Construction of the novel functional proteins composed of calcium-dependent lectin and bioactive peptides
| Project/Area Number |
12660082
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | Nagasaki University |
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Principal Investigator |
HATAKEYAMA Tomomitsu Nagasaki University, Faculty of Engineering, Associate Professor, 工学部, 助教授 (50228467)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2001: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2000: ¥2,700,000 (Direct Cost: ¥2,700,000)
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| Keywords | lectin / carbohydrate-binding prtein / calcium / invertebrate / self-defense / hemolysin / toxin / X-ray crystallography / カルシウムイオン / 両親媒性ペプチド / 溶血活性 / 抗菌活性 / クローニング / タンパク質コンジュゲート |
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| Research Abstract |
A novel protein composed of rat mannose-binding lectin (MBL) and bee venom peptide melittin (Mel-MBL), was expressed in Escherichia coll cells using the plasmid PET-32a. The protein expressed as a fusion protein with thioredoxin (Trx-Mel-MBL) was then cleaved with enterokinase to produce Mel-MBL. When Mel-MBL was incubated with erythrocytes and bacterial cells, hemolytic as well as antibacterial activities were observed. These activities were found to be dependent on the carbohydrate-binding ability ofMBL. The marine invertebrate Cucumaria echinata contains the hemolytic lectin CEL-III. The amino acid sequence of this protein suggested that N-terminal two-thirds are carbohydrate binding domains and C-terminal one-third is a putative oligomerization domain. Several synthetic peptides having the parts of the amino acid sequence of the C-terminal domain were produced, and their properties were examined. As a result, P332 corresponding to the sequence beginning at position 332 exhibited strong antibacterial activity for Gram-positive bacteria, especially Staphylococcus aureus. This activity was found to result from the membrane perturbing ability of this peptide, leading to the increase in permeabilization of the inner membrane of the bacteria. X-ray crystallographic analysis of the C type lectin CEL-I in C. echinata was also performed in order to investigate the carbohydrate-recognition mechanism of this lectin. From the analysis of CEL-I and its complex with N-acetylgalactosamine (GalNAc), it was revealed that CEL I recognizes GalNAc through binding with Ca^<2+>, and very high specificity was enabled by hydrogen bonds formed between carbonyl oxygen and the side chain ofarginine 1 15 of CEL-I. These results could facilitate the designing of novel lectins with altered carbohydrate-binding specificity.
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Report
(3 results)
Research Products
(15 results)
