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.