Molecular and biological study on sialidase
| Project/Area Number |
02671018
|
|---|
| Research Category |
Grant-in-Aid for General Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Research Field |
Biological pharmacy
|
|---|
| Research Institution | Niigata College of Pharmacy |
|---|
Principal Investigator |
UDA Yutaka Ph.D Niigata College of Pharmacy Professor, 薬学部, 教授 (90013937)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
HIRAIWA Masao Ph.D Niigata College of Pharmacy Assistant, 薬学部, 助手 (40148127)
|
|---|
| Project Period (FY) |
1990 – 1991
|
| Project Status |
Completed (Fiscal Year 1991)
|
| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1991: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1990: ¥1,200,000 (Direct Cost: ¥1,200,000)
|
|---|
| Keywords | sialidase / alpha-galactosidase B / alpha-n-acetylgalactosaminidase / cDNA cloning / prosaposin / シアリダ-ゼ / αーガラクトシダ-ゼB / αーNーアセチルガラクトサミニダ-ゼ / cDNAクロ-ニング |
|---|
| Research Abstract |
In the process of molecular cloning of cDNA for proteins associated with a purified human placental sialidase fraction, we discovered one of the proteins with apparent molecular weight of 46 kDa is in reality alpha-N-acetylgalactosaminidase. The full length cDNA, pcD-HS1204, codes for 358 amino acids with the first 17 residues representing a putative signal peptide. The predicted amino acid sequence shows striking homology with human alpha-galactosidase A and yeast alpha-galactosidase. The substrate specificities as well as the behavior of the 46 kDa protein on hydroxylapatite chromatography confirmed that the 46 kDa protein is in reality alpha-N-acetylgalactosaminidase.
Two species of cDNAs for human alpha-N-acetylgalactosaminidase were isolated from a human fibroblast cDNA library. The two species differ each other by a 70 bp insertion in the coding region. Transient expression study in COS cells demonstrated that only the cDNA without the 70 bp insertion expressed alpha-N-acetylgalac
… More
tosaminidase activity. Analysis of mRNA species utilizing polymerase chain reaction revealed that the majority of the mRNA does not contain the 70 bp insertion, and the mRNA containing the 70 bp insertion is present only in a minor amount in human brain.
The activation mechanism of human placental sialidase was studied. We observed that not only the crude enzyme preparation but also the partially purified preparation of sialidase was activated by the incubation at 37ﾟC in pH 4.8 medium. The activation inhibited by amastatin which is an inhibitor of aminopeptidase A and leucine aminopeptidase. The partially purified sialidase preparation used for the activation released leucine and glutamic acid but not arginine, lysine and alanine from methylcoumarlamide(MCA) derivatives of amino acid. Zinc ion significantly depressed the activation of sialidase and also inhibited the hydrolysis of leucine-MCA. The activation of sialidase seems to be caused by an endogeneous protease, probably aminopeptidase.
Sialidase isolated from human placenta is associated with several protein components, which are thought to form an aggregated complex during isolation of sialidase. One of the proteins of 60 kDa was recently identified by Potier et al. as a sialidase protein; this protein also cross-reacted with anti-prosaposin antibodies. We have isolated this protein and from the following evidence identified it as a heavy chain component of immunoglobulin G and not sialidase or a derivative of prosaposin. On gel filtration HPLC, sialidase activity and the 60 kDa protein were clearly separated from one another. The 60 kDa protein cross-reacted not only with antibodies raised against human saposine A,C, and D, but also with second antibody (goat anti-rabbit immunoglobulin G antibody) alone. This 60 kDa protein strongly cross-reacted with anti-human immunoglobulin G antibodies. The sequence of the initial 15 amino acids from the N-terminus of the 60 kDa protein was identical to the sequence of an immunoglobulin G heavy chain protein Tie(gamma1). Less
|
Report
(3 results)
Research Products
(27 results)
