Analysis of the mechanisms of beta-amyloid accumulation by 68kDa serine protease with beta-secretase activity

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(1) Establishment of the efficient preparation method for the 68kDa serine protease and its related enzymes utilizing antibody-specific and ligand-specific affinity chromatography.(2) Establishment of an in vitro system mimicking human brain extracellular matrix to analyze functions of these proteases.(3) Analysis of aggregation and degradation of natural Abeta-harboring substrates. Highmolecular weight aggregated product was most efficiently generated when C-terminal substrates were co-incubated with collagen type 4. On the other hand, N-terminal substrates with extracellular domains of APP were excellent substrates for this protease, and it seems that the efficiency of proteolysis is related to the glycoconjugate binding do main within beta-amyloid.(4) Analysis using sugar-modifying enzymes revealed that human brain 68kDa serine protease contains heparan sulfate giycoconjugates. This finding implies that both substrate and protease are modified by glycoconjugates, suggesting their significance in specificity and topological restriction of proteolysis in vivo.(5) Structural analysis of cDNA encoding the protease.(6) Future prospects : Several proteases with similar biochemical properties were prepared from human brain by antibody-specific and ligand-specific affinity chromatography. Further study of these enzymes seems to be important in view of a series of serine proteases participating in blood coagulation. Isolation and identification of specific inhibitors are also of interest to understand the functions of these proteases.