Limited proteolytic processing of the mature form of cathepsin D in human and mouse brain: postmortem stability of enzyme structure and activity.
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 3.30 and 4.2), Km 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 degrees 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 stimulated postmortem conditions for 0.5-4.2 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.
Compaine
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Laboratories for Molecular Neuroscience, McLean Hospital, Harvard Medical School, Belmont, MA 02178, USA.
Schein
J D
JD
Tabb
J S
JS
Mohan
P S
PS
Nixon
R A
RA
D000368:Aged; D000375:Aging / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002402:Cathepsin D / Q000737:chemistry* / Q000378:metabolism*; D004355:Drug Stability; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D008810:Mice, Inbred C57BL; D009447:Neuroblastoma / Q000378:metabolism / Q000473:pathology; D010447:Peptide Hydrolases / Q000378:metabolism*; D011180:Postmortem Changes; D013997:Time Factors; D014407:Tumor Cells, Cultured
D010447:Peptide Hydrolases; D002402:Cathepsin D