The effect of carbohydrate removal on stability and activity of saposin B.
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.
Hiraiwa
M
M
Department of Neurosciences, University of California, San Diego, La Jolla 92093-0634.
Soeda
S
S
Martin
B M
BM
Fluharty
A L
AL
Hirabayashi
Y
Y
O'Brien
J S
JS
Kishimoto
Y
Y
D002241:Carbohydrates / Q000737:chemistry*; D002553:Cerebroside-Sulfatase / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D005677:G(M1) Ganglioside / Q000378:metabolism; D005776:Gaucher Disease / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D006031:Glycosylation; D006801:Humans; D006868:Hydrolysis; D049231:Saposins; D049128:Sphingolipid Activator Proteins; D013154:Spleen / Q000737:chemistry; D013329:Structure-Activity Relationship; D013433:Sulfoglycosphingolipids / Q000378:metabolism; D001616:beta-Galactosidase / Q000378:metabolism
D002241:Carbohydrates; D006023:Glycoproteins; C483345:PSAP protein, human; D049231:Saposins; D049128:Sphingolipid Activator Proteins; D013433:Sulfoglycosphingolipids; D005677:G(M1) Ganglioside; D002553:Cerebroside-Sulfatase; D001616:beta-Galactosidase