2001 Fiscal Year Final Research Report Summary
Prostaspanin: A novel member of tetraspanin superfamily specifically expressed in the prostate
| Project/Area Number |
12671538
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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 |
Urology
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| Research Institution | Kagawa Medical University |
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Principal Investigator |
NISHI Nozomu Kagawa Medical University, Dept. of Endocrinology, Research Associate, 医学部, 助手 (10145047)
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| Co-Investigator(Kenkyū-buntansha) |
TAKETA Shigeo Kagawa Medical University, Dept. of Urology, Assistant Professor, 医学部・附属病院, 講師 (10227027)
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| Project Period (FY) |
2000 – 2001
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| Keywords | prostate / androgen / tetraspanin / subtraction / growth |
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| Research Abstract |
We have isolated cDNA clones which are specifically expressed during androgen-dependent regrowth of rat ventral prostate by using PCR-based subtractive hybridization technique. One of the clones, AIEG322 (tentatively designated as prostaspanin), showed significant sequence homology with those of the members of tetraspanin superfamily. Among rat tissues examined, highlevels of prostaspanin mRNA expression was detected in the prostate and seminal vesicle. To investigate physiological function of prostaspanin in the prostate and to evaluate the possibility of utilization of the serum prostaspanin concentration as a marker of prostatic cancer, we set the highest priority for preparing anti-prostaspanin antibodies. Prostaspanin cDNAs were amplified from cDNAs derived from rat and human prostates, and the expression vectors for rat and human prostaspanin with a GST- and a hexahistidine-tag were prepared. The recombinant proteins were expressed in E. coli. However, the expressed proteins could not be solubilized, because of the formation of inclusion bodies. Due to the difficulties in using the recombinant proteins as antigens, we started to produce anti-oligopeptide antibodies. The use of an synthetic oligopeptide, FTQVWNTTM, corresponding to one of the extracellular domains of prostapanin (EC2), resulted in the production of high titer antiserum in rabbits. The antiserum recognized recombinant rat and human prostaspanin in Western blot analysis. Western blot analysis using rat tissue extracts and membrane fractions, however, showed that the antiserum could not react with native prostaspnin. Glycosylation at the aspargine residue in the nativ prostaspanin may explain the observation. Currently, we are trying to prepare anti-prostaspanin antibodies using a synthetic oligopeptide corresponding to different regions of the protein.
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