Microglial cathepsin B: an immunological examination of cellular and secreted species.
The cysteine proteinase cathepsin B (CB) was isolated from immortalized murine BV-2 microglial cells and examined via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting to establish physicochemical properties of CB from what is generally considered the resident CNS macrophage. Microglial proteases have been implicated in several pathological processes occurring in the CNS, including neurodegeneration. Murine microglial CB was observed to consist of two major single-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 observed in some microglial preparations. The major CB isozymes in microglia differed from those observed in murine liver and brain, which consisted of both single- and double-chain CB variants of 31 and 24-25 kDa/5 kDa, 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 species when incubated at pH 5.5 but remained stable with respect to molecular 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 species as monitored by immunoblotting. Both pro-CB and 32-kDa single-chain CB species were observed extracellularly following lipopolysaccharide activation of BV-2 microglial cells.
Ryan
R E
RE
Department of Molecular Biology, Mayo Clinic, Jacksonville, Florida 32224, USA.
Sloane
B F
BF
Sameni
M
M
Wood
P L
PL
D000818:Animals; D001921:Brain / Q000201:enzymology; D002401:Cathepsin B / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000378:metabolism; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007526:Isoelectric Point; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D008297:Male; D051379:Mice; D009457:Neuroglia / Q000201:enzymology*; D011480:Protease Inhibitors / Q000494:pharmacology
D003470:Culture Media; D004792:Enzyme Precursors; D007527:Isoenzymes; D011480:Protease Inhibitors; D002401:Cathepsin B