LYSOSOMAL PROTEOLYSIS OF PROSAPOSIN, THE PRECURSOR OF SAPOSINS (SPHINGOLIPID ACTIVATOR PROTEINS) - ITS MECHANISM AND INHIBITION BY GANGLIOSIDE

Saposins A, B, C, and D, which are required for the enzymatic hydrolys is of sphingolipids by specific lysosomal hydrolases, are produced by proteolytic processing of their common precursor protein, prosaposin. Our previous observation suggested that lysosomal cathepsin D may be i nvolved in the proteolysis of prosaposin. Herein we report the involve ment of cathepsin D in the proteolytic processing of prosaposin. An an tibody against human placental cathepsin D blocked the proteolytic act ivity toward prosaposin in a human testicular lysosomal protease mixtu re (glycoprotein fraction). On immunoblot analysis using a monoclonal antibody against human saposin C, cathepsin D showed a similar proteol ytic pattern as that of a human testicular glycoprotein fraction and h ydrolyzed prosaposin into products of 48 and 29 kDa. The K-m and V-max values were 0.9 mu M and 167 nmol/h/mg, respectively. N-Terminal sequ ence analysis indicated that the 48-kDa band was a mixture of two tris aposins, including domains for saposins A, B, and C and saposins B, C, and D, respectively. A similar study also showed that the 29-kDa band contained two disaposins, including domains for saposins A and B and saposins C and D, respectively. By longer treatment with cathepsin D, disaposins were further processed into mature saposin A and small frag ments (14.5-17.5 kDa) containing individual saposins and portions of i nterdomain sequences. These small fragments were no longer processed b y cathepsin D, but trimmed to fragments having similar molecular sizes (10.5-11.5 kDa) to those of mature saposins by a rat lysosome prepara tion. These findings indicated that cathepsin D is involved in the mat uration of saposins but that, in addition to cathepsin D, other protea ses appear to be involved in the maturation of saposin B, C, and D in lysosomes. Gangliosides, which specifically form complexes with prosap osin and saposins, inhibit proteolysis of prosaposin by cathepsin D. T his finding indicates that prosaposin may be protected from lysosomal proteolysis by forming a complex with gangliosides in vivo. (C) 1997 A cademic Press.