Inspection of the conserved regions of plant chitinases participating in yieldin activity : Site-directed mutagenesis and computer-aided prediction of a three-dimensional structure
| Project/Area Number |
14540592
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
植物生理
|
|---|
| Research Institution | Nagoya University |
|---|
Principal Investigator |
KATOU Kiyoshi Nagoya University, Graduate School of Science, Professor, 理学研究科, 教授 (00109258)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
TAKAHASHI Koji Nagoya University, Graduate School of Science, Assistant Professor, 理学研究科, 助手 (40283379)
KIDO Nobuo Nagoya University, Graduate School of Science, Associate Professor, 理学研究科, 助教授 (80161511)
|
|---|
| Project Period (FY) |
2002 – 2003
|
| Project Status |
Completed (Fiscal Year 2003)
|
| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2003: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2002: ¥2,000,000 (Direct Cost: ¥2,000,000)
|
|---|
| Keywords | Elongation growth / Cell wall / Acid growth theory / Yield threshold tension / Yieldin / Chitinase / Site-directed mutagenesis / Monoclonal antibody / 伸長生長 / 臨界降伏張 / 機能阻害 / 部位特異変異 |
|---|
| Research Abstract |
Yieldin, a cell wall protein purified from elongating hypocotyls of cowpea, decreases the yield threshold tension (y) of the wall in an acidic condition. We constructed a high-level expression system of recombinant yieldin (rYLD) in Eseherichia coli. rYLD restored the acid-induced changes of y of heat-denatured glycerinated hollow cylinders (GHCs) of cowpea hypocotyls. Yieldin shares over 40% identity in the amino acid sequence with plant chitinases belonging to the glycosyl hydrolase family 18, and it has two conserved consensus regions involving a putative active site. However, no chitinase activity could be detected for yieldin. A computer-aided prediction of a 3D structure suggested that yieldin has a single domain with a (β/α)_8 barrel fold as concanavalin B and hevamine. We investigated effects of substitution of a glutamic acid residue in the conserved consensus regions of yieldin by site-directed mutagenesis. The resulted rYLD (E128Q) lost the yieldin activity in the stress-strain experiments. Substitutions of other amino acid residues located in the two consensus regions conserved in the family also resulted in loosening the yieldin activity. However, rYLD having site-directed mutation in other than the consensus regions was difficult to be expressed. Thus the role of the conserved region in the regulatory activity of yield threshold tension remained to be clarified Monoclonal antibodies against rYLD were successfully produced. One of them was found to inhibit the function of yieldin. We are now investigating its antigenic determinant.
|
Report
(3 results)
Research Products
(15 results)
