title: Preliminary evidence for a processing error in the biosynthesis of Gaucher activator in mucolipidosis disease types II and III.
abstract: Activator protein (AP), which stimulated fibroblast sphingomyelinase activity, was isolated from the spleen of a patient with Gaucher's disease type I by the combined techniques of heat and alcohol denaturation, DEAE-cellulose column chromatography, gel filtration, preparative polyacrylamide-gel electrophoresis and decyl-agarose chromatography. Urea/sodium dodecyl sulphate (SDS)/polyacrylamide-gel electrophoresis showed two bands, one with an Mr of approx. 3,000 and the other with an Mr of 5,000-6,500. Similarly, SDS/polyacrylamide-gel electrophoresis performed in the absence of urea revealed the presence of two components, one of which adsorbed to a concanavalin A (Con A) column. Both components stimulated sphingomyelinase activity. On a non-denaturing polyacrylamide gel containing Triton X-100, four major components, two of which bound to Con A, were detected with the dye Stains-All. Cross-reacting material (CRM) to polyclonal Gaucher spleen AP antibodies was detected in normal fibroblasts and in fibroblasts from patients with sphingomyelinase and beta-glucocerebrosidase deficiency states (Niemann-Pick and Gaucher's diseases respectively). CRM in normal fibroblasts adsorbed to Con A columns and had the same mobility on SDS/polyacrylamide-gel electrophoresis as Con A-adsorbing Gaucher spleen AP. Normal AP was not observed in mucolipidosis type II (I-cell disease) fibroblasts; instead, extracts from these cells revealed the presence of two closely migrating bands with higher Mr values than normal fibroblast CRM. Furthermore, extracts of media from I-cell fibroblast cultures, but not from control or Gaucher fibroblast cultures, contained AP activity towards sphingomyelinase and beta-glucocerebrosidase. Fibroblasts from a patient with mucolipidosis type III (pseudo-Hurler polydystrophy) showed an intermediate pattern consisting of normal as well as the higher-Mr CRM. Our data provide evidence for the existence of AP in cultured skin fibroblasts and suggest that these proteins may be targetted to the lysosome by post-translational modification in a similar manner to that reported for lysosomal enzymes.
mesh_terms: D002478:Cells, Cultured; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000378:metabolism; D006023:Glycoproteins / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D006801:Humans; D007106:Immune Sera; D007163:Immunosorbent Techniques; D009081:Mucolipidoses / Q000378:metabolism*; D049231:Saposins; D013154:Spleen / Q000378:metabolism
chemical_list: D006023:Glycoproteins; D007106:Immune Sera; D049231:Saposins