| Project/Area Number |
07670291
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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 |
寄生虫学(含医用動物学)
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| Research Institution | Juntendo University School of Medicine |
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Principal Investigator |
YAMASAKI Hiroshi Department of Parasitology, Juntendo University School of Medicine, Lecturer, 医学部, 講師 (00138207)
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| Project Period (FY) |
1995 – 1996
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| Project Status |
Completed (Fiscal Year 1996)
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| Budget Amount *help |
¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 1996: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1995: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Keywords | Fasciola sp. / cysteine protease / cathepsin L / gene structure / recombinant procathepsin L / protease activity / in vitro processing / カテプシンL遺伝子 / PCR / TATAbox |
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| Research Abstract |
(1) Structure and organization of Fasciola cathepsin L precursor genes.
Two distinct genes encoding cathepsin L from adult Fasciola sp.worms were amplified by polymerase chain reaction using oligonucleotide primers synthesized on the basis of nucleotide sequences of two cDNAs encoding Fasciola cathepsin L.PCR products amplified using Fasciola genomic DNA as a template were approximately 1.9 and 1.8 kb in size. Both Fasciola cathepsin L genes consist of 4 exons and 3 introns, but each exon did not correspond to functional units of the enzymes as well as other eukariotic cysteine proteases. Both genes showed high homologies, but remarked differences was seen in the sizes and nucleotide sequences of the third introns. Southern blot analysis revealed that Fasciola cathepsin L genes exist as multigene family not tandem repeats, in contrast to mammalian cathepsin L gene is a single copy. The transcripts of two cathepsin L genes were approximately 1.1 kb in size by northern blot analysis and d
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id not contain spliced leader sequences in their 5'-ends.
(2) Expression and in vitro processing of recombinant Fasciola procathepsin L.
The Fasciola procathepsin L was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase, and the processing mechanism of the procathepsin L to mature cathepsinL was studied. The insoluble fusion protein was solubilized in 8M urea and refolded at pH 10.7 in the pre sence of reduced and oxidized glutathiones. The 38kDa-procathepsin L was obtained by cleavage he fusion protein with thrombin. The recombinant procathepsin L was processed to an enzymatically active 26kDa-cathepsin L at pH4.5-5.5. The processing was completely inhibited by an irreversible cysteine protease inhibitor. These results indicate that in vitro processing of an inactive procathepsin L to the active cathepsin L occur by pH-dependent autocatalytic mechanism. NH2-terminal amino acid sequence of the processed form was different form that of the native cathepsin L produced in Fasciola worm, suggesting that further processing may occur in vivo. On the other hand, the recombinant cathepsin L deleted the entire propeptide resulted in complete loss of enzyme activity, suggesting that the propeptide region of the procathepsin L is essential for the folding and/or refolding of the functional cathepsin L. Less
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