The effect of carbohydrate removal on stability and activity of saposin B.

Abstract

Saposin B is involved in the hydrolysis of sulfatides, GM1 ganglioside, globotriaosylceramide, and several other sphingolipids and glycerolipids by lysosomal hydrolases. Saposin B is one of four small glycoproteins (saposins) derived from prosaposin. The carbohydrate chain of saposin B was removed and deglycosylated saposin B was characterized and compared with native saposin B. Deglycosylated saposin B stimulated the enzymatic hydrolysis of ganglioside GM1 by acid beta-galactosidase and sulfatide by arylsulfatase A to the same extent as native saposin B. In addition deglycosylated saposin B bound sulfatide and GM1 ganglioside identical to native saposin B. The stability of native saposin B to proteolytic digestion was unchanged by deglycosylation. Neither native saposin B nor deglycosylated saposin B were hydrolyzed by trypsin, endoproteinase Glu-C (V-8), chymotrypsin, or a mixture of acid proteases isolated from human testis. Unlike its effect on metabolic stability, the carbohydrate chain appears to affect folding of saposin B. When native and deglycosylated saposin B were reduced under denaturing conditions and refolded under identical conditions examination of the refolded products indicated that each protein was refolded in a qualitatively different way. A human mutation in saposin B-deficient metachromatic leukodystrophy, in which its glycosylation site is eliminated, has been reported. Our observations suggest that instability of the mutated saposin B is not due to the absence of a protective effect of the carbohydrate chain on proteolysis, but is likely due to aberrant folding resulting from the absence of a carbohydrate chain.

• Authors: M Hiraiwa, S Soeda, B M Martin, A L Fluharty, Y Hirabayashi, J S O'Brien, Y Kishimoto
• Journal: Archives of biochemistry and biophysics (Arch Biochem Biophys) Vol. 303 Issue 2 Pg. 326-31 (Jun 1993) ISSN: 0003-9861 [Print] United States
• PMID: 8099782 (Publication Type: Journal Article, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Carbohydrates
• Glycoproteins
• PSAP protein, human
• Saposins
• Sphingolipid Activator Proteins
• Sulfoglycosphingolipids
• G(M1) Ganglioside
• Cerebroside-Sulfatase
• beta-Galactosidase

Topics

• Carbohydrates (chemistry)
• Cerebroside-Sulfatase (metabolism)
• Chromatography, High Pressure Liquid
• Drug Stability
• Electrophoresis, Polyacrylamide Gel
• G(M1) Ganglioside (metabolism)
• Gaucher Disease (metabolism)
• Glycoproteins (chemistry, metabolism, pharmacology)
• Glycosylation
• Humans
• Hydrolysis
• Saposins
• Sphingolipid Activator Proteins
• Spleen (chemistry)
• Structure-Activity Relationship
• Sulfoglycosphingolipids (metabolism)
• beta-Galactosidase (metabolism)