Limited proteolytic processing of the mature form of cathepsin D in human and mouse brain: Postmortem stability of enzyme structure and activity

Abstract

The mature form of cathepsin D (Cat D), purified to homogeneity from postmortem human brain or mouse brain, behaved as a 42-kDa protein in its native state but revealed additional proteolytic processing under denaturing conditions. Human brain Cat D was composed of a 30–32 kDa heavy chain and a protein doublet consisting of 14 and 15 kDa light chains. Mouse Cat D, which closely resembled the human enzyme in amino acid composition, existed mainly as the uncleaved 42-kDa protein, but up to 40% existed as a complex of 30–32 kDa and 12–14 kDa chains. The 3 : 1 ratio of light to heavy (30–32 kDa) chains suggested processing of some 30-kDa chains. Cleavage of the 42-kDa chain could not be induced autolytically. Human brain Cat D had a 2–3-fold higher specific activity than the mouse enzyme but shared other properties, including similar biphasic pH optima (peaks at pH 3.0 and 4.2), K values for methemoglobin and inhibitor profiles. Human Cat D displayed the same polypeptide chain composition when purified from brains differing in postmortem interval (3–28 h). Fresh SH-SY5Y human neuroblastoma cells analyzed on Western blots with anti-Cat D antibodies also displayed only cleaved forms of mature Cat D. Furthermore, brain Cat D isolated from mice stored after death for 5, 15 or 30 h at 25°C contained the same molar ratios of cleaved and uncleaved enzyme found in fresh mouse brain. Cat D activity was stable in human brains with postmortem intervals up to 27 h and stored frozen for up to 3 years. Similarly, total Cat D activity was essentially unchanged in brains of mice subjected to simulated postmortem conditions for 0.5–42 h, although 20% of the total soluble brain protein became insoluble during this postmortem interval. These results demonstrate a remarkable postmortem stability of Cat D and strongly suggest that limited proteolytic cleavage of mature brain Cat D is an in vivo event, the extent of which varies markedly in different species.