Identification of the binding and activating sites of the sphingolipid activator protein, saposin C, with glucocerebrosidase.
Saposin C is a sphingolipid activator protein of 8.5 kDa that activates lysosomal glucocerebrosidase. Previously, we synthesized and characterized a synthetic full-length human saposin C protein that displays 85% of the activity of the native saposin C. In this study we use shorter synthetic peptides derived from the saposin C sequence to map binding and activation sites. By determining the activity and kinetic constant (Kact) values of these peptides, we have identified two functional domains, each comprising a binding site adjacent to or partially overlapping with an activation site. Domains 1 and 2 are located within amino acid positions 6-34 and 41-60, respectively. The activation sites span residues 27-34 and 41-49, whereas binding sites encompass residues 6-27 and 45-60. Peptides containing the sequences of either domain displayed 90% of the activity of the full-length synthetic saposin C. Domain 2, however, bound to glucocerebrosidase by at least an order of magnitude more strongly than domain 1. Binding sites within these domains contain sequences that are excellent candidates for forming amphipathic helical structures. Competition assays demonstrated that the binding of one domain to glucocerebrosidase prevents binding of the other domain, and that saposin A and saposin C bind to the same sites on glucocerebrosidase. A model predicting a saposin C:glucocerebrosidase complex with a stoichiometry of 4:2, respectively, is presented.
Weiler
S
S
Department of Pediatrics, University of Southern California School of Medicine, Children's Hospital of Los Angeles 90027, USA.
Kishimoto
Y
Y
O'Brien
J S
JS
Barranger
J A
JA
Tomich
J M
JM
D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D017124:Conserved Sequence; D004789:Enzyme Activation; D005962:Glucosylceramidase / Q000378:metabolism*; D006023:Glycoproteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008247:Lysosomes / Q000201:enzymology; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D049231:Saposins; D013107:Sphingolipids / Q000378:metabolism
D006023:Glycoproteins; D010455:Peptides; D049231:Saposins; D013107:Sphingolipids; D005962:Glucosylceramidase