CATHEPSIN-G - LOCALIZATION IN HUMAN CEREBRAL-CORTEX AND GENERATION OFAMYLOIDOGENIC FRAGMENTS FROM THE BETA-AMYLOID PRECURSOR PROTEIN

Amyloid deposits in Alzheimer's disease, Down's syndrome and aged brai n are composed largely of A beta protein, which is generated by proteo lytic processing of beta-amyloid precursor protein. Proteases responsi ble for liberating the A beta protein from the precursor have not yet been identified. Here, we examined the ability of cathepsin G, a chymo trypsin-like protease, to cleave two protease substrates: (i) a fluoro genic hexapeptide, whose sequence spans the cleavage site in the precu rsor for generating the A beta NH2-terminus, and (ii) recombinant huma n beta-amyloid precursor protein purified from a baculovirus expressio n system. Unlike two other members of the chymotrypsin family, catheps in G readily degraded the hexapeptide. Furthermore, cathepsin G cleave d the beta-amyloid precursor protein to generate several breakdown pro ducts, including a prominent 11,500 mol. wt fragment immunoreactive wi th antibodies directed against the COOH-terminus of the protein. This COOH-terminal fragment co-migrated using two-dimensional isoelectric f ocusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis with C-100, a recombinant COOH-terminal segment of the beta-amyloid precur sor, whose NH2-terminus is one residue upstream of the NH2-terminus of the A beta domain. We also examined the localization of cathepsin G i n human brain. The distribution of cathepsin G-containing cells was ex amined by immunohistochemistry in the temporal cortex of both Alzheime r's and aged control samples. Cathepsin G-like immunoreactivity was co ntained specifically within neutrophils. As visualized by double-label ing with antibodies to cathepsin G and Factor VIII, neutrophils were m ost frequently found within meningeal or cortical blood vessels. In ad dition, occasional neutrophils could be identified without an apparent vascular surround, in the brain parenchyma. By simultaneous labeling with antibodies to cathepsin G and A beta protein, neutrophils were al so sometimes found associated with both parenchymal and vessel amyloid deposits; however, these associations were rare. These findings indic ate that cathepsin G is capable of cleaving the beta-amyloid precursor protein to liberate the free NH2-terminus of the A beta protein and m ay have access to areas where this material is deposited in Alzheimer' s disease. However, since there is no physical association between neu trophils and deposited amyloid and no increase in the number of neutro phils in an Alzheimer's brain, cathepsin G seems to be an unlikely med iator of amyloid deposition in this disease.