MICROGLIAL CATHEPSIN-B - AN IMMUNOLOGICAL EXAMINATION OF CELLULAR ANDSECRETED SPECIES

The cysteine proteinase cathepsin B (CB) was isolated from immortalize d murine BV-2 microglial cells and examined via sodium dodecyl sulfate -polyacrylamide gel electrophoresis and immunoblotting to establish ph ysicochemical properties of CB from what is generally considered the r esident CNS macrophage. Microglial proteases have been implicated in s everal pathological processes occurring in the CNS, including neurodeg eneration. Murine microglial CB was observed to consist of two major s ingle-chain species of 32 and 34 kDa, with pls of 5.5-5.2 and 5.1-4.5, respectively. In addition, a minor 24-kDa CB species was also observe d in some microglial preparations. The major CB isozymes in microglia differed from those observed in murine liver and brain, which consiste d of both single- and double-chain CB variants of 31 and 24-25 kDa/5 k Da, respectively, with pl values of 5.5-4.5. A microglial pro-CB of 37 kDa was also isolated, which could be processed to the 34-kDa single- chain CB species. Cystatin was observed to prevent pro-CB processing, whereas E-64 and leupeptin were only partially inhibitory. The 37-kDa pro-CB species was observed to undergo processing into the 34-kDa CB s pecies when incubated at pH 5.5 but remained stable with respect to mo lecular mass when incubated at pH 7.0. In contrast, the 34-kDa single- chain CB species was observed to autodegrade when incubated at pH 7.0, whereas incubation at pH 5.5 did not affect the integrity of the spec ies as monitored by immunoblotting. Both pro-CB and 32-kDa single-chai n CB species were observed extracellularly following lipopolysaccharid e activation of BV-2 microglial cells.