600,000) Fiscal Year 2000: ¥2,000,000 (Direct Cost: ¥2,000,000)
Keywords	Dictyostelium / differentiation factor / glycoprotein / protein structure / sugar chains / 細胞性粘菌
Research Abstract	In the previous study, we have purified a factor inducing prespore cell differentiation with an apparent molecular mass of 106 kDa on SDS-PAGE, which we named psi-factor (prespore-cell-inducing factor). We analysed four partial amino acid sequences of the purified psi-factor, and synthesized an oligonucleotide corresponding to one partial amino acid sequence. By using it as a probe, we isolated cDNA clones encoding psi-factor. They contained the full-length psi-factor open reading frame with 1674 mucleotides, which encoded a protein of 557 amino acid residues. The amino acid sequence shows that the factor is a novel protein containing both proline- and cysteine- rich regions. The analysis of the N-terminal sequence of the purified psi-factor indicates that the first 19 amino acid residues are severed and the matured secreted psi-factor consists of 538 amino acid residues with a molecular mass of 60 kDa. This value is much smaller than the apparent molecular mass estimated by SDS-PAGE.. The difference may come from addition of sugar chains to the protein. The amino acid sequence shows six possible Asn-glycosylation sites, Asn-Xaa-Ser/Thr motifs. When psi-factor was treated with endoglycosidase H, its apparent molecular mass decreased on SDS-PAGE. It was strongly bound with ConA-agarose. These results indicated that psi-factor is a glycoprotein with Asn-linked carbohydrates. Knockout mutant Ax2 strains of the psi-factor gene were prepared by targeted integration. Rescuing the mutant strains by transforming actin 15-psi-factor expression vector caused overproduction of psi-factor protein and higher prespore-cell inducing activity. These results show that psi-factor is one of important differentiation factors in Dictyosteliium discoideum.