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Lysosomal Proteolysis of Prosaposin, the Precursor of Saposins (Sphingolipid Activator Proteins): Its Mechanism and Inhibition by Ganglioside

Hiraiwa, Masao, Martin, Brian M., Kishimoto, Yasuo, Conner, Gregory E., Tsuji, Shoji, O'Brien, John S.·1997·Archives of Biochemistry and Biophysics
DOI 10.1006/abbi.1997.9958
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FIG. 1 Proteolytic degradation of prosaposin. (a) Effect of an antihuman cathepsin D antibody on the proteolytic activity in human testicular glycoprotein fraction. Prosaposin (0.5 m g) was incubated in the absence or presence of control or immunoabsorbed human testicular glycoprotein fraction (2 m g protein). After 1 h incubation at 37 7 C, the digests were subjected to SDS-PAGE (10% gel) and followed by immunoblot using a monoclonal antibody against saposin C. (b) Proteolysis of prosaposin by human cathepsin D. Prosaposin (0.5 m g) was digested with a human testicular glycoprotein fraction (2 m g protein) or purified human cathepsin D (20 ng protein) for 60 min at 37 7 C and followed by immunoblot as described in (a). (c) Kinetic study of the digestion of prosaposin by cathepsin D. Prosaposin (5-20 pmol) was incubated with cathepsin D (100 ng) for 60 min at 37 7 C and followed by immunoblot as described in (a). The quantitation of prosaposin was performed by an Image Analyzer.
FIG. 1 Proteolytic degradation of prosaposin. (a) Effect of an antihuman cathepsin D antibody on the proteolytic activity in human testicular glycoprotein fraction. Prosaposin (0.5 m g) was incubated in the absence or presence of control or immunoabsorbed human testicular glycoprotein fraction (2 m g protein). After 1 h incubation at 37 7 C, the digests were subjected to SDS-PAGE (10% gel) and followed by immunoblot using a monoclonal antibody against saposin C. (b) Proteolysis of prosaposin by human cathepsin D. Prosaposin (0.5 m g) was digested with a human testicular glycoprotein fraction (2 m g protein) or purified human cathepsin D (20 ng protein) for 60 min at 37 7 C and followed by immunoblot as described in (a). (c) Kinetic study of the digestion of prosaposin by cathepsin D. Prosaposin (5-20 pmol) was incubated with cathepsin D (100 ng) for 60 min at 37 7 C and followed by immunoblot as described in (a). The quantitation of prosaposin was performed by an Image Analyzer.CC-BY-NC-ND · From the PDF
Image 2
CC-BY-NC-ND · From the PDF
Image 3
CC-BY-NC-ND · From the PDF
FIG. 4 An overall proteolytic processing scheme of prosaposin. Domain structure of prosaposin with the proteolytic cleavage sites is shown in the upper section. Signal peptide is shown as a solid box and saposin domains are depicted as shaded boxes. Sites of proteolytic cleavage are shown by bar with N-terminal amino acids found in the products.
FIG. 4 An overall proteolytic processing scheme of prosaposin. Domain structure of prosaposin with the proteolytic cleavage sites is shown in the upper section. Signal peptide is shown as a solid box and saposin domains are depicted as shaded boxes. Sites of proteolytic cleavage are shown by bar with N-terminal amino acids found in the products.CC-BY-NC-ND · From the PDF
FIG. 5 Effect of ganglioside GM1 concentration on the proteolysis of prosaposin. (a) Prosaposin (25 pmol) was preincubated with different amounts of ganglioside GM1 (10 pmol-1 nmol) to form a complex and then added to 20 ng of cathepsin D. After incubation for 1 h at 37 7 C, the digests were analyzed as described in the legend to Fig. 1a. (b) Prosaposin-ganglioside GM1 complex (25 pmol/250 pmol) was incubated with rat lysosome preparation for 2 h at 37 7 C. The digests were analyzed as described in the legend to Fig. 1a. (c) The amounts of prosaposin hydrolyzed and trisaposins and disaposins yielded were quantitated by an Image Analyzer. n00 , Prosaposin hydrolyzed; , trisaposins; , disaposins.
FIG. 5 Effect of ganglioside GM1 concentration on the proteolysis of prosaposin. (a) Prosaposin (25 pmol) was preincubated with different amounts of ganglioside GM1 (10 pmol-1 nmol) to form a complex and then added to 20 ng of cathepsin D. After incubation for 1 h at 37 7 C, the digests were analyzed as described in the legend to Fig. 1a. (b) Prosaposin-ganglioside GM1 complex (25 pmol/250 pmol) was incubated with rat lysosome preparation for 2 h at 37 7 C. The digests were analyzed as described in the legend to Fig. 1a. (c) The amounts of prosaposin hydrolyzed and trisaposins and disaposins yielded were quantitated by an Image Analyzer. n00 , Prosaposin hydrolyzed; , trisaposins; , disaposins.CC-BY-NC-ND · From the PDF
FIG. 6 Effects of ganglioside GM1 on the activity of cathepsin D toward hemoglobin. Hydrolytic activity of the purified cathepsin D (20 ng) toward denatured hemoglobin in the presence or absence of ganglioside GM1 was determined by the procedure described by Takahashi and Tang (22). After digestion, resultant trichloroacetic acid-soluble peptides were measured at 280 nm. The activity toward prosaposin in the absence or presence of ganglioside GM1 was measured by the procedure described in the legend to Fig. 5a. s , Hemoglobin; l , hemoblobin / ganglioside GM1; j , prosaposin / ganglioside GM1.
FIG. 6 Effects of ganglioside GM1 on the activity of cathepsin D toward hemoglobin. Hydrolytic activity of the purified cathepsin D (20 ng) toward denatured hemoglobin in the presence or absence of ganglioside GM1 was determined by the procedure described by Takahashi and Tang (22). After digestion, resultant trichloroacetic acid-soluble peptides were measured at 280 nm. The activity toward prosaposin in the absence or presence of ganglioside GM1 was measured by the procedure described in the legend to Fig. 5a. s , Hemoglobin; l , hemoblobin / ganglioside GM1; j , prosaposin / ganglioside GM1.CC-BY-NC-ND · From the PDF
FIG. 7 Effects of various sphingolipids and phospholipids on the proteolysis of prosaposin. Prosaposin (0.5 m g) was preincubated with various lipids (10 nmol) for 30 min in 20 m l of 0.1 M sodium acetate buffer, pH 4.0, containing 0.1 M NaCl. After addition of 20 ng cathepsin D, the mixtures were incubated for 2 h at 37 7 C and analyzed by immunoblot as described in the legend to Fig. 1a.
FIG. 7 Effects of various sphingolipids and phospholipids on the proteolysis of prosaposin. Prosaposin (0.5 m g) was preincubated with various lipids (10 nmol) for 30 min in 20 m l of 0.1 M sodium acetate buffer, pH 4.0, containing 0.1 M NaCl. After addition of 20 ng cathepsin D, the mixtures were incubated for 2 h at 37 7 C and analyzed by immunoblot as described in the legend to Fig. 1a.CC-BY-NC-ND · From the PDF

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Metadata

Authors
Hiraiwa, M., Martin, B. M., Kishimoto, Y., Conner, G. E., Tsuji, S., & O'Brien, J. S.
Venue
Archives of Biochemistry and Biophysics
Publisher
Elsevier BV
Published
1997-05
Volume
341
Issue
1
Pages
17-24
ISSN
0003-9861
DOI
10.1006/abbi.1997.9958
Cited by
120
References
0
Language
en
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CoChat. (2026). Citation check: Lysosomal Proteolysis of Prosaposin, the Precursor of Saposins (Sphingolipid Activator Proteins): Its Mechanism and Inhibition by Ganglioside. https://app.cochat.ai/papers/10.1006/abbi.1997.9958
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Hiraiwa, M., Martin, B. M., Kishimoto, Y., Conner, G. E., Tsuji, S., & O'Brien, J. S. (1997). Lysosomal Proteolysis of Prosaposin, the Precursor of Saposins (Sphingolipid Activator Proteins): Its Mechanism and Inhibition by Ganglioside. Archives of Biochemistry and Biophysics. https://doi.org/10.1006/abbi.1997.9958