Cathepsin G: Localization in human cerebral cortex and generation of amyloidogenic fragments from the β-amyloid precursor protein

Abstract

Amyloid deposits in Alzheimer's disease, Down's syndrome and aged brain are composed largely of Aβ protein, which is generated by proteolytic processing of β-amyloid precursor protein. Proteases responsible for liberating the Aβ protein from the precursor have not yet been identified. Here, we examined the ability of cathepsin G, a chymotrypsin-like protease, to cleave two protease substrates: (i) a fluorogenic hexapeptide, whose sequence spans the cleavage site in the precursor for generating the Aβ NH 2-terminus, and (ii) recombinant human β-amyloid precursor protein purified from a baculovirus expression system. Unlike two other members of the chymotrypsin family, cathepsin G readily degraded the hexapeptide. Furthermore, cathepsin G cleaved the β-amyloid precursor protein to generate several breakdown products, including a prominent 11,500 mol. wt fragment immunoreactive with antibodies directed against the COOH-terminus of the protein. This COOH-terminal fragment co-migrated using two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis with C-100, a recombinant COOH-terminal segment of the β -amyloid precursor, whose NH 2-terminus is one residue upstream of the NH 2-terminus of the Aβ domain. We also examined the localization of cathepsin G in human brain. The distribution of cathepsin G-containing cells was examined by immunohistochemistry in the temporal cortex of both Alzheimer's and aged control samples. Cathepsin G-like immunoreactivity was contained specifically within neutrophils. As visualized by double-labeling with antibodies to cathepsin G and Factor VIII, neutrophils were most frequently found within meningeal or cortical blood vessels. In addition, occasional neutrophils could be identified without an apparent vascular surround, in the brain parenchyma. By simultaneous labeling with antibodies to cathepsin G and Aβ protein, neutrophils were also sometimes found associated with both parenchymal and vessel amyloid deposits; however, these associations were rare. These findings indicate that cathepsin G is capable of cleaving the β-amyloid precursor protein to liberate the free NH 2-terminus of the Aβ protein and may have access to areas where this material is deposited in Alzheimer's disease. However, since there is no physical association between neutrophils and deposited amyloid and no increase in the number of neutrophils in an Alzheimer's brain, cathepsin G seems to be an unlikely mediator of amyloid deposition in this disease.