title: Lysosomal proteolysis of prosaposin, the precursor of saposins (sphingolipid activator proteins): its mechanism and inhibition by ganglioside.
abstract: Saposins A, B, C, and D, which are required for the enzymatic hydrolysis 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 involved in the proteolysis of prosaposin. Herein we report the involvement of cathepsin D in the proteolytic processing of prosaposin. An antibody against human placental cathepsin D blocked the proteolytic activity toward prosaposin in a human testicular lysosomal protease mixture (glycoprotein fraction). On immunoblot analysis using a monoclonal antibody against human saposin C, cathepsin D showed a similar proteolytic pattern as that of a human testicular glycoprotein fraction and hydrolyzed prosaposin into products of 48 and 29 kDa. The Km and Vmax values were 0.9 microM and 167 nmol/h/mg, respectively. N-Terminal sequence analysis indicated that the 48-kDa band was a mixture of two trisaposins, 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 fragments (14.5-17.5 kDa) containing individual saposins and portions of interdomain sequences. These small fragments were no longer processed by cathepsin D, but trimmed to fragments having similar molecular sizes (10.5-11.5 kDa) to those of mature saposins by a rat lysosome preparation. These findings indicated that cathepsin D is involved in the maturation of saposins but that, in addition to cathepsin D, other proteases appear to be involved in the maturation of saposin B, C, and D in lysosomes. Gangliosides, which specifically form complexes with prosaposin and saposins, inhibit proteolysis of prosaposin by cathepsin D. This finding indicates that prosaposin may be protected from lysosomal proteolysis by forming a complex with gangliosides in vivo.
authors.affiliation: Department of Neurosciences, School of Medicine, University of California at San Diego, La Jolla 92093, USA. mhiraiwa@ucsd.edu
authors.forename: M
authors.initials: M
authors.lastname: Hiraiwa
authors.forename: B M
authors.initials: BM
authors.lastname: Martin
authors.forename: Y
authors.initials: Y
authors.lastname: Kishimoto
authors.forename: G E
authors.initials: GE
authors.lastname: Conner
authors.forename: S
authors.initials: S
authors.lastname: Tsuji
authors.forename: J S
authors.initials: JS
authors.lastname: O'Brien
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology; D002402:Cathepsin D / Q000276:immunology / Q000378:metabolism*; D002554:Cerebrosides / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism*; D004789:Enzyme Activation; D005260:Female; D005732:Gangliosides / Q000494:pharmacology*; D006023:Glycoproteins / Q000378:metabolism* / Q000494:pharmacology; D006454:Hemoglobins / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000201:enzymology / Q000378:metabolism; D008247:Lysosomes / Q000201:enzymology* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D051381:Rats; D017208:Rats, Wistar; D049231:Saposins; D017421:Sequence Analysis; D013433:Sulfoglycosphingolipids / Q000494:pharmacology
chemical_list: D000906:Antibodies; D002554:Cerebrosides; D005732:Gangliosides; D006023:Glycoproteins; D006454:Hemoglobins; C483345:PSAP protein, human; D010446:Peptide Fragments; D011498:Protein Precursors; C483347:Psap protein, rat; D049231:Saposins; D013433:Sulfoglycosphingolipids; D010450:Endopeptidases; D002402:Cathepsin D