| Budget Amount *help |
¥6,800,000 (Direct Cost: ¥6,800,000)
Fiscal Year 1990: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1989: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1988: ¥4,400,000 (Direct Cost: ¥4,400,000)
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| Research Abstract |
A novel protease (PSGPase), capable of depolymerizing highly glycosylated polyprotein [cf., K Kitajima et al. (1986) J Biol. Chem 261, 5262-5269 ; Mr 200x10^3 PSGP] to 9-kDa PSGP[see S. Inoue & Y. Inoue (1986) J Biol. Chem 261, 5256-5261] upon fertilization of rainbow trout eggs, was isolated and purified 2100-fold from the unfertilized eggs. The enzyme apparently catalyzes the "prolinedirected pre-aspartyl cleavage" at temperature between 10 and 20^0C and only at ionic strength below 40 mM. The specificity may be compared with the cleavage of N-terminal to a pair of basic residues or , proline-directed monobasic arginyl cleavage, known for the processing enzymes acting on precursors of regulatory peptides. PSGPase is colocalized, as an inactive state within asubcellular compartment (cortical alveolus) of the unfertilized eggs together with its physiological substrates, 200-kDa PSGP. Upon fertilization exocytosis of the cortical alveoli occurred and their contents were translocated to
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perivitelline space where the polyprotein structure of 200-kDa PSGP was converted to the repeating unit (9-kDa PSGP) by PSGPase of which activity might be regulated by salt concentration.
Inhibitor studies were carried out on the purified PSGPase. The PSGPase activity was inhibited almost completely by PCMB at 1mM, but the cysteine-protease inhibitor E-46 had little or no effect on PSGPase activity and the presence of reducing agents such as dithiothreitol in all reaction media is not necessary for enzyme activity. These results collectively suggest PSGPase not to be a thiol-protease. Although the presence of 1 mM of phenylmethanesulfonyl fluoride (PMSF) had almost no effect on PSGPase activity, diisopropyl phosphorofluoridate (DFP), at high concentrations interfered with the PSGPase activity, indicating PSGPase likely to be a serine-protease. None of the metalloprotease inhibitors blocked the degradation of 200-kDa PSGP.
In 1978, Inoue and Iwasaki discovered a novel class of acidic glycoproteins in the unfertilized eggs of rainbow trout. The most striking feature of these glycoproteins is the presence of oligo (ploy) sialyl chains in the prosthetic carbohydrate groups, and we named them polysialoglycoproteins (PSGP). We have shown that PSGP is a ubiquitous components of both mature and immature oocytes of all species of salmonid fishes studied. The present study was first undertaken to elicit antibodies against polysialic acids simply because alpha-2, 8-linked homopolymer of N-acetylneuraminic acid (NeuAc) structure occurring in the group B meningococci (Neisseria meningitidis is pathogenic. It is also well known that alpha-2, 8-linked poly (NeuAc) is extremely poor immunogen in various animals including human. Rainbow trout egg PSGP contains exclusively N-glycolylneuraminic acid (NeuGc) , so that we used it as the immunogen to elicit alpha-2, 8-linked poly (NeuGc) antibodies in rabbits and BALB/c mouse. We continued the antigen injection for a long period (up to 16 months), but there was no detectable antibodies in the serum of all animals used when tested by a double immunodiffusion procedure and by enzyme-linked immunosorbent assays. The observed poor immunogenicity is again ascribed, at least in part, to the presence of molecules having similar or identical structures in the host animals so that immunization would likely be suppressed by immune tolerance. Recently, we succeeded in eliciting rainbow trout egg PSGP antiserum in chicken.
We also examined the immunoreactivity of alpha-2, 8-linked poly (NeuGc) chains against a unique antiserum obtained by J. B. Robbins (N. I. H.) after taking for years by immunizing a horse using formalin-treated group B meningococci as immunogen. The antiserum (H. 46) containg polyclonal IgM antibodies to bind alpha-2, 8-linked poly (NeuAc) has been proven to form immunopricipitin bands against alpha-2, 8-linked poly (NeuGc). It can be concluded that H. 46 binds specifically to alpha-2, 8-linked poly (NeuAc) and can only be used as a probe for poly (NeuAc). In this respect, our results on chicken, though preliminary, may provide specific antibody against poly (NeuGc) which may possibly be used as a diagnostic tool for detecting poly (NeuGc) in a variety of animal tissues and species. Less
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